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Misfolding of Cu, Zn superoxide dismutase (SOD1) variants may lead to protein aggregation and ultimately amyotrophic lateral sclerosis (ALS). The mechanism and protein conformational changes during this process are complex and remain unclear. To study SOD1 variants aggregation at the molecular level and in solution, we chemically induced aggregation of a mutant variant (G93A SOD1) with trifluoroethanol (TFE) and used both native mass spectrometry (MS) to analyze the intact protein and Fast Photochemical Oxidation of Proteins (FPOP) to characterize the structural changes induced by TFE. We found partially unfolded G93A SOD1 monomers prior to oligomerization and identified regions of the N-terminus, C-terminus, and strands β5, β6 accountable for the partial unfolding. We propose that exposure of hydrophobic interfaces of these unstructured regions serves as a precursor to aggregation. Our results provide a possible mechanism and molecular basis for ALS-linked SOD1 misfolding and aggregation.

Understanding the thermodynamic mechanisms of adaptation of biomacromolecules to high hydrostatic pressure can help shed light on how piezophilic organisms can survive at pressures reaching over 1000 atmospheres. Interaction of proteins with nucleic acids is one of the central processes that allow information flow encoded in the sequence of DNA. Here, we report the results of a study on the interaction of cold shock protein B from Bacillus subtilis (CspB-Bs) with heptadeoxythymine template (pDT7) as a function of temperature and hydrostatic pressure. Experimental data collected at different CspB-Bs:pDT7 ratios were analyzed using a thermodynamic linkage model that accounts for both protein unfolding and CspB-Bs:pDT7 binding. The global fit to the model provided estimates of the stability of CspB-Bs, ΔGProto, the volume change upon CspB-Bs unfolding, ΔVProt, the association constant for CspB-Bs:pDT7 complex, Kao, and the volume changes upon pDT7 single-stranded DNA (ssDNA) template binding, ΔVBind. The protein, CspB-Bs, unfolds with an increase in hydrostatic pressure (ΔVProt < 0). Surprisingly, our study showed that ΔVBind < 0, which means that the binding of CspB-Bs to ssDNA is stabilized by an increase in hydrostatic pressure. Thus, CspB-Bs binding to pDT7 represents a case of linked equilibrium in which folding and binding react differently upon an increase in hydrostatic pressure: protein folding/unfolding equilibrium favors the unfolded state, while protein-ligand binding equilibrium favors the bound state. These opposing effects set a "maximum attainable" pressure tolerance to the protein-ssDNA complex under given conditions.

Many proteins in cells and in the extracellular matrix assemble into force-bearing networks, and some proteins clearly transduce mechanical stimuli into biochemical signals. Although structural mechanisms remain poorly understood, the designs of such proteins enable mechanical forces to either inhibit or facilitate interactions of protein domains with other proteins, including small molecules and enzymes, including proteases and kinases. Here, we review some of the structural proteins and processes that exhibit distinct modes of force-dependent signal conversion.

The G98R mutation in αA-crystallin is associated with the onset of pre-senile cataract and is characterized biochemically by an increased oligomeric mass, altered chaperone function, and loss of structural stability over time. Thus far, it is not known whether the inherent instability caused by gain of charge mutation could be rescued by a compensatory loss of charge mutation elsewhere on the protein. To answer this question, we investigated whether αA-G98R-mediated instability could be rescued through suppressor mutations by introducing site-specific ‘compensatory’ mutations in αA-G98R crystallin – αA-R21Q/G98R, αA-G98R/R116C, and αA-R157Q/G98R. The recombinant proteins were expressed, purified, characterized, and evaluated by circular dichroism (CD), intrinsic fluorescence and bis-ANS binding studies. Chaperone-like activities of recombinant proteins were assessed using alcohol dehydrogenase (ADH) and insulin as unfolding substrates. Far-UV CD studies revealed an increased α-helical content in αA-G98R in comparison to αA-WT, αA-R21Q, R157Q, and the double-mutants, αA-R21Q/G98R, and αA-R157Q/G98R. Compared to αA-WT, αA-R21Q, and αA-G98R, the double mutants showed an increased intrinsic tryptophan fluorescence, whereas the highest hydrophobicity (bis-ANS binding) was shown by αA-G98R. Introduction of a second mutation in αA-G98R reduced its bis ANS-binding activity. Both αA-R21Q/G98R and αA-R157Q/G98R showed greater chaperone-like activity against ADH aggregation than αA-G98R. However, among the three G98R mutants, only αA-R21Q/G98R protected ARPE-19 cells from H(2)O(2)-induced cytotoxicity. These results suggest that the lost chaperone-like activity of αA-G98R-crystallin can be rescued by another targeted mutation and that substitution of αA-R21Q-crystallin at the N-terminal region can rescue a deleterious mutation in the conserved α-crystallin domain of the protein.

Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from the insoluble fraction of cataractous lenses and also causes protein aggregation

SET and MYND domain-containing protein 3 (SMYD3) is a lysine methyltransferase that plays a central role in a variety of cancer diseases, exerting its pro-oncogenic activity by methylation of key proteins, of both nuclear and cytoplasmic nature. However, the role of SMYD3 in the initiation and progression of cancer is not yet fully understood and further biochemical characterization is required to support the discovery of therapeutics targeting this enzyme. We have therefore developed robust protocols for production, handling, and crystallization of SMYD3 and biophysical and biochemical assays for clarification of SMYD3 biochemistry and identification of useful lead compounds. Specifically, a time-resolved biosensor assay was developed for kinetic characterization of SMYD3 interactions. Functional differences in SMYD3 interactions with its natural small molecule ligands SAM and SAH were revealed, with SAM forming a very stable complex. A variety of peptides mimicking putative substrates of SMYD3 were explored in order to expose structural features important for recognition. The interaction between SMYD3 and some peptides was influenced by SAM. A nonradioactive SMYD3 activity assay using liquid chromatography-mass spectrometry (LC-MS) analysis explored substrate features of importance also for methylation. Methylation was notable only toward MAP kinase kinase kinase 2 (MAP3K2_K-260)-mimicking peptides, although binary and tertiary complexes were detected also with other peptides. The analysis supported a random bi-bi mechanistic model for SMYD3 methyltransferase catalysis. Our work unveiled complexities in SMYD3 biochemistry and resulted in procedures suitable for further studies and identification of novel starting points for design of effective and specific leads for this potential oncology target.

