Different misfolding mechanisms converge on common conformational changes

Prion diseases are caused by misfolding and aggregation of the prion protein (PrP). Pathogenic mutations such as Y218N and E196K are known to cause Gerstmann-Sträussler-Scheinker syndrome and Creutzfeldt-Jakob disease, respectively. Here we describe molecular dynamics simulations of these mutant proteins to better characterize the detailed conformational effects of these sequence substitutions. Our results indicate that the mutations disrupt the wild-type native PrP^C structure and cause misfolding. Y218N reduced hydrophobic packing around the X-loop (residues 165–171), and E196K abolished an important wild-type salt bridge. While differences in the mutation site led PrP mutants to misfold along different pathways, we observed multiple traits of misfolding that were common to both mutants. Common traits of misfolding included: 1) detachment of the short helix (HA) from the PrP core; 2) exposure of side chain F198; and 3) formation of a nonnative strand at the N-terminus. The effect of the E196K mutation directly abolished the wild-type salt bridge E196-R156, which further destabilized the F198 hydrophobic pocket and HA. The Y218N mutation propagated its effect by increasing the HB-HC interhelical angle, which in turn disrupted the packing around F198. Furthermore, a nonnative contact formed between E221 and S132 on the S1-HA loop, which offered a direct mechanism for disrupting the hydrophobic packing between the S1-HA loop and HC. While there were common misfolding features shared between Y218N and E196K, the differences in the orientation of HB and HC and the X-loop conformation might provide a structural basis for identifying different prion strains.     

Salt bridge as a gatekeeper against partial unfolding

Salt bridges are frequently observed in protein structures. Because the energetic contribution of salt bridges is strongly dependent on the environmental context, salt bridges are believed to contribute to the structural specificity rather than the stability. To test the role of salt bridges in enhancing structural specificity, we investigated the contribution of a salt bridge to the energetics of native‐state partial unfolding in a cysteine‐free version of Escherichia coli ribonuclease H (RNase H*). Thermolysin cleaves a protruding loop of RNase H^* through transient partial unfolding under native conditions. Lys86 and Asp108 in RNase H^* form a partially buried salt bridge that tethers the protruding loop. Investigation of the global stability of K86Q/D108N RNase H^* showed that the salt bridge does not significantly contribute to the global stability. However, K86Q/D108N RNase H^* is greatly more susceptible to proteolysis by thermolysin than wild‐type RNase H^* is. The free energy for partial unfolding determined by native‐state proteolysis indicates that the salt bridge significantly increases the energy for partial unfolding by destabilizing the partially unfolded form. Double mutant cycles with single and double mutations of the salt bridge suggest that the partially unfolded form is destabilized due to a significant decrease in the interaction energy between Lys86 and Asp108 upon partial unfolding. This study demonstrates that, even in the case that a salt bridge does not contribute to the global stability, the salt bridge may function as a gatekeeper against partial unfolding that disturbs the optimal geometry of the salt bridge.     

A Comprehensive Alanine-Scanning Mutagenesis Study Reveals Roles for Salt Bridges in the Structure and Activity of Pseudomonas aeruginosa Elastase

The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (k _cat/K _m), half-lives (t _1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.     

Contribution of salt bridges near the surface of a protein to the conformational stability

