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TDP-43 is an essential RNA-binding protein forming aggregates in almost all cases of sporadic amyotrophic lateral sclerosis (ALS) and many cases of frontotemporal lobar dementia (FTLD) and other neurodegenerative diseases. TDP-43 consists of a folded N-terminal domain with a singular structure, two RRM RNA-binding domains, and a long disordered C-terminal region which plays roles in functional RNA regulatory assemblies as well as pernicious aggregation. Evidence from pathological mutations and seeding experiments strongly suggest that TDP-43 aggregates are pathologically relevant through toxic gain-of-harmful-function and/or harmful loss-of-native-function mechanisms. Recent, but not early, microscopy studies and the ability of TDP-43 aggregates to resist harsh treatment and to seed new pathological aggregates in vitro and in cells strongly suggest that TDP-43 aggregates have a self-templating, amyloid-like structure. Based on the importance of the Gln/Asn-rich 341–367 residue segment for efficient aggregation of endogenous TDP-43 when presented as a 12X-repeat and extensive spectroscopic and computational experiments, we recently proposed that this segment adopts a beta-hairpin structure that assembles in a parallel with a beta-turn configuration to form an amyloid-like structure. Here, we propose that this conformer is stabilized by an especially strong class of hypercooperative hydrogen bonding unique to Gln and Asn sidechains. The clinical existence of this conformer is supported by very recent LC-MS/MS characterization of TDP-43 from ex vivo aggregates, which show that residues 341–367 were protected in vivo from Ser phosphorylation, Gln/Asn deamidation and Met oxidation. Its distinct pattern of SDS-PAGE bands allows us to link this conformer to the exceptionally stable seed of the Type A TDP-43 proteinopathy., This work was supported by Grants SAF2013-49179-C2-2-R (DVL) and EU JPND AC14/00037 (DVL) and EU JPND RiModFTD, Italy, Ministero della Sanita’ (EB), and the Thierry Latran Foundation REHNPALS (EB).

The capacity of three designed duodecamer peptides with the low diversity sequence: H1ϕ2I3K4I5D6G7K8ϕ9I10K11H12 where ϕ is His, Phe or Trp, to adopt a β-hairpin conformation was studied using NMR spectroscopy. Whereas KIAβH, the variant with His at positions two and nine, is disordered, KIAβF, the peptide with Phe at these positions, adopts a small population of β-hairpin. A high population of β-hairpin structure was detected for KIAβW, the variant with Trp. Utilizing NMR data, the structure of KIAβW was solved and it reveals a β-hairpin stabilized by hydrophobic interactions between Ile residues on one face and Trp–Trp and cation–π interactions on the opposite face. Upon adding ATP, these peptides show chemical shift changes indicative of ATP binding. The binding of ATP to KIAβW shows a KD ≈ 20 μM at pH 5, 5 °C and has a 1:1 stoichiometry. The KIAβW-ATP complex was determined using NMR spectroscopy and reveals the adenine ring sandwiched between the two Trp indole rings and that ATP binding induces important conformational changes in His1, Trp2, Lys4, Trp9 and Lys11 in the β-hairpin. The implications of these results for the hypothetic presence of β-hairpins and amyloids alongside RNAs on the prebiotic Earth are discussed.

The I27 module from human cardiac titin has become a standard in protein nanomechanics. A proline-scanning study of its ¿mechanical clamp¿ found three mechanically hypomorphic mutants and a paradoxically hypermorphic one (Y9P). Both types of mutants have been commonly used as substrates of several protein unfoldase machineries in studies relating protein mechanostability to translocation or degradation rates. Using AFM-based single-molecule force spectroscopy, polyprotein engineering and steered molecular dynamics simulations we show that, unexpectedly, the mechanostability of the Y9P variant is comparable to the wild type. Furthermore, the NMR analysis of homomeric polyproteins of this variant suggests that these constructs may induce slight structural perturbations in the monomer, and enables us to explain some minor differences in this variant¿s properties; namely the abolishment of the mechanical unfolding intermediate and a reduced thermal stability. Our results clarify a previously reported paradoxical result in protein nanomechanics and contribute to refine our toolbox for understanding the unfolding mechanism used by translocases and degradation machines.

International audience; We have conjugated the tetraazacyclododecane-tetraacetate (DOTA) chelator to Pittsburgh compound B (PiB) forming negatively charged lanthanide complexes, Ln(L4), with targeting capabilities towards aggregated amyloid peptides. The amphiphilic Gd(L4) chelate undergoes micellar aggregation in aqueous solution, with a critical micellar concentration of 0.68 mM, lower than those for the neutral complexes of similar structure. A variable temperature (17)O NMR and NMRD study allowed the assessment of the water exchange rate, k ex (298) = 9.7 × 10(6) s(-1), about the double of GdDOTA, and for the description of the rotational dynamics for both the monomeric and the micellar forms of Gd(L4). With respect to the analogous neutral complexes, the negative charge induces a significant rigidity of the micelles formed, which is reflected by slower and more restricted local motion of the Gd(3+) centers as evidenced by higher relaxivities at 20-60 MHz. Surface Plasmon Resonance results indicate that the charge does not affect significantly the binding strength to Aβ1-40 [K d = 194 ± 11 μM for La(L4)], but it does enhance the affinity constant to human serum albumin [K a = 6530 ± 68 M(-1) for Gd(L4)], as compared to neutral counterparts. Protein-based NMR points to interaction of Gd(L4) with Aβ1-40 in the monomer state as well, in contrast to neutral complexes interacting only with the aggregated form. Circular dichroism spectroscopy monitored time- and temperature-dependent changes of the Aβ1-40 secondary structure, indicating that Gd(L4) stabilizes the random coil relative to the α-helix and β-sheet. TEM images confirm that the Gd(L4) complex reduces the formation of aggregated fibrils.

Transactive response DNA-binding protein 43 kDa (TDP-43) is an RNA transporting and processing protein whose aberrant aggregates are implicated in neurodegenerative diseases. The C-terminal domain of this protein plays a key role in mediating this process. However, the N-terminal domain (residues 1-77) is needed to effectively recruit TDP-43 monomers into this aggregate. In the present study, we report, for the first time, the essentially complete (1) H, (15) N and (13) C NMR assignments and the structure of the N-terminal domain determined on the basis of 26 hydrogen-bond, 60 torsion angle and 1058 unambiguous NOE structural restraints. The structure consists of an ¿-helix and six ß-strands. Two ß-strands form a ß-hairpin not seen in the ubiquitin fold. All Pro residues are in the trans conformer and the two Cys are reduced and distantly separated on the surface of the protein. The domain has a well defined hydrophobic core composed of F35, Y43, W68, Y73 and 17 aliphatic side chains. The fold is topologically similar to the reported structure of axin 1. The protein is stable and no denatured species are observed at pH 4 and 25 °C. At 4 kcal·mol(-1) , the conformational stability of the domain, as measured by hydrogen/deuterium exchange, is comparable to ubiquitin (6 kcal·mol(-1) ). The ß-strands, ¿-helix, and three of four turns are generally rigid, although the loop formed by residues 47-53 is mobile, as determined by model-free analysis of the (15) N{(1) H}NOE, as well as the translational and transversal relaxation rates.