Increased protein solubility is known to correlate with an increase in the proportion of lysine over arginine residues. Previous work has shown that the aggregation propensity of a single-chain variable fragment (scFv) does not correlate with its conformational stability or native-state protein-protein interactions. Here we test the hypothesis that aggregation is driven by the colloidal stability of partially unfolded states, studying the behaviour of scFv mutants harbouring single or multiple site-specific arginine/lysine mutations in denaturing buffers. In 6 M guanidine hydrochloride (GdmCl) or 8 M urea, repulsive protein-protein interactions were measured for the wild-type and lysine enriched (4RK) scFvs on account of weakened short-ranged attractions and increased excluded volume, in contrast to the arginine enriched (7KR) scFv which demonstrated strong reversible association. In 3 M GdmCl, the minimum concentration at which the scFvs were unfolded, the hydrodynamic radius of 4RK remained constant but increased for the wild-type and especially for 7KR. Individually swapping lysine back to arginine in 4KR indicated that the observed aggregation propensity of arginine in denaturing conditions was non-specific. Interestingly, one such swap generated a scFv with especially low aggregation rates under low/high ionic strength and denaturing buffers; molecular modelling identified hydrogen-bonding between the arginine side chain and main chain peptide groups, stabilising the structure. The arginine/lysine ratio is not routinely considered in biopharmaceutical scaffold design, or current amyloid prediction methods. This work therefore suggests a simple method to increase the stability of a biopharmaceutical protein against aggregation.

Transmembrane helices dominate the landscape for many membrane proteins. Often flanked by interfacial aromatic residues, these transmembrane helices also contain loops and inter-helix segments, which could help in stabilizing a transmembrane orientation. Using (2)H-NMR spectroscopy to monitor bilayer incorporated model GWALP23 family peptides, we address systematically the issue of helix fraying in relation to the dynamics and orientation of closely similar individual transmembrane helices. Adjacent to a core transmembrane helix, we inserted aromatic (Phe, Trp, Tyr, His) or non-aromatic residues (Ala, Gly) into positions 4 and 5, to examine the side-chain dependency of the transmembrane orientation, dynamics and helix integrity (extent and location of unraveling). Incorporation of (2)H-alanine labels enables one to assess the helicity of the core sequence and the peptide termini. For most of the helices, we observed substantial unwinding involving at least 3 residues at both ends. For the unique case of histidine at positions 4 and 5, an extended N-terminal unwinding was observed up to residue 7. For further investigation regarding the onset of fraying, we employed A(4,5)GWALP23 with (2)H labels at residues 4 and 5 and found that the number of terminal residues involved in the unwinding depends on bilayer thicknesses and helps to govern the helix dynamics. The combined results enable us to compare and contrast the extent of fraying for each related helix, as reflected by the deviation of experimental (2)H quadrupolar splitting magnitudes of juxta-terminal alanines A3 and A21 from those represented by an ideal helix geometry.

Aggregation of transthyretin (TTR) causes TTR amyloidoses. The native TTR tetramer (a dimer of dimers) is stabilized by packing of phenylalanine 87 (F87) into a hydrophobic cavity of a neighboring protomer across the strong dimer interface. X-ray structures at acidic pH show that the side chain of F87 can be displaced from its binding pocket, but the resultant solution conformations remain unknown. Here we used 19F nuclear magnetic resonance (NMR) and 19F-labeled C10S-S85C TTR to characterize two local conformations of the loop containing F87. At neutral pH, F87 packs correctly into the interprotomer cavity in the dominant conformational state (93% population, T) whereas the remaining minor population is a mispacked tetramer (T*). The population of T* can be enhanced in heterotetramers by mixing C10S-S85C TTR with increasing molar ratios of A120L TTR, where a bulky leucine residue is introduced to disfavor the T state by steric hindrance. Exchange between the T and T* states in the presence of A120L is mediated by subunit exchange from the C10S-S85C tetramer. Compared to the TTR tetramer in which the dimers are correctly packed, mispacking of one or both dimer pairs leads to an increase in the urea unfolding rate of 4-fold or at least 15-fold, respectively. Consistent acid-mediated tetramer dissociation was observed by 19F NMR aggregation assays. Our results highlight the important role of the interprotomer F87 side chain packing in determining the kinetic stability of the TTR tetramer; mispacking of F87 in the T* state predisposes it for rapid dissociation and entry into the aggregation pathway.

The properties of enzymes packaged within the coat protein shell of virus-like particles (VLPs) were studied to provide a comprehensive assessment of such factors. Such entrainment did not seem to perturb enzyme function, but it did significantly enhance enzyme stability against several denaturing stimuli such as heat, organic solvents, and chaotropic agents. This improvement in performance was found to be general and independent of the number of independent subunits required and of the number of catalytically active enzymes packaged. Packaged enzymes were found by measurements of intrinsic tryptophan fluorescence to retain some of their native folded structure even longer than their catalytic activity, suggesting that protein folding is a significant component of the observed catalytic benefits. While we are unable to distinguish between kinetic and thermodynamic effects - including inhibition of enzyme unfolding, acceleration of refolding, and biasing of folding equilibria - VLP packaging appears to represent a useful general strategy for the stabilization of enzymes that operate on diffusible substrates and products.

The sterile α motif, also called the SAM domain, is known to form homo or heterocomplexes that modulate diverse biological functions through regulation of specific protein-protein interactions. The MAPK pathway of budding yeast Saccharomyces cerevisiae comprises a three-tier kinase system akin to mammals. The MAPKKK Ste11 protein of yeast contains a homodimer SAM domain, which is critical for transmitting cues to the downstream kinases. The structural stability of the dimeric Ste11 SAM is maintained by hydrophobic and ionic interactions at the interfacial amino acids. Urea induced equilibrium unfolding process of the Ste11 SAM domain is cooperative without evidence of any intermediate states. The native state H/D exchange under sub-denaturing conditions is a useful method for the detection of intermediate states of proteins. In the present study, we investigated the effect of ionic strength on the conformational stability of the dimer using the H/D exchange study. The hydrogen exchange behavior of the Ste11 dimer under physiological salt concentration reveals two metastable partially folded intermediate states, which may be generated by a sequential and cooperative unfolding of the five helices of the fold. These intermediates appear to be the dominant species for the dynamic and reversible unfolding of the Ste11 SAM domain, underlining a significant pathway for its folding kinetics via hydrophobic collapse. In contrast, higher ionic concentration eliminates this cooperativity between the stabilizing pairs of helices. Ministry of Education (MOE) Accepted version This work was supported by grants from The Ministry of Education (MOE), Singapore.

Hereditary apolipoprotein A-I (apoA-I) amyloidosis is a life-threatening incurable genetic disorder whose molecular underpinnings are unclear. In this disease, variant apoA-I, the major structural and functional protein of high-density lipoprotein, is released in a free form, undergoes an α-helix to intermolecular cross-β-sheet conversion along with a proteolytic cleavage, and is deposited as amyloid fibrils in various organs, which can cause organ damage and death. Glu34Lys is the only known charge inversion mutation in apoA-I that causes human amyloidosis. To elucidate the structural underpinnings of the amyloidogenic behavior of Glu34Lys apoA-I, we generated its recombinant globular N-terminal domain (residues 1-184) and compared the conformation and dynamics of its lipid-free form with those of two other naturally occurring apoA-I variants, Phe71Tyr (amyloidogenic) and Leu159Arg (non-amyloidogenic). All variants showed reduced structural stability and altered aromatic residue packing. The greatest decrease in stability was observed in the non-amyloidogenic variant, suggesting that amyloid formation is driven by local structural perturbations at sensitive sites. Molecular dynamics simulations revealed local helical unfolding and suggested that transient opening of the Trp72 side chain induced mutation-dependent structural perturbations in a sensitive region, including the major amyloid hot spot residues Leu14-Leu22. We posit that a shift from the "closed" to the "open" orientation of the Trp72 side chain modulates structural protection of amyloid hot spots, suggesting a previously unknown early step in the protein misfolding pathway.

