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15 pags., 8 figs., 3 tabs., The phosphotransferase system (PTS), a metabolic pathway formed by five proteins, modulates the use of sugars in bacteria. The second protein in the chain is the histidine phosphocarrier, HPr, with the binding site at His15. The HPr kinase/phosphorylase (HPrK/P), involved in the bacterial use of carbon sources, phosphorylates HPr at Ser46, and it binds at its binding site. The regulator of sigma D protein (Rsd) also binds to HPr at His15. We have designed fragments of HPr, growing from its N-terminus and containing the His15. In this work, we obtained three fragments, HPr38, HPr58 and HPr70, comprising the first thirty-eight, fifty-eight and seventy residues of HPr, respectively. All fragments were mainly disordered, with evidence of a weak native-like, helical population around the binding site, as shown by fluorescence, far-ultraviolet circular dichroism, size exclusion chromatography and nuclear magnetic resonance. Although HPr38, HPr58 and HPr70 were disordered, they could bind to: (i) the N-terminal domain of first protein of the PTS, EIN; (ii) Rsd; and, (iii) HPrK/P, as shown by fluorescence and biolayer interferometry (BLI). The association constants for each protein to any of the fragments were in the low micromolar range, within the same range than those measured in the binding of HPr to each protein. Then, although acquisition of stable, native-like secondary and tertiary structures occurred at the last residues of the polypeptide, the ability to bind protein partners happened much earlier in the growing chain. Binding was related to the presence of the native-like structure around His15., This research has been funded by la Consellería de Innovación, Universidades, Ciencia y Sociedad Digital (Generalitat Valenciana, Valencian Regional Government) [CIAICO 2021/0135 to JLN]. Some of the NMR equipment used in this work has been funded by Generalitat Valenciana (Spain) and co-financed with ERDF funds (OP ERDF of Generalitat Valenciana (Spain) 2014–2020). The funders did not have any role during acquisition and analyses of experiments or in the publication of this manuscript.

11 pags, 6 figs, 3 tabs., Background: The phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. It is formed by a protein cascade in which the first two proteins are general (namely enzyme I, EI, and the histidine phosphocarrier protein, HPr) and the others are sugar-specific permeases; the active site of HPr is His15. The HPr kinase/phosphorylase (HPrK/P), involved in the use of carbon sources in Gram-positive, phopshorylates HPr at a serine. The regulator of sigma D protein (Rsd) also binds to HPr. We are designing specific fragments of HPr, which can be used to interfere with those protein-protein interactions (PPIs), where the intact HPr intervenes. Methods: We obtained a fragment (HPr48) comprising the first forty-eight residues of HPr. HPr48 was disordered as shown by fluorescence, far-ultraviolet (UV) circular dichroism (CD), small angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR)., Results: Secondary structure propensities, from the assigned backbone nuclei, further support the unfolded nature of the fragment. However, HPr48 was capable of binding to: (i) the N-terminal region of EI, EIN; (ii) the intact Rsd; and, (iii) HPrK/P, as shown by fluorescence, far-UV CD, NMR and biolayer interferometry (BLI). The association constants for each protein, as measured by fluorescence and BLI, were in the order of the low micromolar range, similar to those measured between the intact HPr and each of the other macromolecules., Conclusions: Although HPr48 is forty-eight-residue long, it assisted antibiotics to exert antimicrobial activity., General significance: HPr48 could be used as a lead compound in the development of new antibiotics, or, alternatively, to improve the efficiency of existing ones, This research was funded by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN, RTI2018-101969-J-I00 to AF]. Some of the NMR equipment used in this work have been funded by Generalitat Valenciana (Spain) and co-financed with ERDF funds (OP ERDF of Comunitat Valenciana (Spain) 2014-2020).

The conserved C-terminal end segment of troponin I (TnI) plays a critical role in regulating muscle relaxation. This function is retained in the isolated C-terminal 27 amino acid peptide (residues 184-210) of human cardiac TnI (HcTnI-C27): When added to skinned muscle fibers, HcTnI-C27 reduces the Ca2+-sensitivity of activated myofibrils and facilitates relaxation without decreasing the maximum force production. However, the underlying mechanism of HcTnI-C27 function is unknown. We studied the conformational preferences of HcTnI-C27 and a myopathic mutant, Arg192His, (HcTnI-C27-H). Both peptides were mainly disordered in aqueous solution with a nascent helix involving residues from Trp191 to Ile195, as shown by NMR analysis and molecular dynamics simulations. The population of nascent helix was smaller in HcTnI-C27-H than in HcTnI-C27, as shown by circular dichroism (CD) titrations. Fluorescence and isothermal titration calorimetry (ITC) showed that both peptides bound tropomyosin (alpha Tm), with a detectably higher affinity (similar to 10 mu M) of HcTnI-C27 than that of HcTnI-C27-H (similar to 15 mu M), consistent with an impaired Ca2+-desensitization effect of the mutant peptide on skinned muscle strips. Upon binding to aTm, HcTnI-C27 acquired a weakly stable helix-like conformation involving residues near Trp191, as shown by transferred nuclear Overhauser effect spectroscopy and hydrogen/deuterium exchange experiments. With the potent Ca2+-desensitization effect of HcTnI-C27 on skinned cardiac muscle from a mouse model of hypertrophic cardiomyopathy, the data support that the C-terminal end domain of TnI can function as an isolated peptide with the intrinsic capacity of binding tropomyosin, providing a promising therapeutic approach to selectively improve diastolic function of the heart.

23 pags., 5 figs., 3 tabs. -- This article belongs to the Special Issue Structural, Functional and Folding Strategies of Oligomeric Proteins, The histidine phosphocarrier protein (HPr) kinase/phosphorylase (HPrK/P) modulates the phosphorylation state of the HPr protein, and it is involved in the use of carbon sources by Gram-positive bacteria. Its X-ray structure, as concluded from crystals of proteins from several species, is a hexamer; however, there are no studies about its conformational stability, and how its structure is modified by the pH. We have embarked on the conformational characterization of HPrK/P of Bacillus subtilis (bsHPrK/P) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, small-angle X-ray-scattering (SAXS) and dynamic light-scattering (DLS)). bsHPrK/P was mainly a hexamer in solution at pH 7.0, in the presence of phosphate. The protein had a high conformational stability, with an apparent thermal denaturation midpoint of ~70 °C, at pH 7.0, as monitored by fluorescence and CD. The protein was very pH-sensitive, precipitated between pH 3.5 and 6.5; below pH 3.5, it had a molten-globule-like conformation; and it acquired a native-like structure in a narrow pH range (between pH 7.0 and 8.0). Guanidinium hydrochloride (GdmCl) denaturation occurred through an oligomeric intermediate. On the other hand, urea denaturation occurred as a single transition, in the range of concentrations between 1.8 and 18 µM, as detected by far-UV CD and fluorescence., This research was funded by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN, BIO2016-78020-R to ACA and CTQ2017-85425-P to JGHC]; JGHC thanks Fundación Séneca, Región de Murcia for funding (20933/PI/18).

11 pags., 6 figs., 2 tabs., Neurofibromin-1 (NF1) is a large, multidomain tumour suppressor encoded by the NF1 gene. The gene is mutated in neurofibromatosis type I, a disease characterized by malignant tumours of the nervous system and benign neurofibromas. The best-known activity of NF1 is the down-regulation of the mitogen-activated protein kinase pathway via its three-hundred-residue-long GTPase-activating protein (GAP) domain (the so-called GAP-related domain (NF1-GRD)). The NF1-GRD stimulates Ras GTPase activity in turning off signalling. Despite this activity, NF1-GRD has been demonstrated to bind to other different proteins, such as SPRED1 or MC1R. We have embarked on the biophysical and conformational characterization of NF1-GRD in solution by using several spectroscopic (namely fluorescence and circular dichroism (CD)) and biophysical techniques (namely size exclusion chromatography (SEC) and differential scanning calorimetry (DSC)). This biophysical characterization is crucial in deciphering NF1-GRD interactome and in finding biochemical features, modulating possible protein interactions. The native-like structure of NF1-GRD (as monitored by intrinsic fluorescence and far-UV CD) was strongly pH-dependent showing a pH-titration causing a substantial increase in its helicity. NF1-GRD had a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by intrinsic and ANS fluorescence, and CD. Chemical denaturations showed that NF1-GRD unfolded through an intermediate which has a substantial amount of solvent-exposed hydrophobic patches., This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN, BFU2016-78232-P to AVC]; Andalusian Regional Government [“Endocrinology and Metabolism Group” CTS- 202 to SMR]; Diputacion General de Aragon [“Protein targets and Bioactive Compounds group” E45-20R to AVC].