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Amyloid fibrils are exceptionally stable oligomeric structures with extensive, highly cooperative H-bonding networks whose physical origin remains elusive. While nonpolar systems benefit from both H-bonds and hydrophobic interactions, we found that highly polar sequences containing glutamine and asparagine amino acid residues form hyperpolarized H-bonds. This feature, observed by density functional theory calculations, encodes the origin of these polar oligomers' high stability. These results are explained in a theoretical model for complex amyloid assembly based on two different types of cooperative effects resulting from highly delocalized electrons, one of which is always present in both polar and hydrophobic systems. Experimental electric conductivity measurements, ThT fluorescence enhancement, and NMR spectroscopy support this proposal and reveal the conditions for disassembly.

The first life on Earth is believed to have been based on RNA, but might have taken advantage of amino acids and short peptides which form readily under conditions like those of the primitive Earth. We have shown that simple peptides adopt specifically folded four-helix bundle structures that can recognize and cleave RNA. Here, to explore the limits of conformational specificity, we characterize a simpler peptide composed of just Lys, Ile, Ala, and Gly called KIA7I. Using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations, we find kinks in the helices of KIA7I and multiple C-terminal conformations. These results suggest that the C-terminal Ile residue does not completely occupy the hydrophobic pocket that is filled by aromatic side-chains in well-folded KIA7 variants. The capacity of arenes to fill this cavity was tested. Using NMR, we show that benzene and phenol can bind KIA7I, but do not bind the well-folded variant KIA7W or hen egg white lysozyme. Benzene also binds Aβ 1-40, a mostly disordered polypeptide implicated in Alzheimer's disease. 8-Anilinonaphthalene-1-sulfonate (ANS) fluorescence is further enhanced in the presence of both KIA7I and arenes relative to KIA7I alone. This ANS fluorescence enhancement is stronger for smaller and less polar arenes and less ordered KIA variants. These results suggest that arenes are not confined to the pocket, but penetrate and loosen the hydrophobic core of KIA7I.

The C-terminal β-hairpin of RNase A contains a turn with a cis Asn113-Pro114 peptide bond. Pioneering pulsed HX experiments have shown that the C-terminal β-hairpin forms early during refolding. This is puzzling since the Asn113-Pro114 bond is predominately trans at this stage and this conformation destabilizes the native monomer. RNase A, when refolded at high concentration, forms a series of 3D domain-swapped oligomers. In the oligomers formed by C-terminal β-strand swapping, Asn113-Pro114 is trans and permits the formation of a new intersubunit β-sheet. We hypothesize that oligomeric species with trans Asn113-Pro114 may form during refolding. Such species could account for the HX results while comfortably accommodating Asn113-Pro114 in the trans conformation. Here, we test this hypothesis by employing chromatographic methods to detect oligomers forming in refolding conditions and find significant amounts of dimer. We propose that a 3D domain-swapped dimeric intermediate provides a minor alternative pathway for RNase A refolding.

RNase A forms 3D domain-swapped oligomers with novel enzymatic and biological activities. We study how crowding agents and osmolytes affect the formation and dissociation of RNase A oligomers. The crowding agents Ficoll and dextran were found to enhance oligomer formation, whereas the stabilizers sodium sulfate, glycine and trimethylammonium oxide (TMAO) do not. In contrast, TMAO significantly slows RNase A dimer dissociation, while the effect of Ficoll is small. These results lead us to propose that the mechanisms of oligomer formation and dissociation are different. In the RNase A “C-dimer”, the C-terminal β-strand is swapped between two subunits. The loop preceding this β-strand adopts a β-sheet which has been proposed to resemble amyloid structurally. Hydrogen/deuterium (H/D) exchange of the RNase A C-dimer reveal that the H-bonds formed between the swapped C-terminal β-strand and the other subunit are strong. Their rupture may be crucial for C-dimer dissociation. In contrast, H-bonds formed by Asn 113 in the novel β-sheet adopted by the hinge loop in the C-dimer are not strongly protected. Besides the fundamental insights obtained, the results represent a technical advance for obtaining increased oligomer yields and storage lifetimes.

KIA7, a peptide with a highly restricted set of amino acids (Lys, Ile, Ala, Gly and Tyr), adopts a specifically folded structure. Some amino acids, including Lys, Ile, Ala, Gly and His, form under the same putative prebiotic conditions, whereas different conditions are needed for producing Tyr, Phe and Trp. Herein, we report the 3D structure and conformational stability of the peptide KIA7H, which is composed of only Lys, Ile, Ala, Gly and His. When the imidazole group is neutral, this 20-mer peptide adopts a four-helix bundle with a specifically packed hydrophobic core. Therefore, one-pot prebiotic proteins with well-defined structures might have arisen early in chemical evolution. The Trp variant, KIA7W, was also studied. It adopts a 3D structure similar to that of KIA7H and its previously studied Tyr and Phe variants, but is remarkably more stable. When tested for ribonucleolytic activity, KIA7H, KIA7W and even short, unstructured peptides rich in His and Lys, in combination with Mg(++), Mn(++) or Ni(++) (but not Cu(++), Zn(++) or EDTA) specifically cleave the single-stranded region in an RNA stem-loop. This suggests that prebiotic peptide-divalent cation complexes with ribonucleolytic activity might have co-inhabited the RNA world.

Protein self-recognition is essential in many biochemical processes and its study is of fundamental interest to understand the molecular mechanism of amyloid formation. Ribonuclease A (RNase A) is a monomeric protein that may form several oligomers by 3D domain swapping of its N-terminal α-helix, C-terminal β-strand, or both. RNase A oligomerization is induced by 40% acetic acid, which has been assumed to mildly unfold the protein by detaching the terminal segments and consequently facilitating intersubunit swapping, once the acetic acid is removed by lyophilization and the protein is redissolved in a benign buffer. Using UV difference, near UV circular dichroism, folding kinetics, and multidimensional heteronuclear NMR spectroscopy, the conformation of RNase A in 40% acetic acid and in 8 M urea has been characterized. These studies demonstrate that RNase A is chiefly unfolded in 40% acetic acid; it partially retains the native helices, whereas the β-sheet is fully denatured and all X-Pro peptide bonds are predominantly in the trans conformation. Refolding occurs via an intermediate, IN, with non-native X-Pro peptide bonds. IN is known to be populated during RNase A refolding following denaturation in concentrated solutions of urea or guanidinium chloride, and we find that urea- or GdmCl-denatured RNase A can oligomerize during refolding. By revealing the importance of a chiefly denaturated state and a refolding intermediate with non-native X-Pro peptide bonds, these findings revise the model for RNase A oligomerization via 3D domain swapping and have general implications for amyloid formation. © 2010 American Chemical Society., This work was funded by Grants CTQ2007-68014-C02-02, BFU 2006-15543-C02-02/BMC, and CTQ2008-00080/BQU from the Spanish “Ministerio de Educación y Ciencia”.