We report time-resolved measurements of the coupled protein-water modes of solvated ubiquitin during protein folding. Kinetic terahertz absorption (KITA) spectroscopy serves as a label-free technique for monitoring large scale conformational changes and folding of proteins subsequent to a sudden T-jump. We report here KITA measurements at an unprecedented time resolution of 500 ns, a resolution 2 orders of magnitude better than those of any previous KITA measurements, which reveal the coupled ubiquitin-solvent dynamics even in the initial phase of hydrophobic collapse. Complementary equilibrium experiments and molecular simulations of ubiquitin solutions are performed to clarify non-equilibrium contributions and reveal the molecular picture upon a change in structure, respectively. On the basis of our results, we propose that in the case of ubiquitin a rapid (

Defining the role of non-native interactions in directing the course of protein folding or unfolding reactions has been a difficult challenge. In particular, the extent to which such interactions play a productive role by stabilizing the structures of transition states (TSs) found on the folding and unfolding pathways of proteins is not known. On the contrary, it is thought that the TSs are expanded forms of the N state stabilized by native interactions, and it is not known whether non-native interactions can modulate TS structure. In this study of the unfolding of the SH3 domain of PI3 kinase using a microsecond mixing methodology, partial non-native structure formation is shown to occur initially during unfolding. The TS of this partial "folding during unfolding" reaction is more compact than the N state: the apparent rate constant of Trp53 burial during this reaction decreases with an increase in denaturant concentration. Kinetic studies of the unfolding of mutant variants suggest that the unusually compact TS is stabilized by interactions not present in N and that these non-native interactions are hydrophobic in nature. It was determined that mutation could be used to tune the degree of compaction in the TS.

Protein glycosylation has been shown to have a variety of site-specific and glycan-specific effects, but so far, the molecular logic that leads to such observations has been elusive. Understanding the structural changes that occur and being able to correlate those with the physical properties of the glycopeptide are valuable steps toward being able to predict how specific glycosylation patterns will affect the stability of glycoproteins. By systematically comparing the structural features of the O-glycosylated carbohydrate-binding module of a Trichoderma reesei-derived Family 7 cellobiohydrolase, we were able to develop a better understanding of the influence of O-glycan structure on the molecule's physical stability. Our results indicate that the previously observed stabilizing effects of O-glycans come from the introduction of new bonding interactions to the structure and increased rigidity, while the decreased stability seemed to result from the impaired interactions and increased conformational flexibility. This type of knowledge provides a powerful and potentially general mechanism for improving the stability of proteins through glycoengineering.

Mobile loops located at the active site entrance in enzymes often participate in conformational changes required to shield the reaction from bulk solvent, to control the access of the substrate to the active site, and to position residues for substrate binding and catalysis. In d-arginine dehydrogenase from Pseudomonas aeruginosa (PaDADH), previous crystallographic data suggested that residues 45-47 in the FAD-binding domain and residues 50-56 in the substrate-binding domain in loop L1 could adopt two distinct conformations. In this study, we have used molecular dynamics, kinetics, and fluorescence spectroscopy on the S45A and A46G enzyme variants of PaDADH to investigate the impact of mutations in loop L1 on the catalytic function of the enzyme. Molecular dynamics showed that the mutant enzymes have probabilities of being in open conformations that are higher than that of wild-type PaDADH of loop L1, yielding an increased level of solvent exposure of the active site. In agreement, the flavin fluorescence intensity was ∼2-fold higher in the S45A and A46G enzymes than in wild-type PaDADH, with a 9 nm bathochromic shift of the emission band. In the variant enzymes, the k

The interaction between cytochrome c (Cyt c) and cardiolipin (CL) plays a vital role in the early stages of apoptosis. The binding of CL to Cyt c induces a considerable increase in its peroxidase activity that has been attributed to the partial unfolding of the protein, dissociation of the Met80 axial ligand, and formation of non-native conformers. Although the interaction between Cyt c and CL has been extensively studied, there is still no consensus regarding the conformational rearrangements of Cyt c that follow the protein-lipid interaction. To rationalize the different results and gain better insight into the Cyt c-CL interaction, we have studied the formation of the CL complex of the horse heart wild-type protein and selected mutants in which residues considered to play a key role in the interaction with CL (His26, His33, Lys72, Lys73, and Lys79) have been mutated. The analysis was conducted at both room temperature and low temperatures via ultraviolet-visible absorption, resonance Raman, and electron paramagnetic resonance spectroscopies. The trigger and the sequence of CL-induced structural variations are discussed in terms of disruption of the His26-Pro44 hydrogen bond. We unequivocally identify the sixth ligand in the partially unfolded, non-native low-spin state that Cyt c can adopt following the protein-lipid interaction, as a His ligation, ruling out the previously proposed involvement of a Lys residue or an OH- ion.

Mge1, a yeast homologue of Escherichia coli GrpE, is an evolutionarily conserved homodimeric nucleotide exchange factor of mitochondrial Hsp70. Temperature-dependent reversible structural alteration from a dimeric to a monomeric form is critical for Mge1 to act as a thermosensor. However, very limited information about the structural component or amino acid residue(s) that contributes to thermal sensing of Mge1/GrpE is available. In this report, we have identified a single point mutation, His167 to Leu (H167L), within the hinge region of Mge1 that confers thermal resistance to yeast. Circular dichroism, cross-linking, and refolding studies with recombinant proteins show that the Mge1 H167L mutant has increased thermal stability compared to that of wild-type Mge1 and also augments Hsp70-mediated protein refolding activity. While thermal denaturation studies suggest flexibility in the mutant, ionic quenching studies and limited proteolysis analysis reveal a relatively more rigid structure compared to that of the wild type. Intriguingly, Thermus thermophilus GrpE has a leucine at the corresponding position akin to the Mge1 mutant, and thermophilus proteins are well-known for their rigidity and hydrophobicity. Taken together, our results show that the yeast Mge1 H167L mutant functionally and structurally mimics T. thermophilus GrpE.