18 pags., 4 figs., 5 tabs. -- This article belongs to the Special Issue Folding and Design of α-Helical Proteins and Peptides: Theory Meets Nanomaterials, Biotechnology and Health, The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr is mainly disordered. We attempted to improve the affinity of HPr to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. The refore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function., This research was funded by the Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) (RTI2018-097991-B-I00 to J.L.N., BFU2016-78232-P to A.V.-C., BES-2017-080739 to D.O.-A., and RTI2018-101969-J-I00 to A.F.). The NMR equipment used in this work was funded by Generalitat Valenciana (Spain) and cofinanced with ERDF funds (OP ERDF of Comunitat Valenciana (Spain) 2014–2020).

12 pags, 7 figs. -- Supplementary material includes five figures containing: (i) the fluorescence spectrum of the mixture containing prothymosin α and ARM-PKP1 (Fig. S1); (ii) the fluorescence titration curve of SAMp73 and ARM-PKP1 in the presence of 100 mM NaCl (Fig. S2); (iii) the 2D 15N, 1H- HSQC-NMR spectra of isolated SAMp73 and in the presence of ARM-PKP1 in 100 mM NaCl; (iv) binding of ARM-PKP1 and SAMp73 as monitored by ITC in 100 mM of NaCl (Fig. S4); and (v) the fluorescence titrations of the fragments α1α2 and α4α5 with ARM-PKP1 (Fig. S5). Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbagen.2021.129914, Plakophilin 1 (PKP1) is a component of desmosomes, which are key structural components for cell-cell adhesion, and can also be found in other cell locations. The p53, p63 and p73 proteins belong to the p53 family of transcription factors, playing crucial roles in tumour suppression. The α-splice variant of p73 (p73α) has at its C terminus a sterile alpha motif (SAM); such domain, SAMp73, is involved in the interaction with other macromolecules., This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN; BFU2016-78232-P to AVC; BES-2017-080739 to DOA]; Fondo de Investigaciones Sanitarias from Instituto de Salud Carlos III, and European Union (ERDF/ESF, Investing in your future') [PI15/00663 and PI18/00349 to OA]; PAIDI program, Group BIO309 (Junta de Andalucia) to MEF-V; Diputacion General de Aragon [Protein Targets and Bioactive Compounds Group E45_20R to AVC, and Digestive Pathology Group B25_20R to OA]; and Centro de Investigacion Biomedica en Red en Enfermedades Hepaticas y Digestivas (CIBERehd).

Numerous carrier proteins intervene in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin &alpha, with several human isoforms, among them, importin &alpha, 3 (Imp&alpha, 3) features a particularly high flexibility. The protein NUPR1L is an intrinsically disordered protein (IDP), evolved as a paralogue of nuclear protein 1 (NUPR1), which is involved in chromatin remodeling and DNA repair. It is predicted that NUPR1L has a nuclear localization sequence (NLS) from residues Arg51 to Gln74, in order to allow for nuclear translocation. We studied in this work the ability of intact NUPR1L to bind Imp&alpha, 3 and its depleted species, ∆Imp&alpha, 3, without the importin binding domain (IBB), using fluorescence, isothermal titration calorimetry (ITC), circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular docking techniques. Furthermore, the binding of the peptide matching the isolated NLS region of NUPR1L (NLS-NUPR1L) was also studied using the same methods. Our results show that NUPR1L was bound to Imp &alpha, 3 with a low micromolar affinity (~5 &mu, M). Furthermore, a similar affinity value was observed for the binding of NLS-NUPR1L. These findings indicate that the NLS region, which was unfolded in isolation in solution, was essentially responsible for the binding of NUPR1L to both importin species. This result was also confirmed by our in silico modeling. The binding reaction of NLS-NUPR1L to ∆Imp&alpha, 3 showed a larger affinity (i.e., lower dissociation constant) compared with that of Imp&alpha, 3, confirming that the IBB could act as an auto-inhibition region of Imp&alpha, 3. Taken together, our findings pinpoint the theoretical predictions of the NLS region in NUPR1L and, more importantly, suggest that this IDP relies on an importin for its nuclear translocation.

Several carrier proteins are involved in protein transport from the cytoplasm to the nucleus in eukaryotic cells. One of those is importin &alpha, of which there are several human isoforms, among them, importin &alpha, 3 (Imp&alpha, 3) has a high flexibility. The protein NUPR1, a nuclear protein involved in the cell-stress response and cell cycle regulation, is an intrinsically disordered protein (IDP) that has a nuclear localization sequence (NLS) to allow for nuclear translocation. NUPR1 does localize through the whole cell. In this work, we studied the affinity of the isolated wild-type NLS region (residues 54&ndash, 74) of NUPR1 towards Imp&alpha, 3 and several mutants of the NLS region by using several biophysical techniques and molecular docking approaches. The NLS region of NUPR1 interacted with Imp&alpha, 3, opening the way to model the nuclear translocation of disordered proteins. All the isolated NLS peptides were disordered. They bound to Imp&alpha, 3 with low micromolar affinity (1.7&ndash, 27 &mu, M). Binding was hampered by removal of either Lys65 or Lys69 residues, indicating that positive charges were important, furthermore, binding decreased when Thr68 was phosphorylated. The peptide phosphorylated at Thr68, as well as four phospho-mimetic peptides (all containing the Thr68Glu mutation), showed the presence of a sequential NN(i,i + 1) nuclear Overhauser effect (NOE) in the 2D-1H-NMR (two-dimensional&ndash, proton NMR) spectra, indicating the presence of turn-like conformations. Thus, the phosphorylation of Thr68 modulates the binding of NUPR1 to Imp&alpha, 3 by a conformational, entropy-driven switch from a random-coil conformation to a turn-like structure.

Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS type I is regulated by protein-protein interactions with a 65-residue-long protein (IF7). IF7 binds initially to GS through residues at its N terminus. In this work, we studied the conformational preferences of the N-terminal region of IF7 (IF7pep, residues Ala7-Ala29), its binding to GS and its functional properties. Isolated IF7pep populated a nascent helix in aqueous solution. IF7pep was bound to GS with an affinity constant of 0.4 μM, and a 1:1 stoichiometry. IF7pep did not inactivate GS, suggesting that there were other IF7 regions important to carry out the inactivating function. Binding of IF7pep to GS was electrostatically-driven and it did not follow a kinetic two-state model.