Ribonuclease A forms a series of oligomers by 3D domain swapping, a possible mechanism for amyloid formation. Using experimental data, the Ribonuclease oligomerization process is analyzed to obtain estimates of individual equilibrium and microscopic rate constants. The results suggest several novel insights into Ribonuclease oligomer formation: (i) two dimers may combine to yield tetramers, (ii) the lower abundance of the cyclic trimer could be ascribed to the cis conformation of its Asn113-Pro114 peptide bonds, (iii) oligomers become the dominant species at very high protein concentrations or upon applying a modest tenfold increase in the equilibrium constants (iv) the rate constants for trimer and tetramer formation are faster than those of dimer formation and (v) glycosylation affects the relative populations of different trimer and tetramer species. By mass spectrometry, oligomers as large as tetradecamers are detected. These results are consistent with the proposal that 3D domain swapping is a mechanism for amyloid formation.

Eosinophil cationic protein (ECP)/human RNase 3, a member of the RNase A family, is a remarkably cytotoxic protein implicated in asthma and allergies. These activities are probably due to ECP's ability to interact with and disrupt membranes and depend on two Trp, 19 Arg, and possibly an extremely high conformational stability. Here, we have used NMR spectroscopy to assign essentially all 1H, 15N, and backbone 13C resonances, to solve the 3D structure in aqueous solution and to quantify its residue-level stability. The NMR solution structure was determined on the basis of 2316 distance constraints and is well-defined (backbone RMSD = 0.81 A). The N-terminus and the loop composed of residues 114–123 are relatively well-ordered; in contrast, conformational diversity is observed for the loop segments 17–22, 65–68, and 92–95 and most exposed sidechains. The side chain NH groups of the two Trp and 19 Arg showed no significant protection against hydrogen/deuterium exchange. The most protected NH groups belong to the first and last two β-strands, and curiously, the first α-helix. Analysis of their exchange rates reveals a strikingly high global stability of 11.8 kcal/mol. This value and other stability measurements are used to better quantify ECP's unfolding thermodynamics. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 1018–1028, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

9 pags, 6 figs, The effect of strongly destabilizing mutations, I106A and V108G of Ribonuclease A (RNase A), on its structure and stability has been determined by NMR. The solution structures of these variants are essentially equivalent to RNase A. The exchange rates of the most protected amide protons in RNase A (35°C), the I106A variant (35°C), and the V108G variant (10°C) yield stability values of 9.9, 6.0, and 6.8 kcal/mol, respectively, when analyzed assuming an EX2 exchange mechanism. Thus, the destabilization induced b y these mutations is propagated throughout the protein. Simulation of RNase A hydrogen exchange indicates that the most protected protons in RNase A and the V108G variant exchange via the EX2 regime, whereas those of I106A exchange through a mixed EX1 + EX2 process. It is striking that a single point mutation can alter the overall exchange mechanism. Thus, destabilizing mutations joins high temperatures, high pH and the presence of denaturating agents as a factor that induces EX1 exchange in proteins. The calculations also indicate a shift from the EX2 to the EX1 mechanism for less protected groups within the same protein. This should be borne in mind when interpreting exchange data as a measure of local stability in less protected regions. © 2008 by the Biophysical Society., This work was supported by the Spanish Ministerio de Educación y Ciencia (BFU2005-01855/BMC, BFU2006-15543-CO2-02).

Obtaining highly specific and active ribonuclease activities is an important goal with numerous medical and biochemical applications. As a step toward more active and specific ribonucleases, we describe the preparation and the enzymatic and structural properties of RNase S monomers and dimers conjugated to DNA and PNA molecules. Poly(dT)n (2'-oligodeoxyribonucleotides, n = 8, 15) and t8 peptide nucleic acid (PNA) chains have been conjugated to the S-peptide of ribonuclease S. Monomers and dimers of the conjugated enzyme have been obtained and characterized by 1H NMR spectroscopy, showing that DNA or PNA conjugation does not alter the native structure of ribonuclease S. The oligonucleotide-conjugated RNase S monomer and dimer show significant activity against single-stranded RNA and very low/negligible hydrolysis of double-stranded poly(A).poly(U). In contrast, the t8-conjugated RNase S monomer and dimer show substantial activity against both ssRNA and dsRNA. These results highlight the importance of positive charges near but not in the active site in enhancing activity against dsRNA and reveal the promise of PNA-RNase conjugates for modulating RNase activity.

We show that temperature is an important parameter for the sensitivity of saturation transfer difference (STD) spectroscopy. A decreased intensity of STD signals is observed for lactose binding to growth-regulatory galectin7 (p53-induced gene 1), as well as for nucleotide binding to annexin A6, when the temperature is increased from 281 to 298-310 K. Opposite temperature effects on STD intensity are observed for S-peptide binding to S-protein to reconstitute RNase S. However, the STD signals for tryptophan binding to downstream regulatory element antagonist modulator of the human prodynorphin gene (DREAM)are relatively unaffected between 281 and 298 K. The known kinetics of the binding of ATP by the uncoupling protein from brown adipose tissue mitochondria (UCP1) predicted an observable STD at 310 K, but rapid sample degradation limits the experiments to much lower temperatures. Temperature strongly influences the kinetics and affinity constant of various types of complex formation and in so doing influences the observed STD effects. Therefore, temperature can be exploited to facilitate the optimization of STD-based applications, and at the same time minimize the number of test samples. STD-based screening protocols to detect new target-specific compounds may yield a larger number of potential ligands if screened at various temperatures.

© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Metal complexes are increasingly explored as imaging probes in amyloid peptide related pathologies. We report the first detailed study on the mechanism of interaction between a metal complex and both the monomer and the aggregated form of Aß1-40 peptide. We have studied lanthanide(III) chelates of two PiB-derivative ligands (PiB=Pittsburgh compound B), L1 and L2, differing in the length of the spacer between the metal-complexing DO3A macrocycle (DO3A= 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid) and the peptide-recognition PiB moiety. Surface plasmon resonance (SPR) and saturation transfer difference (STD) NMR spectroscopy revealed that they both bind to aggregated Aß1-40 (KD=67-160 ¿M), primarily through the benzothiazole unit. HSQC NMR spectroscopy on the 15N-labeled, monomer Aß1-40 peptide indicates nonsignificant interaction with monomeric Aß. Time-dependent circular dichroism (CD), dynamic light scattering (DLS), and TEM investigations of the secondary structure and of the aggregation of Aß1-40 in the presence of increasing amounts of the metal complexes provide coherent data showing that, despite their structural similarity, the two complexes affect Aß fibril formation distinctly. Whereas GdL1, at higher concentrations, stabilizes ß-sheets, GdL2 prevents aggregation by promoting ¿-helical structures. These results give insight into the behavior of amyloid-targeted metal complexes in general and contribute to a more rational design of metal-based diagnostic and therapeutic agents for amyloid- associated pathologies.