Knotted and slipknotted proteins are topologically complex. Understanding their folding and unfolding mechanism has attracted considerable interest. Here we combined protein engineering, single-molecule optical tweezers, and steered molecular dynamics (SMD) simulations to investigate the mechanical unfolding and folding of a slipknotted protein pyruvoyl-dependent arginine decarboxylase (PADC). In its slipknotted structure, PADC contains a long threaded loop (85 residues), which is almost twice the size of the knotting loop. When stretched from its N- and C-termini, the majority of PADC can be readily unfolded in a two-state manner, and the slipknotted structure was untied. A small percentage of PADC unfolded following a three-state pathway involving the formation of an unfolding intermediate state. These unfolding intermediate states showed a broad distribution of contour length increments, suggesting that they did not have a well-defined specific structure. SMD simulations revealed the main free energy barrier to the unfolding of PADC and suggested that the unfolding intermediate states may originate from the frication of polypeptide chain sliding during the process of pulling the threaded loop out of the knotting loop. Upon relaxation, a small percentage of the unfolded and untied PADC polypeptide chain can refold back to its native slipknotted conformation, but a large fraction can only reach a misfolded state. Our results revealed the complexity of the mechanical unfolding and refolding of a slipknotted protein with a long threaded loop.

Interferon response suppression by the respiratory syncytial virus relies on two unique nonstructural proteins, NS1 and NS2, that interact with cellular partners through high-order complexes. We hypothesized that two conserved proline residues, P81 and P67, participate in the conformational change leading to oligomerization. We found that the molecular dynamics of NS1 show a highly mobile C-terminal helix, which becomes rigid upon in silico replacement of P81. A soluble oligomerization pathway into regular spherical structures at low ionic strengths competes with an aggregation pathway at high ionic strengths with an increase in temperature. P81A requires higher temperatures to oligomerize and has a small positive effect on aggregation, while P67A is largely prone to aggregation. Chemical denaturation shows a first transition, involving a high fluorescence and ellipticity change corresponding to both a conformational change and substantial effects on the environment of its single tryptophan, that is strongly destabilized by P67A but stabilized by P81A. The subsequent global cooperative unfolding corresponding to the main β-sheet core is not affected by the proline mutations. Thus, a clear link exists between the effect of P81 and P67 on the stability of the first transition and oligomerization/aggregation. Interestingly, both P67 and P81 are located far away in space and sequence from the C-terminal helix, indicating a marked global structural dynamics. This provides a mechanism for modulating the oligomerization of NS1 by unfolding of a weak helix that exposes hydrophobic surfaces, linked to the participation of NS1 in multiprotein complexes. Fil: Conci, Julieta. 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 Fil: Álvarez Paggi, Damián Jorge. 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 Fil: de Oliveira, Guilherme A. P.. Universidade Federal do Rio de Janeiro; Brasil Fil: Pagani, Talita Duarte. Universidade Federal do Rio de Janeiro; Brasil Fil: Esperante, Sebastian. 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 Fil: Borkosky, Silvina Susana. 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 Fil: Aran, Martin. 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 Fil: Alonso, Leonardo Gabriel. 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 Fil: Mohana Borges, Ronaldo. Universidade Federal do Rio de Janeiro; Brasil 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

Mitochondrial cytochrome c is a highly conserved protein in eukaryotes. Certain functions of cytochrome c have been tuned during evolution. For instance, the intrinsic peroxidase activity of human cytochrome c is much lower than that of the yeast counterpart. Structural studies on K72A yeast iso-1-cytochrome c [McClelland, L. J., et al. (2014) Proc. Natl. Acad. Sci. USA, 111, 6648-6653] revealed that residues 81 and 83 in Ω-loop D (residues 70-85) may be gatekeeper residues for the peroxidase activity linked to intrinsic apoptosis. Amino acids at both positions evolve to more sterically demanding amino acids. We hypothesized that residues 81 and 83 evolved such that steric constraints at these positions tune down the peroxidase activity of human cytochrome c. To test this hypothesis, I81A and V83G variants of human cytochrome c were prepared. Our results show that the I81A substitution significantly influences the thermodynamics and kinetics of access to alternate conformers of human cytochrome c, while the V83G substitution has a more modest effect on these properties. The I81A variant also shows a significant enhancement in peroxidase activity, particularly below pH 7, whereas the V83G variant has a similar peroxidase activity to wild-type human cytochrome c. However, neither variant increases the peroxidase activity of human cytochrome c to the level of yeast iso-1-cytochrome c, indicating that other substructures of cytochrome c are also involved in tuning the intrinsic peroxidase activity of mitochondrial cytochrome c.

Temperature-sensitive (Ts) mutants are important tools for understanding the role of essential gene(s), but their molecular basis is not well understood. We use CcdB ( Controller of Cell Death protein B) as a model system to explore the effects of Ts mutations on protein stability, folding, and ligand binding. Previously isolated Ts CcdB mutants fall broadly into two categories, namely, buried site (

There has been widespread interest in studying early intermediate states and their roles in protein folding. The interest in intermediate states has been further emphasized in the recent literature because of their implications for protein aggregation. Unfortunately, direct kinetic characterization of intermediates has been difficult because of the limited time resolutions offered by the kinetic techniques and the heterogeneity of the folding and aggregation landscape. Even in equilibrium experiments, the characterization of intermediate states could be difficult because (a) their populations in equilibrium could be low and/or (b) they lack any specific biochemical or biophysical signatures for their identification. In this paper, we have used fluorescence correlation spectroscopy to study the nature of a low-pH intermediate state of the intestinal fatty acid binding protein, a small protein with predominantly β-sheet structure. Our results have shown that the pH 3 intermediate diffuses faster than the folded protein and has strong helix forming propensity. These behaviors support Lim's hypothesis according to which even an entirely β-sheet protein would form helical bundles at the early stage. Using dynamic light scattering and thioflavin T binding measurements, we have observed that the pH 3 intermediate is prone to aggregation. We believe that early helix formation is the result of a local effect, which originates from the interaction of the neighboring amino acids around the hydrophobic core residues. This early intermediate reorganizes subsequently, and this structural reorganization is initiated by the destabilizing interactions induced by the distant residues, unfavorable entropic costs, and steric constraints of the hydrophobic side chains. Mutational analyses show further that the increase in the hydrophobicity in the hydrophobic core region increases the population of the α-helical intermediate, enhancing the aggregation propensity of the protein, while an identical change, distant from the hydrophobic core, does not show any effect. This study re-emphasizes an overlap between the folding and aggregation landscape of a protein, where the fine-tuning between the local and global effects may be important for the protein to fold efficiently or to aggregate.