Glutamine synthetase (GS) catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia. The activity of Synechocystis sp. PCC 6803 GS is regulated, among other mechanisms, by protein-protein interactions with a 65-residue-long, intrinsically disordered protein (IDP), named IF7. IDPs explore diverse conformations in their free states and, in some cases, in their molecular complexes. We used both nuclear magnetic resonance (NMR) at 11.7 T and small angle X-ray scattering (SAXS) to study the size and the dynamics in the picoseconds-to-nanosecond (ps-ns) timescale of: (i) isolated IF7; and (ii) the IF7/GS complex. Our SAXS findings, together with MD results, show: (i) some of the possible IF7 structures in solution; and, (ii) that the presence of IF7 affected the structure of GS in solution. The joint use of SAXS and NMR shows that movements of each amino acid of IF7 were uncorrelated with those of its neighbors. Residues of IF7 with the largest values of the relaxation rates (R1, R2 and ηxy), in the free and bound species, were mainly clustered around: (i) the C terminus of the protein; and (ii) Ala30. These residues, together with Arg8 (which is a hot-spot residue in the interaction with GS), had a restricted mobility in the presence of GS. The C-terminal region, which appeared more compact in our MD simulations of isolated IF7, seemed to be involved in non-native contacts with GS that help in the binding between the two macromolecules. Ministerio de Economía y Competitividad RTI 2018-097991- BI00

[Background]: Eukaryotic cells have a continuous transit of macromolecules between the cytoplasm and the nucleus. Several carrier proteins are involved in this transport. One of them is importin α, which must form a complex with importin β to accomplish its function, by domain-swapping its 60-residue-long N terminus. There are several human isoforms of importin α; among them, importin α3 has a particularly high flexibility., [Methods]: We studied the conformational stability of intact importin α3 (Impα3) and its truncated form, where the 64-residue-long, N-terminal importin-β-binding domain (IBB) has been removed (ΔImpα3), in a wide pH range, with several spectroscopic, biophysical, biochemical methods and with molecular dynamics (MD)., [Results]: Both species acquired native-like structure between pH 7 and 10.0, where Impα3 was a dimer (with an apparent self-association constant of ~10 μM) and ΔImpα3 had a higher tendency to self-associate than the intact species. The acquisition of secondary, tertiary and quaternary structure, and the burial of hydrophobic patches, occurred concomitantly. Both proteins unfolded irreversibly at physiological pH, by using either temperature or chemical denaturants, through several partially folded intermediates. The MD simulations support the presence of these intermediates., [Conclusions]: The thermal stability of Impα3 at physiological pH was very low, but was higher than that of ΔImpα3. Both proteins were stable in a narrow pH range, and they unfolded at physiological pH populating several intermediate species., [General significance]: The low conformational stability explains the flexibility of Impα3, which is needed to carry out its recognition of complex cargo sequences., This work was supported by Spanish Ministry of Economy and Competitiveness and European FEDER Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-B-I00 to JLN and JG, BFU2016-75471-C2-1-P to CA, PGC2018-094548-B-I00 to AA and BIO2016-78020-R to ACA], Basque Country [IT-1175-19 to AA], GVA and EU-FEDER funds “Una forma de hacer Europa” [GVA-IDIFEDER 2018/020] and Portuguese FCT [UIDB/04565/2020 and SAICTPAC/0019/2015 to AC and MP]. AUC SV assays were performed at the Molecular Interactions Facility at the CIB Margarita Salas. CD-G acknowledges Medical Biochemistry and Biophysics Doctoral Programme (M2B-PhD) FCT reference: SFRH/PD/BD/135154/2017. BR acknowledges the kind hospitality and use of computational resources in the European Magnetic Resonance Center (CERM), Sesto Fiorentino (Florence), Italy.

The assembly of the protein complex of cytochrome c oxidase (COX), which participates in the mitochondrial respiratory chain, requires a large number of accessory proteins (the so-called assembly factors). Human COX assembly factor 3 (hCOA3), also known as MITRAC12 or coiled-coil domain-containing protein 56 (CCDC56), interacts with the first subunit protein of COX to form its catalytic core and promotes its assemblage with the other units. Therefore, hCOA3 is involved in COX biogenesis in humans and can be exploited as a drug target in patients with mitochondrial dysfunctions. However, to be considered a molecular target, its structure and conformational stability must first be elucidated. We have embarked on the description of such features by using spectroscopic and hydrodynamic techniques, in aqueous solution and in the presence of detergents, together with computational methods. Our results show that hCOA3 is an oligomeric protein, forming aggregates of different molecular masses in aqueous solution. Moreover, on the basis of fluorescence and circular dichroism results, the protein has (i) its unique tryptophan partially shielded from solvent and (ii) a relatively high percentage of secondary structure. However, this structure is highly flexible and does not involve hydrogen bonding. Experiments in the presence of detergents suggest a slightly higher content of nonrigid helical structure. Theoretical results, based on studies of the primary structure of the protein, further support the idea that hCOA3 is a disordered protein. We suggest that the flexibility of hCOA3 is crucial for its interaction with other proteins to favor mitochondrial protein translocation and assembly of proteins involved in the respiratory chain.

10 pags., 6 figs., 1 tab., Plakophilin 1 (PKP1) is a member of the armadillo repeat family of proteins. It serves as a scaffold component of desmosomes, which are key structural components for cell–cell adhesion. We have embarked on the biophysical and conformational characterization of the ARM domain of PKP1 (ARM-PKP1) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, analytical ultracentrifugation (AUC), dynamic light scattering (DLS) and differential scanning calorimetry (DSC)). ARM-PKP1 was a monomer in solution at physiological pH, with a low conformational stability, as concluded from DSC experiments and thermal denaturations followed by fluorescence and CD. The presence or absence of disulphide bridges did not affect its low stability. The protein unfolded through an intermediate which has lost native-like secondary structure. ARM-PKP1 acquired a native-like structure in a narrow pH range (between pH 6.0 and 8.0), indicating that its adherent properties might only work in a very narrow pH range., This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/ERDF, EU) [RTI2018-097991-B-I00 to JLN, BIO2016-78020-R to ACA, CTQ2017-85425-P to JGHC] and by the PAIDI program, Group BIO309 (Junta de Andalucía) to MEFV. JGHC thanks Fundación Séneca, Región de Murcia for funding (20933/PI/18).

7 pags., 4 figs., BRMS1 is a 246-residue-long protein belonging to the family of metastasis suppressors. It is a predominantly nuclear protein, although it can also function in the cytoplasm. At its C terminus, it has a region that is predicted to be a nuclear localization sequence (NLS); this region, NLS2, is necessary for metastasis suppression. We have studied in vitro and in silico the conformational preferences in aqueous solution of a peptide (NLS2-pep) that comprises the NLS2 of BRMS1, to test whether it has a preferred conformation that could be responsible for its function. Our spectroscopic (far-UV circular dichroism, DOSY-NMR and 2D-NMR) and computational (all-atom molecular dynamics) results indicate that NLS2-pep was disordered in aqueous solution. Furthermore, it did not acquire a structure even when experiments were performed in a more hydrophobic environment, such as the one provided by 2,2,2-trifluoroethanol (TFE). The hydrodynamic radius of the peptide in water was identical to that of a random-coil sequence, in agreement with both our molecular simulations and other theoretical predictions. Thus, we suggest that NLS2 is a disordered region, with non pre-formed structure, that participates in metastasis suppression., This work was supported by Spanish Ministry of Economy and Competitiveness and European Funds [CTQ2015-64445-R (to JLN)].Partial support was received from Susan G. Komen for the Cure [SAC11037], the National Foundation for Cancer Research and theAmerican Cancer Society [PF-16-227-O1-CSM]. DP-U acknowledges theuse of the“Manuel Rico”NMR laboratory, LMR (CSIC), a Spanish large-scale national NMR facility ICTS R-LRB. BR acknowledges kind hospi-tality and use of computational resources in the European Magnetic Resonance Center (CERM), Sesto Fiorentino (Florence), Italy

15 pags., 6 figs., 3 tabs. -- Open Access funded by Creative Commons Atribution Licence 4.0, p53 is frequently mutated in human cancers. Its levels are tightly regulated by the E3 ubiquitin ligase MDM2. The complex between MDM2 and p53 is largely formed by the interaction between the N-terminal domain of MDM2 and the N-terminal transactivation (TA) domain of p53 (residues 15–29). We investigated the kinetic and thermodynamic basis of the MDM2/p53 interaction by using wild-type and mutant variants of the TA domain. We focus on the effects of phosphorylation at positions Thr18 and Ser20 including their substitution with phosphomimetics. Conformational propensities of the isolated peptides were investigated using in silico methods and experimentally by circular dichroism and H-NMR in aqueous solution. Both experimental and computational analyses indicate that the p53 peptides are mainly disordered in aqueous solution, with evidence of nascent helix around the Ser20-Leu25 region. Both phosphorylation and the phosphomimetics at Thr18 result in a decrease in the binding affinity by ten- to twenty-fold when compared to the wild-type. Phosphorylation and phosphomimetics at Ser20 result in a smaller decrease in the affinity. Mutation of Lys24 and Leu25 also disrupts the interaction. Our results may be useful for further development of peptide-based drugs targeting the MDM2/p53 interaction., This work was supported by Spanish Ministry of Economy and Competitiveness [CTQ 2015–64445-R (to JLN)]. JLN acknowledges the regional government Generalitat Valenciana for a BEST short-stay fellowship in the Department of Pharmacology, Cambridge, UK. LSI acknowledges support of a Senior Fellowship from the Medical Research Foundation (UK).