TDP-43 can form pathological proteinaceous aggregates linked to ALS and FTLD. Within the putative aggregation domain, engineered repeats of residues 341-366 can recruit endogenous TDP-43 into aggregates inside cells; however, the nature of these aggregates is a debatable issue. Recently, we showed that a coil to β-hairpin transition in a short peptide corresponding to TDP-43 residues 341-357 enables oligomerization. Here we provide definitive structural evidence for amyloid formation upon extensive characterization of TDP-43(341-357) via chromophore and antibody binding, electron microscopy (EM), solid-state NMR, and X-ray diffraction. On the basis of these findings, structural models for TDP-43(341-357) oligomers were constructed, refined, verified, and analyzed using docking, molecular dynamics, and semiempirical quantum mechanics methods. Interestingly, TDP-43(341-357) β-hairpins assemble into a novel parallel β-turn configuration showing cross-β spine, cooperative H-bonding, and tight side-chain packing. These results expand the amyloid foldome and could guide the development of future therapeutics to prevent this structural conversion.

© The Royal Society of Chemistry 2015. We show via 3D-heteronuclear NMR spectroscopy that Aβ1-40 adopts a disordered conformational ensemble with fluctuating turns in DMSOd6. Using NMR, we map the binding sites of three water-insoluble aggregation inhibitors to Aβ1-40 in DMSOd6 and discover remarkable differences in Aβ1-40 recognition by a fourth inhibitor in H2O versus DMSOd6.

© 2014 American Chemical Society. Gemini surfactants have important advantages, e.g., low micromolar CMCs and slow millisecond monomer 虠 micelle kinetics, for membrane mimetics and for delivering nucleic acids for gene therapy or RNA silencing. However, as a prerequisite, it is important to characterize interactions occurring between Gemini surfactants and proteins. Here NMR and CD spectroscopies are employed to investigate the interactions of cationic Gemini surfactants with RNase Sa, a negatively charged ribonuclease. We find that RNase Sa binds Gemini surfactant monomers and micelles at pH values above 4 to form aggregates. Below pH 4, where the protein is positively charged, these aggregates dissolve and interactions are undetectable. Thermal denaturation experiments show that surfactant lowers RNase Sa's conformational stability, suggesting that surfactant binds the protein's denatured state preferentially. Finally, Gemini surfactants were found to bind RNA, leading to the formation of large complexes. Interestingly, Gemini surfactant binding did not prevent RNase Sa from cleaving RNA. (Figure Presented).

Amyloidogenic neurodegenerative diseases are one of the most widely investigated groups of human disorders. In the last three decades the understanding of the molecular pathogenesis of this group of disorders has experienced remarkable progress, including the discoveries of the causative genes and proteins involved in Alzheimer's, Parkinson's or Huntington's diseases. The causative link between these so-called 'neurotoxic' proteins, a subset of the class of intrinsically disordered proteins (IDPs), and the development of disease has been well established. However, critical questions of neurodegeneration, such as the nature of the specific neurotoxic conformation, and why different neurons degenerate in different diseases, remain unsolved. In recent years, emerging single-molecule techniques have been used to study single molecules of neurotoxic proteins and to make direct measurements of their conformational polymorphism and fast fluctuations. This chapter aims to provide an overall view of the specific contributions of one of these techniques - single-molecule force spectroscopy - that allows the manipulation and analysis (nanomechanics) of single molecules. These studies on neurotoxic protein nanomechanics have shed light on the nature and behavior of these proteins whose associated diseases have such a high social and health impact. We also indicate the key open questions in this field; the answers to these questions will provide a more complete molecular understanding of neurodegeneration.

Several important human inherited neurodegenerative diseases are caused by >polyQ expansions>, which are aberrant long repeats of glutamine residues in proteins. PolyQ binding peptide 1 (QBP1), whose minimal active core sequence is Trp-Lys-Trp-Trp-Pro-Gly-Ile-Phe, binds to expanded polyQs and blocks their β-structure transition, aggregation and in vivo neurodegeneration. Whereas QBP1 is a widely used, commercially available product, its structure is unknown. Here, we have characterized the conformations of QBP1 and a scrambled peptide (Trp-Pro-Ile-Trp-Lys-Gly-Trp-Phe) in aqueous solution by CD, fluorescence and NMR spectroscopies. A CD maximum at 227 nm suggests the presence of rigid Trp side chains in QBP1. Based on 41 NOE-derived distance constraints, the 3D structure of QBP1 was determined. The side chains of Trp 4 and Ile 7, and to a lesser extent, those of Lys 2, Trp 3 and Phe 8, form a small hydrophobic cluster. Pro 5 and Gly 6 adopt a type II tight turn and Lys 2's ζ-NH3 + is positioned to form a favorable cation-π interaction with Trp 4's indole ring. In contrast, the scrambled QBP1 peptide, which lacks inhibitory activity, does not adopt a preferred structure. These results provide a basis for future structure-based design approaches to further optimize QBP1 for therapy. © 2014 Elsevier Inc. All rights reserved.

The crystal structure of the Sup35 prion segment, GNNQQNY, revealed precise side chain packing and an extensive H-bond network. However, the conformers and stabilizing interactions involved at nascent amyloid formation are still unclear. Here, long molecular dynamics simulations and quantum mechanical calculations have been utilized to study the conformation and energetics of the initial structure that acts to nucleate further growth. Considering all the plausible intermediates that may act as stepping stones, we find that the initial nucleus is a twisted single-layer, three-stranded parallel β-sheet. H-bonds between β-strands in this twisted sheet, some of which differ from those of the crystal structures nontwisted β-strands, are key for the nucleus formation and stability. High level theoretical calculations of these H-bonds energetics can account for this amyloid-like trimers remarkable stability. The intermeshing of facing sheets to form the dry interface provides less stability and would occur between two three-stranded β-sheets without metastable water nanowires. © 2014 American Chemical Society.

We have used NMR methods to characterize the structure and dynamics of ribonuclease Sa in solution. The solution structure of RNase Sa was obtained using the distance constraints provided by 2,276 NOEs and the C6-C96 disulfide bond. The 40 resulting structures are well determined; their mean pairwise RMSD is 0.76 A (backbone) and 1.26 A (heavy atoms). The solution structures are similar to previously determined crystal structures, especially in the secondary structure, but exhibit new features: the loop composed of Pro 45 to Ser 48 adopts distinct conformations and the rings of tyrosines 51, 52, and 55 have reduced flipping rates. Amide protons with greatly reduced exchange rates are found predominantly in interior beta-strands and the alpha-helix, but also in the external 3/10 helix and edge beta-strand linked by the disulfide bond. Analysis of (15)N relaxation experiments (R1, R2, and NOE) at 600 MHz revealed five segments, consisting of residues 1-5, 28-31, 46-50, 60-65, 74-77, retaining flexibility in solution. The change in conformation entropy for RNase SA folding is smaller than previously believed, since the native protein is more flexible in solution than in a crystal.