Initial recognition of promoter DNA by RNA polymerase (RNAP) is proposed to trigger a series of conformational changes beginning with bending and wrapping of the 40-50 bp of DNA immediately upstream of the -35 region. Kinetic studies demonstrated that the presence of upstream DNA facilitates bending and entry of the downstream duplex (to +20) into the active site cleft to form an advanced closed complex (CC), prior to melting of ∼13 bp (-11 to +2), including the transcription start site (+1). Atomic force microscopy and footprinting revealed that the stable open complex (OC) is also highly wrapped (-60 to +20). To test the proposed bent-wrapped model of duplex DNA in an advanced RNAP-λP(R) CC and compare wrapping in the CC and OC, we use fluorescence resonance energy transfer (FRET) between cyanine dyes at far-upstream (-100) and downstream (+14) positions of promoter DNA. Similarly large intrinsic FRET efficiencies are observed for the CC (0.30 ± 0.07) and the OC (0.32 ± 0.11) for both probe orientations. Fluorescence enhancements at +14 are observed in the single-dye-labeled CC and OC. These results demonstrate that upstream DNA is extensively wrapped and the start site region is bent into the cleft in the advanced CC, reducing the distance between positions -100 and +14 on promoter DNA from >300 to

To understand the consequences of macromolecular crowding, studies have largely employed in vitro experiments with synthetic polymers assumed to be both pure and "inert". These polymers alter enzyme kinetics by excluding volume that would otherwise be available to the enzymes, substrates, and products. Presented here is evidence that other factors, in addition to excluded volume, must be considered in the interpretation of crowding studies with synthetic polymers. Dextran has a weaker effect on the Michaelis-Menten kinetic parameters of yeast alcohol dehydrogenase (YADH) than its small molecule counterpart, glucose. For glucose, the decreased Vmax values directly correlate with slower translational diffusion and the decreased Km values likely result from enhanced substrate binding due to YADH stabilization. Because dextran is unable to stabilize YADH to the same extent as glucose, this polymer's ability to decrease Km is potentially due to the nonideality of the solution, a crowding-induced conformational change, or both. Chronoamperometry reveals that glucose and dextran have surprisingly similar ferricyanide diffusion coefficients. Thus, the reduction in Vmax values for glucose is partially offset by an additional macromolecular crowding effect with dextran. Finally, this is the first report that supplier-dependent impurities in dextran affect the kinetic parameters of YADH. Taken together, our results reveal that caution should be used when interpreting results obtained with inert synthetic polymeric agents, as additional effects from the underlying monomer need to be considered.

Kramers rate theory is a milestone in chemical reaction research, but concerns regarding the basic understanding of condensed phase reaction rates of large molecules in viscous milieu persist. Experimental studies of Kramers theory rely on scaling reaction rates with inverse solvent viscosity, which is often equated with the bulk friction coefficient based on simple hydrodynamic relations. Apart from the difficulty of abstraction of the prefactor details from experimental data, it is not clear why the linearity of rate versus inverse viscosity, k ∝ η(-1), deviates widely for many reactions studied. In most cases, the deviation simulates a power law k ∝ η(-n), where the exponent n assumes fractional values. In rate-viscosity studies presented here, results for two reactions, unfolding of cytochrome c and cysteine protease activity of human ribosomal protein S4, show an exceedingly overdamped rate over a wide viscosity range, registering n values up to 2.4. Although the origin of this extraordinary reaction friction is not known at present, the results indicate that the viscosity exponent need not be bound by the 0-1 limit as generally suggested. For the third reaction studied here, thermal dissociation of CO from nativelike cytochrome c, the rate-viscosity behavior can be explained using Grote-Hynes theory of time-dependent friction in conjunction with correlated motions intrinsic to the protein. Analysis of the glycerol viscosity-dependent rate for the CO dissociation reaction in the presence of urea as the second variable shows that the protein stabilizing effect of subdenaturing amounts of urea is not affected by the bulk viscosity. It appears that a myriad of factors as diverse as parameter uncertainty due to the difficulty of knowing the exact reaction friction and both mode and consequences of protein-solvent interaction work in a complex manner to convey as though Kramers rate equation is not absolute.

The intracellular Ca²⁺ concentration is an important regulator of many cellular functions. The small acidic protein calmodulin (CaM) serves as a Ca²⁺ sensor and control element for many enzymes. Nitric oxide synthase (NOS) is one of the proteins that is activated by CaM and plays a major role in a number of key physiological and pathological processes. Previous studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. We have analyzed the structure and dynamics of complexes formed by peptides based on inducible NOS (iNOS) and endothelial NOS (eNOS) with CaM at Ca²⁺ concentrations that mimic the physiological basal (17 and 100 nM) and elevated levels (225 nM) found in mammalian cells using fluorescence techniques and nuclear magnetic resonance spectroscopy. The results show the CaM-NOS complexes have similar structures at physiological and fully saturated Ca²⁺ levels; however, their dynamics are remarkably different. At 225 nM Ca²⁺, the CaM-NOS complexes show overall an increase in backbone dynamics, when compared to the dynamics of the complexes at saturating Ca²⁺ concentrations. Specifically, the N-lobe of CaM in the CaM-iNOS complex displays a lower internal mobility (higher S²) and higher exchange protection compared to those of the CaM-eNOS complex. In contrast, the C-lobe of CaM in the CaM-eNOS complex is less dynamic. These results illustrate that structures of CaM-NOS complexes determined at saturated Ca²⁺ concentrations cannot provide a complete picture because the differences in intramolecular dynamics become visible only at physiological Ca²⁺ levels.

In spite of their structural similarities, epithelial (E-) and neural (N-) cadherin are expressed at different types of synapses and differ significantly in their dimerization kinetics. Recent studies proposed a transient intermediate in E-cadherin as the key requirement for rapid disassembly kinetics of the adhesive dimer. This E-cadherin intermediate comprises four intermolecular ionic and H-bonding interactions between adhesive partners. These interactions are not preserved in N-cadherin except for a basic residue at the 14th position, which could stabilize the intermediate through either H-bonding or ionic interactions with the partner protomer. To investigate the origin of the rapid dimerization kinetics of N-cadherin in the presence of calcium, studies reported here systematically test the role of ionic and H-bonding interactions in dimerization kinetics using R14S, R14A, and R14E mutants of N-cadherin. Analytical size-exclusion chromatographic and bead aggregation studies showed two primary results. First, N-cadherin/R14S and N-cadherin/R14A mutants showed fast assembly and disassembly kinetics in the calcium-saturated state similar to that of wild-type N-cadherin. These results indicate that the fast disassembly of the calcium-saturated dimer of N-cadherin does not require a basic residue at the 14th position. Second, the dimerization kinetics of N-cadherin/R14E were slow in the calcium-saturated state, indicating that negative charge destabilizes the intermediate state. Taken together, these results indicate that the basic residue at the 14th position does not promote rapid dimerization kinetics but that an acidic amino acid in that position significantly impairs dimerization kinetics.