Background: The p53, p63 and p73 proteins belong to the p53 family of transcription factors, playing key roles in tumour suppression. The α-splice variant of p73 (p73α) has at its C terminus a sterile alpha motif (SAM); this domain, SAMp73, formed by five helices (α1 to α5), is thought to mediate in protein-protein interactions. The E3-ligase MDM2 binds to p73 at its N terminus transactivation domain (TA), but it does not promote its degradation via ubiquitination; however, the details of such MDM2/p73 interaction are not fully known. Methods: We studied the binding of SAMp73 with N-terminal MDM2, by several biophysical techniques, namely, fluorescence, far-UV circular dichroism (CD), NMR and bio-layer interferometry (BLI). Results: Our results obtained by fluorescence, T -relaxation measurements and BLI show that there was binding between both proteins with a dissociation constant of ~10 μM. Furthermore, the binding region of SAMp73 involved mainly residues in the major α-helix, α5, and the nearby α4, as shown by HSQC-NMR. The binding was so specific that an isolated peptide comprising α4 and α5 helices of SAMp73, α4α5, did also bind to the N terminus of MDM2, although with weaker affinity than the entire domain. Conclusions: A new interaction between MDM2 and SAMp73 has been found, which could have potential therapeutic applications in cancers involving inactivated p53. General significance: A novel interaction between the C-terminal SAM of p73 and N-terminal MDM2 is described. The interaction could be used to modulate the functions where the wild-type, intact p73 is involved., This work was supported by Spanish Ministry of Economy and Competitiveness and European FEDER Funds [CTQ2015-64445-R to JLN] and Portuguese FCT [FAPESP/20107/2014 and SAICTPAC/0019/2015 to AC and MP]. CD-G acknowledges Medical Biochemistry and Biophysics Doctoral Programme (M2B-PhD) FCT reference: SFRH/PD/BD/135154/2017.

The phosphotransferase system (PTS) controls the preferential use of sugars in bacteria and it is also involved in other processes, such as chemotaxis. It is formed by a protein cascade in which the first two proteins are general (namely, EI and HPr) and the others are sugar-specific permeases. The Rsd protein binds specifically to the RNA polymerase (RNAP) σ factor. We first characterized the conformational stability of Escherichia coli Rsd. And second, we delineated the binding regions of Streptomyces coelicolor, HPr, and E. coli Rsd, by using fragments derived from each protein. To that end, we used several biophysical probes, namely, fluorescence, CD, NMR, ITC and BLI. Rsd had a free energy of unfolding of 15 kcal mol at 25 °C, and a thermal denaturation midpoint of 103 °C at pH 6.5. The affinity between Rsd and HPr was 2 μM. Interestingly enough, the isolated helical-peptides, comprising the third (RsdH3) and fourth (RsdH4) Rsd helices, also interacted with HPr in a specific manner, and with affinities similar to that of the whole Rsd. Moreover, the isolated peptide of HPr, HPr, comprising the active site, His15, also was bound to intact Rsd with similar affinity. Therefore, binding between Rsd and HPr was modulated by the two helices H3 and H4 of Rsd, and the regions around the active site of HPr. This implies that specific fragments of Rsd and HPr can be used to interfere with other protein-protein interactions (PPIs) of each other protein.

The LrtA protein of Synechocystis sp. PCC 6803 intervenes in cyanobacterial post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family of proteins, involved in protein synthesis. In this work, we studied the conformational preferences and stability of isolated LrtA in solution. At physiological conditions, as shown by hydrodynamic techniques, LrtA was involved in a self-association equilibrium. As indicated by Nuclear Magnetic Resonance (NMR), circular dichroism (CD) and fluorescence, the protein acquired a folded, native-like conformation between pH 6.0 and 9.0. However, that conformation was not very stable, as suggested by thermal and chemical denaturations followed by CD and fluorescence. Theoretical studies of its highly-charged sequence suggest that LrtA had a Janus sequence, with a context-dependent fold. Our modelling and molecular dynamics (MD) simulations indicate that the protein adopted the same fold observed in other members of the HPF family (&beta, &alpha, &beta, ) at its N-terminal region (residues 1&ndash, 100), whereas the C terminus (residues 100&ndash, 197) appeared disordered and collapsed, supporting the overall percentage of overall secondary structure obtained by CD deconvolution. Then, LrtA has a chameleonic sequence and it is the first member of the HPF family involved in a self-association equilibrium, when isolated in solution.

This article belongs to the Special Issue Functionally Relevant Macromolecular Interactions of Disordered Proteins., The 191-residue-long LrtA protein of Synechocystis sp. PCC 6803 is involved in post-stress survival and in stabilizing 70S ribosomal particles. It belongs to the hibernating promoting factor (HPF) family, intervening in protein synthesis. The protein consists of two domains: The N-terminal region (N-LrtA, residues 1⁻101), which is common to all the members of the HPF, and seems to be well-folded; and the C-terminal region (C-LrtA, residues 102⁻191), which is hypothesized to be disordered. In this work, we studied the conformational preferences of isolated C-LrtA in solution. The protein was disordered, as shown by computational modelling, 1D-¹H NMR, steady-state far-UV circular dichroism (CD) and chemical and thermal denaturations followed by fluorescence and far-UV CD. Moreover, at physiological conditions, as indicated by several biochemical and hydrodynamic techniques, isolated C-LrtA intervened in a self-association equilibrium, involving several oligomerization reactions. Thus, C-LrtA was an oligomeric disordered protein., This research was funded by Spanish Ministry of Economy and Competitiveness [CTQ2015-64445-R (to J.L.N.) and MAT2015-63704-P (to A.A.), with Fondo Social Europeo (ESF)], and by the Basque Government [IT-654-13 (to A.A.)].

NUPR1 is a protumoral multifunctional intrinsically disordered protein (IDP), which is activated during the acute phases of pancreatitis. It interacts with other IDPs such as prothymosin α, as well as with folded proteins such as the C-terminal region of RING1-B (C-RING1B) of the Polycomb complex; in all those interactions, residues around Ala33 and Thr68 (the ‘hot-spot’ region) of NUPR1 intervene. Its paralogue, NUPR1L, is also expressed in response to DNA damage, it is p53-regulated, and its expression down-regulates that of the NUPR1 gene. In this work, we characterized the conformational preferences of isolated NUPR1L and its possible interactions with the same molecular partners of NUPR1. Our results show that NUPR1L was an oligomeric IDP from pH 2.0 to 12.0, as judged by steady-state fluorescence, circular dichroism (CD), dynamic light scattering, 1D 1H-NMR (nuclear magnetic resonance), and as indicated by structural modelling. However, in contrast with NUPR1, there was evidence of local helical- or turn-like structures; these structures were not rigid, as judged by the lack of sigmoidal behaviour in the chemical and thermal denaturation curves obtained by CD and fluorescence. Interestingly enough, NUPR1L interacted with prothymosin α and C-RING1B, and with a similar affinity to that of NUPR1 (in the low micromolar range). Moreover, NUPR1L hetero-associated with NUPR1 with an affinity of 0.4 µM and interacted with the ‘hot-spot’ region of NUPR1. Thus, we suggest that the regulation of NUPR1 gene by NUPR1L does not only happen at the DNA level, but it could also involve direct interactions with NUPR1 natural partners.