Folding kinetics for phage 434 Cro protein are examined and compared with those reported for lambda(6-85), the N-terminal domain of the repressor of phage lambda. The two proteins have similar all-helical structures consisting of five helices but different stabilities. In contrast to lambda(6-85), sharp and distinct aromatic (1)H NMR signals without exchange broadening characterize the native and urea-denatured 434 Cro forms at equilibrium at 20 degrees C, indicating slow interconversion on the NMR time scale. Stopped-flow fluorescence data using the single 434 Cro tryptophan indicate strongly urea-dependent refolding rates and smaller urea dependencies of the unfolding rates, suggesting a native-like transition state ensemble. Refolding rates are slower and unfolding rates considerably faster at pH 4 than at pH 6. This accounts for the lower stability of 434 Cro at pH 4 and suggests the existence of pH-dependent, possibly salt bridge interactions that are more stabilizing at pH 6. At

Thermodynamic parameters describing the phage 434 Cro protein have been determined by calorimetry and, independently, by far-UV circular dichroism (CD) measurements of isothermal urea denaturations and thermal denaturations at fixed urea concentrations. These equilibrium unfolding transitions are adequately described by the two-state model. The far-UV CD denaturation data yield average temperature-independent values of 0.99 ( 0.10 kcal mol -1 M -1 for m and 0.98 ( 0.05 kcal mol -1 K -1 for ¢Cp,U, the heat capacity change accompanying unfolding. Calorimetric data yield a temperature- independent ¢Cp,U of 0.95 ( 0.30 kcal mol -1 K -1 or a temperature-dependent value of 1.00 ( 0.10 kcal mol -1 K -1 at 25 °C. ¢Cp,U and m determined for 434 Cro are in accord with values predicted using known empirical correlations with structure. The free energy of unfolding is pH-dependent, and the protein is completely unfolded at pH 2.0 and 25 °C as judged by calorimetry or CD. The stability of 434 Cro is lower than those observed for the structurally similar N-terminal domain of the repressor of phage 434 (R1-69) or of phage I (I6-85), but is close to the value reported for the putative monomeric I Cro. Since a protein's structural stability is important in determining its intracellular stability and turnover, the stability of Cro relative to the repressor could be a key component of the regulatory circuit controlling the levels and, consequently, the functions of the two proteins in vivo.

The impact of folding funnels and folding simulations on the way experimentalists interpret results is examined. The image of the transition state has changed from a unique species that has a strained configuration, with a correspondingly high free energy, to a more ordinary folding intermediate, whose balance between limited conformational entropy and stabilizing contacts places it at the top of the free energy barrier. Evidence for a broad transition barrier comes from studies showing that mutations can change the position of the barrier. The main controversial issue now is whether populated folding intermediates are productive on-pathway intermediates or dead-end traps. Direct experimental evidence is needed. Theories suggesting that populated intermediates are trapped in a glasslike state are usually based on mechanisms which imply that trapping would only be extremely short-lived (e.g., nanoseconds) in water at 25 degrees C. There seems to be little experimental evidence for long-lived trapping in monomers, if folding aggregates are excluded. On the other hand, there is good evidence for kinetic trapping in dimers. alpha-Helix formation is currently the fastest known process in protein folding, and incipient helices are present at the start of folding. Fast helix formation has the effect of narrowing drastically the choice of folding routes. Thus helix formation can direct folding. It changes the folding metaphor from pouring liquid down a folding funnel to a train leaving a switchyard with only a few choices of exit tracks.

After the recent discovery of a ribonuclease A unfolding intermediate [Kiefhaber, T., et al. (1995) Nature 375, 513-515], we investigated the unfolding pathway of hen egg white lysozyme. At pH* 4.00 with D2O at 10 degrees C and 6 M guanidinium chloride, unfolding shows a single, slow kinetic phase, with a relaxation time of 3300 s when monitored by circular dichroism (CD). Exchange of the tryptophan indole nitrogen protons shows that buried Trp residues 123, 111, and 108 lose tight packing and become solvent-exposed simultaneously, with a mean relaxation time of 3300 s, similar to the CD-monitored unfolding rate. Unfolding monitored by Trp fluorescence shows, moreover, that 90% of the amplitude change occurs in a slow phase, with a relaxation time of 2400 s. Faster-unfolding phases with minor amplitudes are detected by Trp indole hydrogen exchange and by fluorescence. It is likely that these changes are caused by Trp 62 and Trp 63, active site residues which are not buried in the hydrophobic core. Lysozyme unfolding was further monitored by the histidine 15 C epsilon1 proton, which gives resolved lines for the native and unfolded species in one-dimensional 1H-NMR spectra. The majority of the unfolding reaction, 70%, occurs in a slow phase with a relaxation time of 3600 s, but there is also a rapid unfolding phase; 30% of the His 15 C epsilon1 proton resonance intensity is found at the unfolded chemical shift within tens of seconds after the start of unfolding. The amplitude of the rapid unfolding phase increases proportionally with the concentration of GdmCl denaturant present. These results show that a partially buried residue of lysozyme, histidine 15, takes part in forming an unfolding intermediate similar to the one observed earlier for valine 63 in ribonuclease A. The tryptophan side chains buried in the hydrophobic core of lysoyzme, in contrast, do not participate in forming the unfolding intermediate, as judged by proton chemical shifts. The buried tryptophan residues of dihydrofolate reductase, monitored by 19F-NMR, do participate in forming an unfolding intermediate [Hoeltzli, S. D.,Frieden, C. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9318-9322]; the difference between that study and ours may reside in the greater sensitivity of 19F to the detection of motional differences.

Onconase® FL-G zymogen is a 120 residue protein produced by circular permutation of the native Onconase® sequence. In this construction, the wild type N- and C-termini are linked by a 16 residue segment and new N- and C-termini are generated at wild type positions R73 and S72. This novel segment linking the native N- and C-termini is designed to obstruct Onconase's® active site and encloses a cleavage site for the HIV-1 protease. As a first step towards the resolution of its 3D structure and the study of its structure-function relationships, we report here the nearly complete NMR 1H, 13C and 15N resonance chemical shift assignments at pH 5.2 and 35 C (BMRB deposit no 17973). The results presented here clearly show that the structure of the wild type Onconase ® is conserved in the FL-G zymogen This work was supported by the projects, CTQ2008-0080, CTQ2010-21567-C02-02 and BFU2009-06935/ BMC from MICINN and PUG2008A from the Universitat de Girona

8 pags, 3 pags, Protein aggregation via 3D domain swapping is a complex mechanism which can lead to the acquisition of new biological, benign or also malignant functions, such as amyloid deposits. In this context, RNase A represents a fascinating model system, since by dislocating different polypeptide chain regions, it forms many diverse oligomers. No other protein displays such a large number of different quaternary structures. Here we report a comparative structural analysis between natural and artificial RNase A dimers and bovine seminal ribonuclease, a natively dimeric RNase with antitumor activity, with the aim to design RNase A derivatives with improved pharmacological potential. © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Amyloids are ordered protein aggregates that are typically associated with neurodegenerative diseases and cognitive impairment. By contrast, the amyloid-like state of the neuronal RNA binding protein Orb2 in Drosophila was recently implicated in memory consolidation, but it remains unclear what features of this functional amyloid-like protein give rise to such diametrically opposed behaviour. Here, using an array of biophysical, cell biological and behavioural assays we have characterized the structural features of Orb2 from the monomer to the amyloid state. Surprisingly, we find that Orb2 shares many structural traits with pathological amyloids, including the intermediate toxic oligomeric species, which can be sequestered in vivo in hetero-oligomers by pathological amyloids. However, unlike pathological amyloids, Orb2 rapidly forms amyloids and its toxic intermediates are extremely transient, indicating that kinetic parameters differentiate this functional amyloid from pathological amyloids. We also observed that a well-known anti-amyloidogenic peptide interferes with long-term memory in Drosophila. These results provide structural insights into how the amyloid-like state of the Orb2 protein can stabilize memory and be nontoxic. They also provide insight into how amyloid-based diseases may affect memory processes., This research was supported by funds from SAF2013-49179-C2-1-R JPND_CD_FP-688- 059 (AC14/00037 ISCIII) to MCV, SIMR to KS, SAF2013-49179-C2-2-R JPND_CD_FP-688-059 (AC14/00037 ISCIII) to DVL, BFU2012-36825, S2011/BMD-2457 (Comunidad de Madrid)