α-Crystallin is the archetypical chaperone of the small heat-shock protein family, all members of which contain the so-called “α-crystallin domain” (ACD). This domain and the N- and C-terminal extensions are considered the main functional units in its chaperone function. Previous studies have shown that a 19-residue fragment of the ACD of human αA-crystallin called mini-αA-crystallin (MAC) shows chaperone properties similar to those of the parent protein. Subsequent studies have confirmed the function of this peptide, but no studies have addressed the mechanistic basis for the chaperone function of MAC. Using human γD-crystallin (HGD), a key substrate protein for parent α-crystallin in the ocular lens, we show here that MAC not only protects HGD from aggregation during thermal and chemical unfolding but also binds weakly and reversibly to HGD (Kd ≈ 200–700 μM) even when HGD is in the native state. However, at temperatures favoring the unfolding of HGD, MAC forms a stable complex with HGD similar to parent α-crystallin. Using nuclear magnetic resonance spectroscopy, we identify the residues in HGD that are involved in these two modes of binding and show that MAC protects HGD from aggregation by binding to Phe 56 and Val 132 at the domain interface of the target protein, and residues Val 164 to Leu 167 in the core of the C-terminal domain. Furthermore, we suggest that the low-affinity, reversible binding of MAC on the surface of HGD in the native state is involved in facilitating its binding to both the domain interface and core regions during the early stages of the unfolding of HGD. This work highlights some structural features of MAC and MAC-like peptides that affect their chaperone activity and can potentially be manipulated for translational studies.

Diversity in eukaryotic rRNA structure and function offers possibilities of therapeutic targets. Unlike ribosomes of prokaryotes, eukaryotic ribosomes contain species-specific rRNA expansion segments (ESs) with idiosyncratic structures and functions that are essential and specific to some organisms. Here we investigate expansion segment 7 (ES7), one of the largest and most variable expansions of the eukaryotic ribosome. We hypothesize that ES7 of the pathogenic fungi Candida albicans (ES7CA) could be a prototypic drug target. We show that isolated ES7CA folds reversibly to a native-like state. We developed a fluorescence displacement assay using an RNA binding fluorescent probe, F-neo. F-neo binds tightly to ES7CA with a Kd of 2.5 × 10–9 M but binds weakly to ES7 of humans (ES7HS) with a Kd estimated to be greater than 7 μM. The fluorescence displacement assay was used to investigate the affinities of a library of peptidic aminosugar conjugates (PAs) for ES7CA. For conjugates with highest affinities for E...

Measurements at pH 8 allow evaluation of binding of 100% cardiolipin vesicles to site A of cytochrome c without interference from other known binding sites. Site A encompasses Lys72, Lys73, Lys86, and Lys87, located in or adjacent to Ω-loop D (residues 70-85), which positions Met80 for binding to the heme. Binding of cytochrome c to cardiolipin disrupts Met80 heme binding, permitting peroxidase activity. Binding of cardiolipin to yeast iso-1-cytochrome c versus human cytochrome c is compared to assess how binding of cardiolipin to site A has evolved for cytochrome c from species that do not have a complete intrinsic apoptotic pathway to species that do. Using a nondestructive method of quantifying cardiolipin concentration, highly reproducible binding curves are obtained. The results indicate two sequential structural rearrangements on the surface of 100% cardiolipin vesicles. The first, more modest, structural rearrangement occurs at an exposed (outer leaflet) lipid:protein ratio of 8-10 for both cytochromes c. The second, occurring at higher lipid:protein ratios, causes significant unfolding of cytochrome c and requires a much higher lipid:protein ratio for human versus yeast cytochrome c. Higher lipid:protein ratios enhance the peroxidase activity of cytochrome c, suggesting that human cytochrome c has evolved a more stringent on/off switch for cardiolipin peroxidation in the early stages of apoptosis. For both human and yeast cytochrome c, the K72A mutation has only minor effects on binding to site A, suggesting that other nearby lysines can compensate for the lack of Lys72.

The tumor susceptibility gene-101 coiled coil domain (TSG101cc) is an integral component of the endosomal maturation machinery and cytokinesis, and also interacts with several transcription factors. The TSG101cc has been crystallized as a homotetramer but is known to interact with two of its binding partners as a heterotrimer. To investigate this apparent discrepancy, we examined the solution thermodynamics of the TSG101cc. Here, we use circular dichroism, differential scanning calorimetry, analytical ultracentrifugation, fluorescence, and structural thermodynamic analysis to investigate the structural stability and the unfolding of the TSG101cc. We demonstrate that TSG101cc exists in solution primarily as a tetramer, which unfolds in a two-state manner. Surprisingly, no homodimeric or homotrimeric species were detected. Structural thermodynamic analysis of the homotetrameric structure and comparison with known oligomeric coiled-coils suggests that the TSG101cc homotetramer is comparatively unstable on a per residue basis. Furthermore, the homotrimeric coiled-coil is predicted to be much less stable than the functional heterotrimeric coiled-coil in the endosomal sorting complex required for transport 1 (ESCRT1). These results support a model whereby the tetramer–monomer equilibrium of TSG101 serves as the cellular reservoir of TSG101, which is effectively outcompeted when its binding partners are present and the heteroternary complex can form.

We test the hypothesis that Lys72 suppresses the intrinsic peroxidase activity of human cytochrome c, as observed previously for yeast iso-1-cytochrome c [McClelland et al. PNAS 111, 6648–6653 (2014)]. A 1.25 Å X-ray structure of K72A human cytochrome c shows that the mutation minimally affects structure. Guanidine hydrochloride denaturation demonstrates that the K72A mutation increases global stability by 0.5 kcal/mol. The K72A mutation also increases the apparent pKa of the alkaline transition, a measure of the stability of the heme crevice, by 0.5 units. Consistent with the increase in the apparent pKa, the rate of formation of the dominant alkaline conformer decreases and this conformer is no longer stabilized by proline isomerization. Peroxidase activity measurements show that the K72A mutation increases kcat by 1.6– to 4–fold at pH values from 7 to 10, a larger effect than for the yeast protein. X-ray structures of wild type and K72A human cytochrome c indicate that direct interactions of Lys72 with the far side of Ω-loop D, which are seen in X-ray structures of horse and yeast cytochrome c and could suppress peroxidase activity, are lacking. Instead, we propose that the larger effect of the K72A mutation on peroxidase activity of human versus yeast cytochrome c results from relief of steric interactions between the side chains at positions 72 and 81 (Ile in human versus Ala in yeast), which suppress the dynamics of Ω-loop D necessary for the intrinsic peroxidase activity of cytochrome c.

A crucial event in calcium signaling is the transition of a calcium sensor from the apo (Ca

The fibrillization of α-synuclein (α-syn) is involved in Parkinson's disease, a neurodegenerative disorder that affects four million people in the world. The amino acid sequence 71-82 of this protein (VTGVTAVAQKTV) has appeared to be essential for fibril formation. In the present study, we have investigated the secondary structure and thermal stability of the peptide fragment 71-82, α-syn71-82, as a function of concentration and temperature, as well as its interactions with phospholipid model membranes using various spectroscopic techniques. The data show that α-syn71-82 is mainly disordered in solution with the presence of a few β-sheet structure elements. The peptide reversibly forms intermolecular β-sheets with increasing concentration and decreasing temperature, suggesting that it is subjected to a thermodynamic equilibrium between a monomeric and an oligomeric form. This equilibrium seems to be affected by the presence of zwitterionic membranes. Conversely, the influence of the peptide on zwitterionic lipid bilayers is small and concentration-dependent. By contrast, α-syn71-82 is strongly affected by anionic vesicles. The peptide indeed exhibits a dramatic conformational change, reflecting an extensive and irreversible self-aggregation, the majority of the amino acids being involved in a parallel β-sheet conformation. The aggregates appear to be located near the membrane surface but do not perturb significantly the membrane order. Comparing these results with the literature, it appears that α-syn71-82 shares several general properties and structural similarities with its parent protein. These common points suggest that the sequence 71-82 may overall contribute to the behavior and properties of α-syn.