The alpha splice variant of p73 (p73α), a homologue of the tumour suppressor p53, has at its C terminus a sterile alpha motif (SAM); this domain, SAMp73, is involved in lipid binding and it is thought to mediate in protein-protein interactions. SAMp73 is composed of five helices (α1-α5). In this work, we dissected SAMp73 in fragments encompassing the different helices, to study the conformational stability of the isolated elements of secondary structure. There was no evidence of stable residual helical structure in the isolated α1, α4 and α5 helices in aqueous solution, as shown by 2D-(1)H NMR and far-UV CD spectroscopies; those helices acquired native-like helical structure in the presence of 40% trifluoroethanol (TFE). The population of helical structure in α5 seemed to be driven by the indole moiety of Trp542, and it was enhanced by the presence of α4. On the other hand, helices α2 and 310(α3) had a tendency to self-associate even in TFE-water solutions. However, the short, aggregation-prone 310(α3) helix was key to attain the native-like fold of SAMp73, as suggested by experiments with non-covalent complexes among the peptides.

The alpha splice variant of p73 (p73α), a homologue of the tumour suppressor p53, has at its C terminus a sterile alpha motif (SAM); this domain, SAMp73, is involved in lipid binding and it is thought to mediate in protein-protein interactions. As SAMp73 is a 68-residue-long helical bundle, it could be a good model to study the (2,2,2-trifluoroethanol) TFE-induced conformational transitions of α-helical proteins. Furthermore, as SAMp73 binds to lipids through a well-known polypeptide patch, we can test whether TFE is a good mimic of lipids and membranes. To address those questions, we used several biophysical probes, namely, fluorescence, circular dichroism, 1D, 2D and 3D-NMR spectroscopies, and dynamic light scattering. The TFE-induced conformational transition of SAMp73 was complex, involving several species as detected by the biophysical probes. The last TFE-induced transition occurred at a concentration of TFE of ∼20% (v/v), where the protein lost its compactness. None of those TFE-induced species accumulated during the two-state folding of SAMp73 in aqueous solution. The final state at 40% TFE was highly helical, but its structure was not rigid. For SAMp73, TFE did not properly mimic a membrane-like environment, since at very low TFE concentrations, other residues, together with those known to interact with lipids, were also affected by the co-solvent. Comparison with studies on isolated peptides, comprising the helical regions of SAMp73, suggests that peptides were good models of the intact protein in TFE.

Tyrosine hydroxylase (TyrH) catalyzes the hydroxylation of tyrosine to form 3,4-dihydroxyphenylalanine, the first step in the synthesis of catecholamine neurotransmitters. The protein contains a 159-residue regulatory domain (RD) at its N-terminus that forms dimers in solution; the N-terminal region of RDTyrH (residues 1-71) is absent in the solution structure of the domain. We have characterized the conformational stability of two species of RDTyrH (one containing the N-terminal region and another lacking the first 64 residues) to clarify how that N-terminal region modulates the conformational stability of RD. Under the conditions used in this study, the RD species lacking the first 64 residues is a monomer at pH 7.0, with a small conformational stability at 25 °C (4.7 ± 0.8 kcal mol(-1)). On the other hand, the entire RDTyrH is dimeric at physiological pH, with an estimated dissociation constant of 1.6 μM, as determined by zonal gel filtration chromatography; dimer dissociation was spectroscopically silent to circular dichroism but not to fluoresecence. Both RD species were disordered below physiological pH, but the acquisition of secondary native-like structure occurs at pHs lower than those measured for the attainment of tertiary native- and compactness-like arrangements.

E2 ubiquitin conjugases are ∼ 20 kDa enzymes involved in ubiquitination processes in eukaryotes. The E2s are responsible for the transference of ubiquitin (Ub) to E3 enzymes, which finally transfer Ub to diverse target proteins, labelling them for degradation, localization and regulation. Although their functions are relatively well-characterized, their conformational stabilities are poorly known. In this work, we have used, as a model for our biophysical and binding studies, the E2-C from Carassius auratus (goldfish), a homologue of the human ubiquitin conjugase UbcH10. E2-C ca was a monomeric protein with an elongated shape; moreover, the protein was only marginally stable within a narrow pH range (from 6.0 to 8.0). We also explored the binding of E2-C ca towards non-canonical E3 ligases. Binding of E2-C ca to the C terminus of murine Ring 1B (C-Ring1B), which does not contain the RING finger of the whole Ring1B, occurred with an affinity of ∼ 400 nM, as shown by fluorescence and ITC. Furthermore, binding of E2-C ca to C-Ring1B did not occur at its canonical E2-loops, since residues M43 and F53, far away from those loops, were involved in binding. Thus, the C-Ring1B-interacting region of E2-C ca comprises the first β-strand and nearby residues., We deeply thank Prof. T. Tokumoto the kind gift of the E2-Cca clone. We thank the three referees for discussions, ideas and helpful suggestions, which have improved this manuscript. We thank May García, María del Carmen Fuster and Javier Casanova for technical assistance. This work was supported by the Spanish Ministerio de Ciencia e Innovación (MCINN) (CTQ2011-24393, CSD2008-00005 to JLN), and an intramural BIFI grant. The authors declare they do not have any competing interest.

The availability of enzymes with a high promiscuity/specificity relationship permits the hydrolysis of several substrates with a view to obtaining a certain product or using one enzyme for several productive lines. N-Carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens (Atβcar) has shown high versatility to hydrolyze different N-carbamoyl-, N-acetyl- and N-formyl-amino acids to produce different α, β, γ and δ amino acids. We have calculated the promiscuity index for the enzyme, obtaining a value of 0.54, which indicates that it is a modestly promiscuous enzyme. Atβcar presented the highest probability of hydrolysis for N-carbamoyl-amino acids, being the enzyme more efficient for the production of α-amino acids. We have also demonstrated by mutagenesis, modelling, kinetic and binding experiments that W218 and A359 indirectly influence the plasticity of the enzyme due to interaction with the environment of R291, the key residue for catalytic activity.

The C-terminal domain (CTD) of the capsid protein (CA) of HIV-1 participates both in the formation of CA hexamers and in the joining of hexamers through homodimerization to form the viral capsid. Intact CA and the CTD are able to homodimerize with similar affinity (~15 μM); CTD homodimerization involves mainly an α-helical region. We have designed peptides derived from that helix with predicted higher helical propensities than the wild-type sequence while keeping residues important for dimerization. These peptides showed a higher helicity than that of the wild-type peptide, although not as high as theoretically predicted, and proved to be able to self-associate with apparent affinities similar to that of the whole CTD. However, binding to CTD mainly occurs at the last helical region of the protein. Accordingly, most of those peptides are unable to inhibit CA polymerization in vitro. Therefore, there is a subtle tuning between monomer-monomer interactions important for CTD dimerization and the maximal helical content achieved by the wild-type sequence of the interface.

The cystathionine beta-synthase (CBS) domains function as regulatory motifs in several proteins. Elucidating how CBS domains exactly work is relevant because several genetic human diseases have been associated with mutations in those motifs. Here, we show, for the first time, that a CBS domain binds calf-thymus DNA and E-boxes recognized by transcription factors. We have carried out the DNA-binding characterization of the CBS domain protein MJ0729 from Methanocaldococcus jannaschii by biochemical and spectroscopic techniques. Binding induces conformational changes in the protein, and involves the sole tryptophan residue. The apparent dissociation constant for the E-boxes is ∼10μM. These results suggest that CBS domains might interact with DNA.

The largest group of plant thioredoxins (TRXs) consists of the so-called h-type; their great number raises questions about their specific or redundant roles in plant cells. Pisum sativum thioredoxin h1 (PsTRXh1) and Pisum sativum thioredoxin h2 (PsTRXh2) are both h-type TRXs from pea (Pisum sativum) previously identified and biochemically characterized. While both are involved in redox regulation and show a high-sequence identity (60%), they display different behavior during in vitro and in vivo assays. In this work, we show that these two proteins display different specificity in the capturing of protein targets in vitro, by the use of a new stringent method. PsTRXh2 interacted with classical antioxidant proteins, whereas PsTRXh1 showed a completely different pattern of targeted proteins, and was able to capture a transcription factor. We also showed that the two proteins display very different thermal and chemical stabilities. We suggest that the differences in thermal and chemical stability point to a distinct and characteristic pattern of protein specificity.