Amyloidogenic neurodegenerative diseases are incurable conditions with high social impact that are typically caused by specific, largely disordered proteins. However, the underlying molecular mechanism remains elusive to established techniques. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these >neurotoxic proteins> triggers the pathogenic cascade. We use force spectroscopy and a novel methodology for unequivocal single-molecule identification to demonstrate a rich conformational polymorphism in the monomer of four representative neurotoxic proteins. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the critical monomeric β-conformational change, neurotoxicity, and neurodegeneration. Hence, we postulate that specific mechanostable conformers are the cause of these diseases, representing important new early-diagnostic and therapeutic targets. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt, or reverse multiple neurodegenerative diseases. © 2012 Hervás et al.

1 pags. -- 56th Annual Meeting of the Biophysical-Society, FEB 25-29, 2012, San Diego, CA, Amyloidogenic neurodegenerative diseases are incurable conditions caused by specific largely disordered proteins. However, the underlying molecular mechanism remains elusive. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these ‘‘neurotoxic proteins’’ triggers the pathogenic cascade. Using force spectroscopy with unequivocal singlemolecule identification we demonstrate a rich conformational polymorphism at their monomer level. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the monomeric b-conformational change and neurodegeneration. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt or reverse multiple neurodegenerative diseases

An Arg-rich peptide called RIA7; sequence ac-ARAAAAAIRAIAAIIRAGGY-am, tetramerizes to form a well folded, four helix X-bundle protein. The Arg side chains are solvent exposed and the hydrophobic core is composed of the side chains from some Alas, all the Iles and the C-terminal Tyr. Since Gly, Ala and Ile, and in lesser amounts Arg and Tyr have been reported to form under putative prebiotic Earth conditions, it is plausible that RIA7-like peptides might have formed on the primitive Earth and interacted with RNAs. The interaction of RIA7 with two RNAs was tested and the formation of insoluble aggregates was observed. These results contrast with previous studies of a Lys-rich variant, called KIA7, which promotes the cleavage of RNAs. Their close structural similarity makes RIA7 and KIA7 an excellent system to compare the relative contributions of Arg and Lys to protein conformational stability. NMR-monitored hydrogen/deuterium exchange measurements and CD-monitored thermal denaturation experiments performed at different peptide and salt concentrations reveal that the conformational stabilities of RIA7 and KIA7 are practically the same. This finding has relevance for protein engineering as Lys is frequently replaced by Arg to improve ligand binding and membrane association and penetration. © 2012 Elsevier Inc. All rights reserved.

An understanding of the solution factors contributing to the rate of aggregation of a protein into amyloid oligomers, to the modulation of the conformational state populated prior to aggregation and to the structure/morphology of the resulting oligomers is one of the goals of present research in this field. We have studied the influence of six different salts on the conversion of the N-terminal domain of Escherichia coli HypF (HypF-N) into amyloid-like oligomers under conditions of acidic pH. Our results show that salts having different anions (NaCl, NaClO4, NaI, Na 2SO4) accelerate oligomerization with an efficacy that follows the electroselectivity series of the anions (SO4 2- ≥ ClO4 - > I- > Cl-). By contrast, salts with different cations (NaCl, LiCl, KCl) have similar effects. We also investigated the effect of salts on the structure of the final and initial states of HypF-N aggregation. The electroselectivity series does not apply to the effect of anions on the structure of the oligomers. By contrast, it applies to their effect on the content of secondary structure and on the exposure of hydrophobic clusters of the monomeric precursor state. The results therefore indicate that the binding of anions to the positively charged residues of HypF-N at low pH is the mechanism by which salts modulate the rate of oligomerization and the structure of the monomeric precursor state but not the structure of the resulting oligomers. Overall, the data contribute to rationalize the effect of salts on amyloid-like oligomer formation and to explain the role of charged biological macromolecules in protein aggregation processes.

Gemini surfactants have two polar head groups and two hydrocarbon tails. Compared with conventional surfactants, geminis have much lower (οM vs. mM) critical micelle concentrations and possess slower (ms vs οs) monomer micelle kinetics. The structure of the gemini surfactants studied is [HOCH 2CH 2-, CH 3-, CH 3(CH 2) 15-N +-(CH 2) s-N +-(CH 2) 15CH 3,-CH 3,-CH 2CH 2OH]·2Br - where s=4, 5, or 6. Our objective is to reveal the effect of these cationic gemini surfactants on the structure and stability of two model proteins: Ribonuclease A (RNase A) and Hen Egg White Lysozyme (HEWL). 2D 1H NMR and Circular Dichroism (CD) spectroscopies show that the conformation of RNase A and HEWL is unaffected at low to neutral pH where these proteins are positively charged, although hydrogen exchange shows that RNase A's conformational stability is slightly lowered. At alkaline pH, where these proteins lose their net positive charge, fluorescence and CD spectroscopies and ITC experiments show that they do interact with gemini surfactants, and multiple protein•gemini complexes are observed. Based on the results, we conclude that these cationic gemini surfactants neither interact strongly with nor severely destabilize these well folded proteins in physiological conditions, and we advance that they can serve as useful membrane mimetics for studying the interactions between membrane components and positively charged proteins. © 2011 Elsevier Inc.