The quaternary structure stability of proteins is typically studied under conditions that accelerate their aggregation/unfolding processes on convenient laboratory time scales. Such conditions include high temperature or pressure, chaotrope-mediated unfolding, or low or high pH. These approaches have the limitation of being nonphysiological and that the concentration of the protein in solution is changing as the reactions proceed. We describe a methodology to define the quaternary structure stability of the amyloidogenic homotetrameric protein transthyretin (TTR) under physiological conditions. This methodology expands from a described approach based on the measurement of the rate of subunit exchange of TTR with a tandem flag-tagged (FT₂) TTR counterpart. We demonstrate that subunit exchange of TTR with FT₂·TTR can be analyzed and quantified using a semi-native polyacrylamide gel electrophoresis technique. In addition, we biophysically characterized two FT₂·TTR variants derived from wild-type and the amyloidogenic variant Val122Ile TTR, both of which are associated with cardiac amyloid deposition late in life. The FT₂·TTR variants have similar amyloidogenic potential and similar thermodynamic and kinetic stabilities compared to those of their nontagged counterparts. We utilized the methodology to study the potential of the small molecule SOM0226, a repurposed drug under clinical development for the prevention and treatment of the TTR amyloidoses, to stabilize TTR. The results enabled us to characterize the binding energetics of SOM0226 to TTR. The described technique is well-suited to study the quaternary structure of other human aggregation-prone proteins under physiological conditions.

Many neurodegenerative diseases are linked with formation of amyloid aggregates. It is increasingly accepted that not the fibrils but rather oligomeric species are responsible for degeneration of neuronal cells. Strong evidence suggests that in Parkinson's disease (PD), cytotoxic α-synuclein (αSN) oligomers are key to pathogenicity. Nevertheless, insight into the oligomers' molecular properties remains scarce. Here we show that αSN oligomers, despite a large amount of disordered structure, are remarkably stable against extreme pH, temperature, and even molar amounts of chemical denaturants, though they undergo cooperative unfolding at higher denaturant concentrations. Mutants found in familial PD lead to slightly larger oligomers whose stabilities are very similar to that of wild-type αSN. Isolated oligomers do not revert to monomers but predominantly form larger aggregates consisting of stacked oligomers, suggesting that they are off-pathway relative to the process of fibril formation. We also demonstrate that 4-(dicyanovinyl)julolidine (DCVJ) can be used as a specific probe for detection of αSN oligomers. The high stability of the αSN oligomer indicates that therapeutic strategies should aim to prevent the formation of or passivate rather than dissociate this cytotoxic species.

The 35-residue, C-terminal headpiece subdomain of the protein villin folds to a stable structure on a microsecond time scale and has served as a model system in numerous studies of protein folding. To obtain a convenient spectroscopic probe of the folding dynamics, Kubelka et al. introduced an ionized histidine residue at position 27, with the expectation that it would quench the fluorescence of tryptophan 23 in the folded protein by extracting an electron from the excited indole ring [Kubelka, J., et al. (2003) J. Mol. Biol. 329, 625-630]. Although the fluorescence yield decreased as anticipated when the protein folded, it was not clear that the side chains of the two residues were sufficiently close together for electron transfer to compete effectively with fluorescence. Here, hybrid classical-quantum mechanical molecular dynamics simulations are used to examine the rates of transfer of an electron from the excited tryptophan to various possible acceptors in the modified headpiece and a smaller fragment comprised of residues 21-27 (HP7). The dominant reaction is found to be transfer to the amide group on the carboxyl side of W23 (amide a24). This process is energetically favorable and has a large coupling factor in the folded protein at 280 K but becomes unfavorable as HP7 unfolds at higher temperatures. Changes in electrostatic interactions of the solvent and other parts of the protein with the indole ring and a24 contribute importantly to this change in energy.

Human HtrA1 (high-temperature requirement protein A1) belongs to a conserved family of serine proteases involved in protein quality control and cell fate. The homotrimeric ubiquitously expressed protease has chymotrypsin-like specificity and primarily targets hydrophobic stretches in selected or misfolded substrate proteins. In addition, the enzyme is capable of exerting autolytic activity removing the N-terminal insulin-like growth factor binding protein (IGFBP)/Kazal-like tandem motif without affecting the protease activity. In this study, we have addressed the mechanism governing the autolytic activity and find that it depends on the integrity of the disulphide bonds in the N-terminal IGFBP/Kazal-like domain. The specificity of the autolytic cleavage reveals a high preference for cysteine in the P1 position of HtrA1, explaining the lack of autolysis prior to disulphide reduction. Significantly, the disulphides were reduced by thioredoxin, suggesting that autolysis of HtrA1 in vivo is linked to the endogenous redox balance and that the N-terminal domain acts as a redox-sensing switch.

The insulin hexamer is resistant to degradation and fibrillation, which makes it an important quaternary structure for its in vivo storage in Zn(2+)- and Ca(2+)-rich vesicles in the pancreas and for pharmaceutical formulations. In addition to the two Zn(2+) ions that are required for its formation, three other species, Zn-coordinating anions (e.g., Cl(-)), Ca(2+), and phenols (e.g., resorcinol), bind to the hexamer and affect the subunit conformation and stability. The contributions of these four species to the thermodynamics of insulin unfolding have been quantified by differential scanning calorimetry and thermal unfolding measurements to determine the extent and nature of their stabilization of the insulin hexamer. Both Zn(2+) and resorcinol make a significant enthalpic contribution, while Ca(2+) primarily affects the protein heat capacity (solvation) by its interactions in the central cation-binding cavity, which is modulated by the surrounding subunit conformations. Coordinating anions have a negligible effect on the stability of the hexamer, even though subunits shift to an alternate conformation when these anions bind to the Zn(2+) ions. Finally, Zn(2+) in excess of the two that are required to form the hexamer further stabilizes the protein by additional enthalpic contributions.