Dihydropyrimidinase is involved in the reductive pathway of pyrimidine degradation, catalysing the reversible hydrolysis of the cyclic amide bond (-CO-NH-) of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl-beta-amino acids. This enzyme is an attractive candidate for commercial production of D-aminoacids, which are used in the production of semi-synthetic beta-lactams, antiviral agents, artificial sweeteners, peptide hormones and pesticides. We have obtained the crystal structure of the dihydropyrimidinase from Sinorhizobium meliloti (SmelDhp) in the presence of zinc ions, but we have not been able to obtain good diffracting crystals in its absence. Then, the role of the ion in the structure of the protein, and in its stability, remains to be elucidated. In this work, the stability and the structure of SmelDhp have been studied in the absence and in the presence of zinc. In its absence, the protein acquired a tetrameric functional structure at pH approximately 6.0, which is stable up to pH approximately 9.0, as concluded from fluorescence and CD. Chemical-denaturation occurred via a monomeric intermediate with non-native structure. The addition of zinc caused: (i) an increase of the helical structure, and changes in the environment of aromatic residues; and, (ii) a higher thermal stability. However, chemical-denaturation still occurred through a monomeric intermediate. This is the first hydantoinase whose changes in the stability and in the secondary structure upon addition of zinc are described and explained, and one of the few examples where the zinc exclusively alters the secondary helical structure and the environment of some aromatic residues in the protein, leaving unchanged the quaternary structure.

Xylans are the most abundant polysaccharides forming the plant cell wall hemicelluloses, and they are degraded, among other proteins, by beta-xylosidase enzymes. In this work, the structural and biophysical properties of the family 52 beta-xylosidase from Geobacillus stearothermophilus, XynB2, are described. Size exclusion chromatography, analytical centrifugation, ITC, CD, fluorescence (steady state and ANS-binding) and FTIR were used to obtain the structure, the oligomerization state and the conformational changes of XynB2, as pH, chemical denaturants or temperature were modified. This report describes the first extensive conformational characterization of a family 52 beta-xylosidase. The active protein was a highly hydrated dimer, whose active site was formed by the two protomers, and it probably involved aromatic residues. At low pH, the protein was not active and it populated a monomeric molten-globule-like species, which had a conformational transition with a pK(a) of approximately 4.0. Thermal and chemical-denaturations of the native protein showed hysteresis behaviour. The protein at physiological pH was formed by alpha-helix (30%) and beta-sheet (30%), as shown by CD and FTIR. Comparison with other xylosidases of the same family indicates that the percentages of secondary structure seem to be conserved among the members of the family.

Ammonium is incorporated into carbon skeletons by the sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria. The activity of Synechocystis sp. PCC 6803 GS type I is controlled by protein-protein interactions with two intrinsically disordered inactivating factors (IFs): the 65-residue (IF7) and the 149-residue one (IF17). In this work, we studied both IF7 and IF17 by nuclear magnetic resonance (NMR), and we described their binding to GS by using NMR and biolayer interferometry. We assigned the backbone nuclei of all residues of IF7. Analyses of chemical shifts and the N-{H} NOEs at two field strengths suggest that IF7 region Thr3-Arg13 and a few residues around Ser27 and Phe41 populated helical conformations (although the percentage is smaller around Phe41). The two-dimensional H-N HSQC and CON experiments suggest that IF17 populated several conformations. We followed the binding between GS and IF7 by NMR at physiological pH, and the residues interacting first with IF7 were Gln6 and Ser27, belonging to those regions that appeared to be ordered in the isolated protein. We also determined the k values and k values for the binding of both IF7 and IF17 to GS, where the GS protein was bound to a biosensor. The measurements of the kinetic constants for the binding of IF7 to GS suggest that: (i) binding does not follow a kinetic two-state model (GS+IF7 koffkon[IF7]GS:IF7), (ii) there is a strong electrostatic component in the determined k, and (iii) the binding is not diffusion-limited.

The isolation, purification, biochemical and biophysical characterization of the first reported beta-xylosidase from Geobacillus pallidus are described. The protein has an optimum pH close to 8 and an optimum temperature of 70 degrees C. These biochemical properties agree with those obtained by spectroscopic techniques, namely, circular dichroism (CD), infrared (FTIR) and fluorescence measurements. Thermal denaturation, followed by CD and FTIR, showed an apparent thermal denaturation midpoint close to 80 degrees C. The protein was probably a hydrated trimer in solution with, an elongated shape, as shown by gel filtration experiments. FTIR deconvolution spectra indicated that the protein contains a high percentage of alpha-helix (44%) and beta-sheet (40%). The sequencing of the N terminus and the biochemical features indicate that this new member of beta-xylosidases belongs to the GH52 family. Since there are no reported structural studies of any member of this family, our studies provide the first clue for the full conformational characterization of this protein family.

Hydantoin racemase enzyme plays a crucial role in the reaction cascade known as “hydantoinase process.” In conjunction with a stereoselective hydantoinase and a stereospecific carbamoylase, it allows the total conversion from D,L-5-monosubstituted hydantoins, with a low rate of racemization, to optically pure D- or L-amino acids. Residues Cys76 and Cys181 belonging to hydantoin racemase from Sinorhizobium meliloti (SmeHyuA) have been proved to be involved in catalysis. Here, we report biophysical data of SmeHyuA Cys76 and Cys181 to alanine mutants, which point toward a two-base mechanism for the racemization of 5-monosubstituted hydantoins. The secondary and the tertiary structure of the mutants were not significantly affected, as shown by circular dichroism. Calorimetric and fluorescence experiments have shown that Cys76 is responsible for recognition and proton retrieval of D-isomers, while Cys181 is responsible for L-isomer recognition and racemization. This recognition process is further supported by measurements of protein stability followed by chemical denaturation in the presence of the corresponding compound.

Most proteins contain small cavities that can be filled by replacing cavity-lining residues by larger ones. Since shortening mutations in hydrophobic cores tend to destabilize proteins, it is expected that cavity-filling mutations may conversely increase protein stability. We have filled three small cavities in apoflavodoxin and determined by NMR and equilibrium unfolding analysis their impact in protein structure and stability. The smallest cavity (14 A3) has been filled, at two different positions, with a variety of residues and, in all cases, the mutant proteins are locally unfolded, their structure and energetics resembling those of an equilibrium intermediate of the thermal unfolding of the wild-type protein. In contrast, two slightly larger cavities of 20 A3 and 21 A3 have been filled with Val to Ile or Val to Leu mutations and the mutants preserve both the native fold and the equilibrium unfolding mechanism. From the known relationship, observed in shortening mutations, between stability changes and the differential hydrophobicity of the exchanged residues and the volume of the cavities, the filling of these apoflavodoxin cavities is expected to stabilize the protein by approximately 1.5 kcal mol(-1). However, both urea and thermal denaturation analysis reveal much more modest stabilizations, ranging from 0.0 kcal mol(-1) to 0.6 kcal mol(-1), which reflects that the accommodation of single extra methyl groups in small cavities requires some rearrangement, necessarily destabilizing, that lowers the expected theoretical stabilization. As the size of these cavities is representative of that of the typical small, empty cavities found in most proteins, it seems unlikely that filling this type of cavities will give rise to large stabilizations.

2,2,2-Trifluoroethanol (TFE) effectively destabilizes the otherwise highly stable tetrameric structure of the potassium channel KcsA, a predominantly alpha-helical membrane protein [Valiyaveetil, F. I., Zhou, Y., and MacKinnon, R. (2002) Biochemistry 41, 10771-10777]. Here, we report that the effects on the protein structure of increasing concentrations of TFE in detergent solution include two successive protein concentration-dependent, cooperative transitions. In the first of such transitions, occurring at lower TFE concentrations, the tetrameric KcsA simultaneously increases the exposure of tryptophan residues to the solvent, partly loses its secondary structure, and dissociates into its constituent subunits. Under these conditions, simple dilution of the TFE permits a highly efficient refolding and tetramerization of the protein in the detergent solution. Moreover, following reconstitution into asolectin giant liposomes, the refolded protein exhibits nativelike potassium channel activity, as assessed by patch-clamp methods. Conversely, the second cooperative transition occurring at higher TFE concentrations results in the irreversible denaturation of the protein. These results are interpreted in terms of a protein and TFE concentration-dependent reversible equilibrium between the folded tetrameric protein and partly unfolded monomeric subunits, in which folding and oligomerization (or unfolding and dissociation in the other direction of the equilibrium process) are seemingly coupled processes. At higher TFE concentrations this is followed by the irreversible conversion of the unfolded monomers into a denatured protein form.