The thermal stability and enzymatic activity of bovine pancreatic ribonuclease A (RNase A) have been investigated in the presence of a homologous series of cationic gemini surfactants (alkanediyl-α,ω-bis(hydroxyethyl methyl hexadecyl ammonium bromide)). UV, circular dichorism and fluorescence spectroscopies have been used for this study. The denaturation curves at various surfactant concentrations were analyzed on basis of a two-transition model to obtain values of T m (temperature at the midpoint of denaturation) and ΔH m (enthalpy change at T m) of each transition. The main conclusion of this study is that these cationic gemini surfactants slightly activate and stabilize RNase A below their critical micelle concentrations at pH 5.0. The cationic gemini surfactant with the shorter spacer interacts more efficiently with RNase A than those with longer spacers

10 pags, 6 figs, 1 tab, The protein NA14 is a key adaptor protein mediating the intermolecular interactions of microtubules and Spastin. To gain insight into its structure and function, we have expressed, purified and characterized human NA14 and some variants. NA14 is rather insoluble and tends to oligomerize and form fibrils. Successive mutation of the three Cys and two potentially exposed Leu residues (83 and 93) yielded a water-soluble quintuple variant, named 3CS-2LR. NA14 and its variants have a high helical content as determined by circular dichroism (CD). Based on nuclear magnetic resonance data of the quintuple mutant and the wild-type (wt) protein in the presence of dodecylphosphocholine micelles, the N-(M1-N13) and C-termini (K105-S119) were found to lack preferred structure. The remaining residues (14104) participate in NA14 self-association, probably by forming a parallel coiled-coil structure. We hypothesize that Leu 83 and Leu 93 mediate interactions among NA14, Spastin and microtubules. We have also examined urea and thermal denaturation of the quintuple and other NA14 variants at different pH values by CD. The pH dependence of the conformational stability and the elevated native-state pKa determined for the two conserved Tyr allow us to propose that the NA14 structure may be stabilized by two Glu-COO- ||| HO-Tyr H-bonds, highly conserved in NA14-like proteins in other species. © The Author 2011., This work was supported by the Spanish Ministerio de Ciencia e Innovación, MICINN, (CSD2006-20642, CTQ2010-21567-C02-02, CTQ2008-00080/BQU, and BFU2010-15703/BMC), and by the Comunidad de Madrid (S-GEN-0166-2006).

9 pags, 4 figs, 1 tab, The structural determinants that are responsible for the formation of higher order associations of folded proteins remain unknown. We have investigated the role on the dimerization process of different residues of a domain-swapped dimer human pancreatic ribonuclease variant. This variant is a good model to study the dimerization and swapping processes because dimer and monomer forms interconvert, are easily isolated, and only one dimeric species is produced. Thus, simple models for the swapping process can be proposed. The dimerization (dissociation constant) and swapping propensity have been studied using different variants with changes in residues that belong to different putative molecular determinants of dimerization. Using NMR spectroscopy, we show that these mutations do not substantially alter the overall conformation and flexibility, but affect the residue level stability. Overall, the most critical residues for the swapping process are those of one subunit that interact with the hinge loop of another one-subunit residue, stabilizing it in a conformation that favors the interchange. Tyr25, Gln101, and Pro 19, with Asn17, Ser21, and Ser23, are found to be the most significant; notably, Glu103 and Arg 104, which were postulated to form salt bridges that would stabilize the dimer, are not critical for dimerization. © 2011 Biophysical Society., Work was supported by grants No. BFU2009-06935/BMC, No. CTQ2010-21567-C02-02, and No. CTQ2008-00080/BQU from Ministerio de Ciencia e Innovación (Spain).

10 pags, 5 figs, Eosinophil cationic protein (ECP) is a highly stable, cytotoxic ribonuclease with the ability to enter and disrupt membranes that participates in innate immune defense against parasites but also kills human cells. We have used NMR spectroscopy to characterize the binding of ECP to membrane and heparin mimetics at a residue level. We believe we have identified three Arg-rich surface loops and Trp35 as crucial for membrane binding. Importantly, we have provided evidence that the interaction surface of ECP with heparin mimetics is extended with respect to that previously described (fragment 34-38). We believe we have identified new sites involved in the interaction for the first time, and shown that the N-terminal α-helix, the third loop, and the first and last β-strands are key for heparin binding. We have also shown that a biologically active ECP N-terminal fragment comprising the first 45 residues (ECP1-45) retains the capacity to bind membrane and heparin mimetics, thus neither the ECP tertiary structure nor its high conformational stability are required for cytotoxicity. © 2010 by the Biophysical Society., This work was supported by the Spanish Ministerio de Ciencia e Innovación (CTQ2008-00080/BQU, BIO2008-04487-CO3-02, BFU2006-15543-CO2-01, BFU2009-09371).

6 pags, 4 figs, In trypanosomes, the production of mRNA relies on the synthesis of the spliced leader (SL) RNA. Expression of the SL RNA is initiated at the only known RNA polymerase II promoter in these parasites. In the pathogenic trypanosome, Trypanosoma brucei, transcription factor IIB (tTFIIB) is essential for SL RNA gene transcription and cell viability, but has a highly divergent primary sequence in comparison to TFIIB in well-studied eukaryotes. Here we describe the 2.3 Å resolution structure of the C-terminal domain of tTFIIB (tTFIIBC). The tTFIIBC structure consists of 2 closely packed helical modules followed by a C-terminal extension of 32 aa. Using the structure as a guide, alanine substitutions of basic residues in regions analogous to functionally important regions of the well-studied eukaryotic TFIIB support conservation of a general mechanism of TFIIB function in eukaryotes. Strikingly, tTFIIBC contains additional loops and helices, and, in contrast to the highly basic DNA binding surface of human TFIIB, contains a neutral surface in the corresponding region. These attributes probably mediate trypanosome-specific interactions and have implications for the apparent bidirectional transcription by RNA polymerase II in protein-encoding gene expression in these organisms., This work was supported by National Institutes of Health Grant AI-29478 (to V.B.), American Heart Association Fellowship 0425791T (to J.B.P.), American Heart Association Scientist Development Grant 0735532T (to A.D.), and Ministerio de Educacion y Ciencia (Spain) Grant CTQ2007-68014-C02-02 (to D.V.L.).

Eosinophil cationic protein (ECP)/human RNase 3, a member of the RNase A family, is a remarkably cytotoxic protein implicated in asthma and allergies. These activities are probably due to ECP's ability to interact with and disrupt membranes and depend on two Trp, 19 Arg, and possibly an extremely high conformational stability. Here, we have used NMR spectroscopy to assign essentially all (1)H, (15)N, and backbone (13)C resonances, to solve the 3D structure in aqueous solution and to quantify its residue-level stability. The NMR solution structure was determined on the basis of 2316 distance constraints and is well-defined (backbone RMSD = 0.81 A). The N-terminus and the loop composed of residues 114-123 are relatively well-ordered; in contrast, conformational diversity is observed for the loop segments 17-22, 65-68, and 92-95 and most exposed sidechains. The side chain NH groups of the two Trp and 19 Arg showed no significant protection against hydrogen/deuterium exchange. The most protected NH groups belong to the first and last two beta-strands, and curiously, the first alpha-helix. Analysis of their exchange rates reveals a strikingly high global stability of 11.8 kcal/mol. This value and other stability measurements are used to better quantify ECP's unfolding thermodynamics.