In this study, we demonstrate that U1A-RNA molecular recognition is mediated by a combined mechanism of conformational selection and induced fit. The binding of U1A to RNA has been discussed in the context of induced fit that involves the reorientation of the α-helix in the C-terminal region (Helix-C) of U1A to permit RNA access only when U1A correctly recognizes RNA. However, according to our molecular dynamics simulations, even in the absence of RNA, Helix-C spontaneously reoriented to permit RNA access. Nonetheless, such a conformational change was still incomplete. Helix-C was often partially or even fully unfolded and in an infrequent RNA-accessible conformation, which can be detected using state-of-the-art nuclear magnetic resonance methodology. These results suggest that the formation of an energetically stabilized complex is promoted by specific interactions between U1A and RNA. In conclusion, in the recognition of RNA by U1A protein, we propose a combined mechanism that requires the reorientation of Helix-C and the subsequent contact with RNA through conformational selection, although the stabilization of the U1A-RNA complex is caused by induced fit. We further propose a modification to the conventional assumption regarding the mechanism of U1A-RNA molecular recognition.

We have examined the effect of incubating a monoclonal antibody (mAb) in low (0-2.0 M) concentrations of guanidine hydrochloride (GdnHCl) on the protein's conformation and aggregation during isothermal incubation. In GdnHCl solutions at concentrations from 1.2 to 1.6 M, the mAb was partially unfolded. As demonstrated by fluorescence and circular dichroism spectroscopy, the partially unfolded state of the antibody had perturbed tertiary structure but retained native secondary structure. Furthermore, partial unfolding of the antibody was documented by analytical ultracentrifugation, dynamic light scattering, and limited proteolysis. Subsequent aggregation of the antibody was characterized using size-exclusion chromatography, analytical ultracentrifugation, and dynamic light scattering. Over the entire concentration range (0-2.0 M) of GdnHCl, protein-protein interactions were attractive, as quantified by negative osmotic second virial coefficients measured with static light scattering. However, during isothermal incubation at 37 °C, the aggregation of the antibody was detected only in solutions that induced partial unfolding. Differential scanning calorimetry studies showed that the antibody's CH2 domains were unfolded in antibody molecules that had been incubated in 1.2 M and higher concentrations of GdnHCl. These results suggest that unfolding of the CH2 domains leads to aggregation.

The specific recognition by proteins of G-quadruplex structures provides evidence of a functional role for in vivo G-quadruplex structures. As previously reported, the ribonucleoprotein, hnRNP Al, and it is proteolytic derivative, unwinding protein 1 (UP1), bind to and destabilize G-quadruplex structures formed by the human telomeric repeat d(TTAGGG)n. UP1 has been proposed to be involved in the recruitment of telomerase to telomeres for chain extension. In this study, a detailed thermodynamic characterization of the binding of UP1 to a human telomeric repeat sequence, the d[AGGG(TTAGGG)3] G-quadruplex, is presented and reveals key insights into the UP1-induced unfolding of the G-quadruplex structure. The UP1-G-quadruplex interactions are shown to be enthalpically driven, exhibiting large negative enthalpy changes for the formation of both the Na(+) and K(+) G-quadruplex-UP1 complexes (ΔH values of -43 and -19 kcal/mol, respectively). These data reveal three distinct enthalpic contributions from the interactions of UP1 with the Na(+) form of G-quadruplex DNA. The initial interaction is characterized by a binding affinity of 8.5 × 10(8) M(-1) (strand), 200 times stronger than the binding of UP1 to a single-stranded DNA with a comparable but non-quadruplex-forming sequence [4.1 × 10(6) M(-1) (strand)]. Circular dichroism spectroscopy reveals the Na(+) form of the G-quadruplex to be completely unfolded by UP1 at a binding ratio of 2:1 (UP1:G-quadruplex DNA). The data presented here demonstrate that the favorable energetics of the initial binding event are closely coupled with and drive the unfolding of the G-quadruplex structure.

The anthrax protective antigen (PA) is an 83 kDa protein that is one of three protein components of the anthrax toxin, an AB toxin secreted by Bacillus anthracis. PA is capable of undergoing several structural changes, including oligomerization to either a heptameric or octameric structure called the prepore, and at acidic pH a major conformational change to form a membrane-spanning pore. To follow these structural changes at a residue-specific level, we have conducted initial studies in which we have biosynthetically incorporated 5-fluorotryptophan (5-FTrp) into PA, and we have studied the influence of 5-FTrp labeling on the structural stability of PA and on binding to the host receptor capillary morphogenesis protein 2 (CMG2) using (19)F nuclear magnetic resonance (NMR). There are seven tryptophans in PA, but of the four domains in PA, only two contain tryptophans: domain 1 (Trp65, -90, -136, -206, and -226) and domain 2 (Trp346 and -477). Trp346 is of particular interest because of its proximity to the CMG2 binding interface, and because it forms part of the membrane-spanning pore. We show that the (19)F resonance of Trp346 is sensitive to changes in pH, consistent with crystallographic studies, and that receptor binding significantly stabilizes Trp346 to both pH and temperature. In addition, we provide evidence that suggests that resonances from tryptophans distant from the binding interface are also stabilized by the receptor. Our studies highlight the positive impact of receptor binding on protein stability and the use of (19)F NMR in gaining insight into structural changes in a high-molecular weight protein.

The orange carotenoid protein (OCP) and fluorescence recovery protein (FRP) are present in many cyanobacteria, and regulate an essential photoprotection cycle in an antagonistic manner as a function of light intensity. We characterized the oligomerization states of OCP and FRP by using native mass spectrometry, a technique that has the capability of studying native proteins under a wide range of protein concentrations and molecular masses. We found that dimeric FRP (dFRP) is the predominant state at protein concentrations ranging from 3 μM to 180 μM, and that higher order oligomers gradually form at protein concentrations above this range. The OCP, however, demonstrates significantly different oligomerization behavior. Monomeric OCP (mOCP) dominates at low protein concentrations, with an observable population of dimeric OCP (dOCP). The ratio of dOCP to mOCP, however, increases proportionally with the protein concentration. Higher order OCP oligomers form at protein concentrations beyond 10 μM. Additionally, native mass spectrometry coupled with ion mobility allowed us to measure protein collisional cross sections (CCS) and interrogate the unfolding of different FRP and OCP oligomers. We found that monomeric FRP exhibits a one-stage unfolding process, which could be correlated with its C-terminal bent crystal structure. The structural domain compositions of FRP and OCP are compared and discussed.

Calnuc is a ubiquitously expressed protein of the EF-hand Ca2+-binding superfamily. Previous studies have implicated it in Ca2+-sensitive physiological processes, whereas details of its function and involvement in human diseases are lacking. Drawing upon the sequence homology of calnuc with calreticulin, we propose it functions as a molecular chaperone-like protein. In cells under thermal, chemical [urea and guanidinium chloride (GdmCl)], and acidic stress, calnuc exhibits properties similar to those of established chaperone-like proteins (GRP78, spectrin, and α-crystallin), effectively demonstrated by its ability to suppress aggregation of malate dehydrogenase (MDH), alcohol dehydrogenase, and catalase. Calnuc aids in refolding of MDH with retention of 80% of its enzymatic activity. In HEK293 cells subjected to heat shock, calnuc chaperones luciferase, protecting its activity. Our in vitro and cell culture results establish the ability of calnuc to inhibit fibrillation of insulin and lysozyme and validat...