The type 1 human immunodeficiency virus presents a conical capsid formed by several hundred units of the capsid protein, CA. Homodimerization of CA occurs via its C-terminal domain, CA-C. This self-association process, which is thought to be pH-dependent, seems to constitute a key step in virus assembly. CA-C isolated in solution is able to dimerize. An extensive thermodynamic characterization of the dimeric and monomeric species of CA-C at different pHs has been carried out by using fluorescence, circular dichroism (CD), absorbance, nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), and size-exclusion chromatography (SEC). Thermal and chemical denaturation allowed the determination of the thermodynamic parameters describing the unfolding of both CA-C species. Three reversible thermal transitions were observed, depending on the technique employed. The first one was protein concentration-dependent; it was observed by FTIR and NMR, and consisted of a broad transition occurring between 290 and 315 K; this transition involves dimer dissociation. The second transition (Tm approximately 325 K) was observed by ANS-binding experiments, fluorescence anisotropy, and near-UV CD; it involves partial unfolding of the monomeric species. Finally, absorbance, far-UV CD, and NMR revealed a third transition occurring at Tm approximately 333 K, which involves global unfolding of the monomeric species. Thus, dimer dissociation and monomer unfolding were not coupled. At low pH, CA-C underwent a conformational transition, leading to a species displaying ANS binding, a low CD signal, a red-shifted fluorescence spectrum, and a change in compactness. These features are characteristic of molten globule-like conformations, and they resemble the properties of the second species observed in thermal unfolding.

Glutamine synthetase (GS) is the key enzyme responsible for the primary assimilation of ammonium in all living organisms, and it catalyses the synthesis of glutamine from glutamic acid, ATP, and ammonium. One of the recently discovered mechanisms of GS regulation involves protein-protein interactions with a small 65-residue-long protein named IF7. Here, we study the structure and stability of IF7 and its binding properties to GS, by using several biophysical techniques (fluorescence, circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopies, and gel filtration chromatography) which provide complementary structural information. The findings show that IF7 has a small amount of residual secondary structure, but lacks a well defined tertiary structure, and is not compact. Thus, all of the studies indicate that IF7 is a "natively unfolded" protein. The binding of IF7 to GS, its natural binding partner, occurs with an apparent dissociation constant of K(D) = 0.3 +/- 0.1 microM, as measured by fluorescence. We discuss the implications for the GS regulation mechanisms of IF7 being unfolded.

The folding of a 93-residue protein, the histidine-phosphocarrier protein of Streptomyces coelicolor, HPr, has been studied using several biophysical techniques, namely fluorescence, 8-anilinonaphthalene-1-sulfate binding, circular dichroism, Fourier transform infrared spectroscopy, gel filtration chromatography and differential scanning calorimetry. The chemical-denaturation behaviour of HPr, followed by fluorescence, CD and gel filtration, at pH 7.5 and 25 °C, is described as a two-state process, which does not involve the accumulation of thermodynamically stable intermediates. Its conformational stability under those conditions is ΔG = 4.0 ± 0.2 kcal·mol−1 (1 kcal = 4.18 kJ), which makes the HPr from S. coelicolor the most unstable member of the HPr family described so far. The stability of the protein does not change significantly from pH 7–9, as concluded from the differential scanning calorimetry and thermal CD experiments. Conformational studies at low pH (pH 2.5–4) suggest that, in the absence of cosmotropic agents, HPr does not unfold completely; rather, it accumulates partially folded species. The transition from those species to other states with native-like secondary and tertiary structure, occurs with a pKa = 3.3 ± 0.3, as measured by the averaged measurements obtained by CD and fluorescence. However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8-anilinonaphthalene-1-sulfate binding experiments); or (b) that measured by acquisition of native-like compactness (gel-filtration studies). It seems that acquisition of native-like features occurs in a wide pH range and it cannot be ascribed to a unique side-chain titration. These series of intermediates have not been reported previously in any member of the HPr family.

The sterile alpha motif (SAM) domain is a protein module of approximately 65 to 70 amino acids found in many diverse proteins whose functions range from signal transduction to transcriptional repression. The alpha splice variant of p73 (p73 alpha), a homologue of the tumor suppressor p53, has close to its C-terminus a SAM motif. Here, we report the folding equilibrium properties of the p73 alpha SAM domain (SAMp73) by using different biophysical techniques (circular dichroism, fluorescence, and Fourier transform infrared spectroscopies, and differential scanning calorimetry). Those probes indicate that SAMp73 folds via a two-state mechanism. Fluorescence experiments performed at different pHs showed two titrations: the first one due to an acid residue (with a pK(a) = 4.5 +/- 0.3) and the second due to deprotonation of tyrosine residues. The conformational stability of the protein upon chemical denaturation was determined over the pH range 3 to 10. The maximum conformational stability is DeltaG = 5.7 +/- 0.4 kcal x mol(-1) (at 25 degrees C) and occurs in a broad maximum, with little variation, between pH 6 and 10. The high melting temperature of SAMp73 (T(m) = 93.5 degrees C), despite its moderate conformational stability at 25 degrees C, can be ascribed to its low heat capacity change upon unfolding, DeltaC(p), which is estimated to be around 915 cal x K(-1) x mol(-1) at 25 degrees C and only around 543 cal x K(-1) x mol(-1) at the T(m). The implications of the temperature-dependent nature of DeltaC(p) are discussed in relation to the thermal stability of proteins as opposed to their conformational stability at room temperature.

Proton NMR spectral resonances of thioredoxin m from spinach have been assigned, and its solution structure has been determined on the basis of 1156 nuclear Overhauser effect- (NOE-) derived distance constraints by using restrained molecular dynamics calculations. The average pairwise root-mean-square deviation (RMSD) for the 25 best NMR structures for the backbone was 1.0 +/- 0.1, when the structurally well-defined residues were considered. The N- and C-terminal segments (1-13 and 118-119) and residues 41-49, comprising the active site, are highly disordered. At the time of concluding this work, a crystal structure of this protein was reported, in which thioredoxin m was found to crystallize as noncovalent dimers. Although the solution and crystal structures are very similar, no evidence was found about the existence of dimers in solution, thus confirming that dimerization is not needed for the regulatory activity of thioredoxin m. The spinach thioredoxin m does not unfold by heat in the range 25-85 degrees C, as revealed by thermal circular dichroic (CD) measurements. However, its unfolding free energy (9.1 +/- 0.8 kcal mol(-1), at pH 5.3 and 25 degrees C) could be determined by extrapolating the free energy values obtained at different concentrations of guanidinium chloride (GdmCl). The folding-unfolding process is two-state as indicated by the coincidence of the CD denaturation curves obtained at far and near UV. The H/D exchange behavior of backbone amide protons was analyzed. The slowest-exchanging protons, requiring a global-unfolding mechanism in order to exchange, are those from beta2, beta3, and beta4, the central strands of the beta-sheet, which constitute the main element of the core of the protein. The free energies obtained from exchange measurements of protons belonging to the alpha-helices are lower than those derived from GdmCl denaturation studies, indicating that those protons exchange by local-unfolding mechanisms. Fil: Neira, José L.. Universidad de Miguel Hernández; España Fil: González, Carlos. Consejo Superior de Investigaciones Científicas; España Fil: Toiron, Catherine. Consejo Superior de Investigaciones Científicas; España Fil: de Prat Gay, Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Rico, Manuel. Consejo Superior de Investigaciones Científicas; España

We have analysed the hydrogen/deuterium exchange of the tetramerization domain of human tumour suppressor p53 under mild chemical denaturation conditions, and at different temperatures. Exchange behaviour has been measured for 16 amide protons in the chemical-denaturation studies and for seven protons in the temperature-denaturation studies. The exchange rates are in the range observed for other proteins with similar elements of secondary structure. The slowest-exchange core includes contributions from residues in the alpha helix and the beta sheet. However, only some of the slowest-exchanging protons correspond to residues involved in native interactions in the transient intermediate detected during the folding of this domain. The guanidinium-chloride denaturation curves of all residues seem to merge together, although they are well below the main isotherm of global unfolding. Thus, there is no evidence for several subglobal unfolding units. The activation parameters obtained from the temperature-denaturation experiments are similar to those obtained for monomeric proteins, and well below the global unfolding enthalpy obtained by circular dichroism measurements. Thus, the exchange studies at different denaturant concentrations and temperatures indicate that no particular folding intermediate is populated under those conditions.