RNase A self-associates under certain conditions to form a series of domain-swapped oligomers. These oligomers show high catalytic activity against double-stranded RNA and striking antitumor actions that are lacking in the monomer. However, the dissociation of these metastable oligomers limits their therapeutic potential. Here, a widely used conjugating agent, 1-ethyl-3-(3-dimethylaminoisopropyl) carbodiimide (EDC), has been used to induce the formation of amide bonds between carboxylate and amine groups of different subunits of the RNase A C-dimer. A cross-linked C-dimer which does not dissociate was isolated and was found have augmented enzymatic activity toward double-stranded RNA relative to the unmodified C-dimer. Characterization using chromatography, electrophoresis, mass spectrometry, and NMR spectroscopy revealed that the EDC-treated C-dimer retains its structure and contains one to three novel amide bonds. Moreover, both the EDC-treated C-dimer and EDC-treated RNase A monomer were found to carry an increased number of positive charges (about 6 ± 2 charges per subunit). These additional positive charges are presumably due to adduct formation with EDC, which neutralizes a negatively charged carboxylate group and couples it to a positively charged tertiary amine. The increased net positive charge endowed by EDC adducts likely contributes to the heightened cleavage of double-stranded RNA of the EDC-treated monomer and EDC-treated C-dimer. Further evidence for EDC adduct formation is provided by the reaction of EDC with a dipeptide Ac-Asp-Ala-NH(2) monitored by NMR spectroscopy and mass spectrometry. To determine if EDC adduct formation with proteins is common and how this affects protein net charge, conformation, and activity, four well-characterized proteins, ribonuclease Sa, hen lysozyme, carbonic anhydrase, and hemoglobin, were incubated with EDC and the products were characterized. EDC formed adducts with all these proteins, as judged by mass spectrometry and electrophoresis. Moreover, all suffered conformational changes ranging from slight structural modifications in the case of lysozyme, to denaturation for hemoglobin as measured by NMR spectroscopy and enzyme assays. We conclude that EDC adduct formation with proteins can affect their net charge, conformation, and enzymatic activity.

Pancreatic-type ribonucleases are a family of RNA degrading enzymes that share different degrees of sequence identity but a very similar 3D-structure. The prototype of this family is bovine pancreatic ribonuclease or ribonuclease A. This enzyme has been the object of landmark work on the folding, stability, protein chemistry, catalysis, enzyme-substrate interaction and molecular evolution. In the recent years, the interest in the study of pancreatic-type ribonucleases has increased due to the involvement of some members of this family in special biological functions. In addition, dimeric and also higher oligomeric structures can be attained by the members of this family. The oligomers described structurally to date are mainly formed by 3D-domain swapping, a process which consists of the exchange of identical domains (i.e. identical structural elements, usually the N- and C-termini) between the subunits and is considered to be a mechanism for amyloid-type aggregate formation. This review compares the dimeric and oligomeric structures of different members of the pancreatic-type ribonuclease family which are able to acquire these structures, namely, bovine seminal ribonuclease, ribonuclease A and its human counterpart, human pancreatic ribonuclease. A specific focus is placed on what is known about the structural determinants that lead to the acquisition of a particular oligomeric structure and on the proposed mechanism of 3D-swapping.

Human pancreatic ribonuclease (RNase 1) is expressed in many tissues; has several important enzymatic and biological activities, including efficient cleavage of single-stranded RNA, double-stranded RNA and double-stranded RNA–DNA hybrids, digestion of dietary RNA, regulation of vascular homeostasis, inactivation of the HIV, activation of immature dendritic cells and induction of cytokine production; and furthermore shows potential as an anti-tumor agent. The solution structure and dynamics of uncomplexed, wild-type RNase 1 have been determined by NMR spectroscopy methods to better understand these activities. The family of 20 structures determined on the basis of 6115 unambiguous nuclear Overhauser enhancements is well resolved (pairwise backbone RMSD = 1.07 Å) and has the classic RNase A type of tertiary structure. Important structural differences compared with previously determined crystal structures of RNase 1 variants or inhibitor-bound complexes are observed in the conformation of loop regions and side chains implicated in the enzymatic as well as biological activities and binding to the cytoplasmic RNase inhibitor. Multiple side chain conformations observed for key surface residues are proposed to be crucial for membrane binding as well as translocation and efficient RNA hydrolysis. 15N–1H relaxation measurements interpreted with the standard and our extended Lipari–Szabo formalism reveal rigid regions and identify more dynamic loop regions. Some of the most dynamic areas are key for binding to the cytoplasmic RNase inhibitor. This finding and the important differences observed between the structure in solution and that bound to the inhibitor are indications that RNase 1 to inhibitor binding can be better described by the “induced fit” model rather than the rigid “lock-into-key” mechanism. Translational diffusion measurements reveal that RNase 1 is predominantly dimeric above 1 mM concentration; the possible implications of this dimeric state for the remarkable biological properties of RNase 1 are discussed., This work was supported by projects OTKA NK 68578 from the Hungarian Research Foundation and BFU2005-01855/BMC from the Spanish Ministerio de Educación y Ciencia.

To investigate the pH dependence of the conformational stability of ribonucleases A and T1, urea and guanidine hydrochloride denaturation curves have been determined over the pH range 2-10. The maximum conformational stability of both proteins is about 9 kcal/mol and occurs near pH 4.5 for ribonuclease T1 and between pH 7 and 9 for ribonuclease A. The pH dependence suggests that electrostatic interactions among the charged groups make a relatively small contribution to the conformational stability of these proteins. The dependence of delta G on urea concentration increases from about 1200 cal mol-1 M-1 at high pH to about 2400 cal mol-1 M-1 at low pH for ribonuclease A. This suggests that the unfolded conformations of RNase A become more accessible to urea as the net charge on the molecule increases. For RNase T1, the dependence of delta G on urea concentration is minimal near pH 6 and increases at both higher and lower pH. An analysis of information of this type for several proteins in terms of a model developed by Tanford [Tanford, C. (1964) J. Am. Chem. Soc. 86, 2050-2059] suggests that the unfolded states of proteins in urea and GdnHCl solutions may differ significantly in the extent of their interaction with denaturants. Thus, the conformations assumed by unfolded proteins may depend to at least some extent on the amino acid sequence of the protein.

A protocol for the rapid purification of ribonuclease T1 expressed from a chemically synthesized gene cloned into Escherichia coli is described. QAE ion-exchange and Sephadex G-50 chromatography are used to give over 300 mg (88% yield) of pure ribonuclease T1 from 6 1 of liquid culture in 3 days. We also report a new absorption coefficient for RNase T 1 : E 278 nm 1% = 15.4 .

We show that temperature is an important parameter for the sensitivity of saturation transfer difference (STD) spectroscopy. A decreased intensity of STD signals is observed for lactose binding to growth-regulatory galectin7 (p53-induced gene 1), as well as for nucleotide binding to annexin A6, when the temperature is increased from 281 to 298-310 K. Opposite temperature effects on STD intensity are observed for S-peptide binding to S-protein to reconstitute RNase S. However, the STD signals for tryptophan binding to downstream regulatory element antagonist modulator of the human prodynorphin gene (DREAM)are relatively unaffected between 281 and 298 K. The known kinetics of the binding of ATP by the uncoupling protein from brown adipose tissue mitochondria (UCP1) predicted an observable STD at 310 K, but rapid sample degradation limits the experiments to much lower temperatures. Temperature strongly influences the kinetics and affinity constant of various types of complex formation and in so doing influences the observed STD effects. Therefore, temperature can be exploited to facilitate the optimization of STD-based applications, and at the same time minimize the number of test samples. STD-based screening protocols to detect new target-specific compounds may yield a larger number of potential ligands if screened at various temperatures.