We have studied the biochemical features, the conformational preferences in solution, and the DNA binding properties of human p8 (hp8), a nucleoprotein whose expression is affected during acute pancreatitis. Biochemical studies show that hp8 has properties of the high mobility group proteins, HMG-I/Y. Structural studies have been carried out by using circular dichroism (near- and far-ultraviolet), Fourier transform infrared, and NMR spectroscopies. All the biophysical probes indicate that hp8 is monomeric (up to 1 mm concentration) and partially unfolded in solution. The protein seems to bind DNA weakly, as shown by electrophoretic gel shift studies. On the other hand, hp8 is a substrate for protein kinase A (PKA). The phosphorylated hp8 (PKAhp8) has a higher content of secondary structure than the nonphosphorylated protein, as concluded by Fourier transform infrared studies. PKAhp8 binds DNA strongly, as shown by the changes in circular dichroism spectra, and gel shift analysis. Thus, although there is not a high sequence homology with HMG-I/Y proteins, hp8 can be considered as a HMG-I/Y-like protein.

Native-like complexes of proteins, formed by the association of two complementary fragments comprising the entire sequence of the protein, can be used to gain insight into the stability and folding of the intact protein. We have studied the structural, thermodynamic and kinetic properties of four barnase complexes, with the cleavage site at different positions of the amino-acid chain (CB36, at position 36; CB56, at position 56; CB68, at position 68; and CB79, at position 79). The four barnase complexes have native-like structure as shown by fluorescence, far-and near-UV CD, size-exclusion chromatography and NMR. The NMR characterization indicated that the structural changes were mainly located in regions close to the cleavage site. The main core of the protein was fully formed and the overall structure was similar to that of intact barnase. The thermal and chemical denaturation showed that all complexes were substantially destabilized. CB56 displayed two denaturation transitions, probably because of the presence of partially folded conformations around the cleavage site. The rate constant for the association/folding of fragments decreased with the decreasing length of the C-terminal fragment. Thus, the larger the fragment (and, consequently, the larger the amount of residual native-like structure), the faster the association. These findings are consistent with the proposed model of barnase folding.

The bacterial phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), formed by a cascade of several proteins, mediates the uptake and phosphorylation of carbohydrates, and it is also involved in signal transduction. Its uniqueness in bacteria makes the PTS a target for new antibacterial drugs. These drugs can be obtained from peptides or proteins fragments able to interfere the first step of the protein cascade: the phosphorylation of the HPr protein by the enzyme EI. We designed a peptide comprising the active site and the first α-helix of HPr of S. coelicolor; we also obtained a fragment of HPr by protein engineering methods, comprising the first forty-eight residues and thus, containing the amino acids of the shorter peptide. Both fragments were disordered in aqueous solution, with a similar percentage of helical structure (∼7 %), and an identical free energy of helix formation. In 40 % TFE, both fragments acquired native-like helical structure, stabilized by non-native hydrophobic interactions, as shown by the 2D-NMR assignments of the shorter peptide, and the presence of similar NOE contacts in both fragments. These findings, with the kinetic results in other members of the HPr family, highlight the importance of short- and long-range interactions during the folding reaction of HPr proteins. Based on the residual helical population, hypothesis about the inhibition capacity of the PTS by both fragments are discussed. © 2007 Bentham Science Publishers Ltd.

We have characterised a series of C-terminal fragments of barnase by different biophysical techniques to find out when they acquire secondary and tertiary native-like structure. Fragments B96-110 (which comprises the last 15 residues of the intact protein) up to B37-110 (which involves most of the protein except the two first helices and a loop) were mainly disordered. Only fragment B23-110, which lacks alpha-helix1, showed native-like near and far-UV CD and fluorescence spectra. The intensities of these spectra were lower than those of the full-length protein, which suggests the absence of complete side-chain packing. Urea denaturation followed by fluorescence, far-UV CD and gel-filtration chromatography techniques indicated a co-operative transition only for B23-110. None of the fragments melted co-operatively with temperature. Thus, the formation of secondary and tertiary structure requires most of the polypeptide chain to be present, that is, secondary and tertiary structure are formed in parallel. This agrees with the proposed model for barnase folding, where the residual structure in small fragments is weak and flickering, and it is only consolidated when there are enough tertiary interactions. Thus, the development of structure in the series of C-terminal fragments follows a similar behaviour to that observed in the series of N-terminal fragments of barnase.

We are examining possible roles of native and non-native interactions in early events in protein folding by a systematic analysis of the structures of fragments of proteins whose folding pathways are well characterised. Seven fragments of the 110-residue protein barnase, corresponding to the progressive elongation from its N terminus, have been characterised by a battery of biophysical and spectroscopic methods. Barnase is a multi-modular protein that folds via an intermediate in which the C-terminal region of its major alpha-helix (alpha-helix1, residues Thr6-His18) is substantially formed as is also its anti-parallel beta-sheet, centred around a beta-hairpin (residues Ser92-Leu95). Fragments up to, and including, residues 1-95 (fragment B95), appeared to be mainly disordered, although a small amount of helical secondary structure in each was inferred from far-UV CD experiments, and fluorescence studies indicated some native-like tertiary interactions in B95. The largest fragment (residues 1-105, B105) is compactly folded. The secondary structure in alpha-helix1 in the seven fragments was found by NMR to increase with increasing chain length faster than the build-up of tertiary interactions, indicating that alpha-helix1 is being stabilised by non-native interactions. This behaviour contrasts with that in fragments of the 64-residue chymotrypsin inhibitor 2 (CI2), in which tertiary and secondary structures build up in parallel with increasing length. CI2 consists of a single module of structure that folds without a detectable intermediate. The largest fragment of barnase, B105, has interactions that resemble its folding intermediate, whereas one of the largest fragments of CI2 (residues 1-60) resembles the folding transition state. The folding pathways of both proteins are consistent with a scheme in which there are low levels of native-like secondary structure in the denatured state that become stabilised by long-range interactions as folding proceeds. Neither protein forms a stable fold when lacking the last ten residues at the C terminus. Since at least 20 amino acid residues are bound to the ribosome during protein biosynthesis, these small proteins do not fold until they have left the ribosome, and so the studies of the folding of such proteins in vitro may be relevant to their folding in vivo, especially as the molecular chaperone GroEL binds only weakly to denatured CI2 and does not discernibly alter the folding mechanism of barnase.

Fluorescence and circular dichroism, as analytical spectroscopic techniques, and mass spectrometry as an analytical tool to determine the molecular mass, provide important biophysical approaches in structural virology. Although they do not provide atomic, or near-atomic, details as electron microscopy, X-ray crystallography or nuclear magnetic resonance spectroscopy can do, they do provide important insights into virus particle composition, structure, conformational stability and dynamics, assembly and maturation, and interactions with other viral and cellular biomolecules. They can be used also to investigate the molecular determinants of virus particle structure and properties, and the changes induced in them by external factors. In this chapter, I describe the physical bases of these three techniques, and some examples on how they have helped us to understand virus particle structure and physicochemical properties.