Salt bridges play important roles in the conformational stability of proteins. However, the effect of a surface salt bridge on the stability remains controversial even today; some reports have shown little contribution of a surface salt bridge to stability, whereas others have shown a favorable contribution. In this study, to elucidate the net contribution of a surface salt bridge to the conformational stability of a protein, systematic mutant human lysozymes, containing one Glu to Gln (E7Q) and five Asp to Asn mutations (D18N, D49N, D67N, D102N, and D120N) at residues where a salt bridge is formed near the surface in the wild-type structure, were examined. The thermodynamic parameters for denaturation between pH 2.0 and 4.8 were determined by use of a differential scanning calorimeter, and the crystal structures were analyzed by X-ray crystallography. The denaturation Gibbs energy (DeltaG) of all mutant proteins was lower than that of the wild-type protein at pH 4, whereas there was little difference between them near pH 2. This is caused by the fact that the Glu and Asp residues are ionized at pH 4 but protonated at pH 2, indicating a favorable contribution of salt bridges to the wild-type structure at pH 4. Each contribution was not equivalent, but we found that the contributions correlate with the solvent inaccessibility of the salt bridges; the salt bridge contribution was small when 100% accessible, while it was about 9 kJ/mol if 100% inaccessible. This conclusion indicates how to reconcile a number of conflicting reports about role of surface salt bridges in protein stability. Furthermore, the effect of salts on surface salt bridges was also examined. In the presence of 0.2 M KCl, the stability at pH 4 decreased, and the differences in stability between the wild-type and mutant proteins were smaller than those in the absence of salts, indicating the compensation to the contribution of salt bridges with salts. Salt bridges with more than 50% accessibility did not contribute to the stability in the presence of 0.2 M KCl.     

Interactions across the interface contribute the stability of homodimeric 3α-hydroxysteroid dehydrogenase/carbonyl reductase

The dimerization of 3α-hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. Substitution of alanine, lysine and serine for D249 decreased catalytic efficiency 30, 1400 and 1.4-fold, and lowered the melting temperature 6.9, 5.4 and 7.6 °C, respectively. The mutated enzymes have the dimeric species but the equilibrium between monomer and dimer for these mutants varies from each other, implying that these residues might contribute differently to the dimer stability. Thermal and urea-induced unfolding profiles for wild-type and mutant enzymes appeared as a two-state transition and three-state transition, respectively. In addition, mutation on D249 breaks the salt bridges and causes different effects on the loss of enzymatic activity for D249A, D249K and D249S mutants in the urea-induced unfolding profiles. Hence, D249 at the dimeric interface in 3α-HSD/CR is essential for conformational stability, oligomeric integrity and enzymatic activity.     

Molecular Origin of the Stability Difference in Four Shark IgNAR Constant Domains

Improving the stability of antibodies for manufacture and shelf life is one of the main focuses of antibody engineering. One stabilization strategy is to perform specific mutations in human antibodies based on highly stable antibodies in other species. To identify the key residues for mutagenesis, it is necessary to understand the roles of these residues in stabilizing the antibody. Here, we use molecular dynamics simulations to study the molecular origin of the four shark immunoglobulin new antigen receptors constant domains (C1–C4). According to the unfolding pathways and the conformational free energy surfaces in 8 M urea at 380 K, the C2 domain is the most stable, followed by C4, C1, and C3, which agrees with the experimental findings. The C1 and C3 domains follow a common unfolding pathway in which the unfolding starts from the edge strands, particularly strand g, and then gradually progresses to the inner strands. Detailed structural analysis of the C2 domain reveals a “sandwich-like” R339-E322-R341 salt-bridge cluster on strand g, which grants ultrahigh stability to the C2 domain. We further design two sets of mutations by mutating E322 to alanine or setting all atomic charges in E322 to zero to break the salt-bridge cluster in the C2 domain, which confirms the importance of the salt bridges in stability. In the C4 domain, the D80-K104 salt bridge on strand g also strengthens the stability. On the other hand, in the C1 and C3 domains, there is no salt bridge on strand g. In addition to the salt bridges, the overall hydrophobicity score of the hydrophobic core is also positively correlated with the domain stability. Our findings provide a detailed microscopic picture of the molecular origin of the four shark immunoglobulin new antigen receptors constant domains that not only explains the differences in their structural stability but also provides important insights into future antibody design.     

The salt dependence of the interferon regulatory factor 1 DNA binding domain binding to DNA reveals ions are localized around protein and DNA

The equilibrium dissociation constant of the DNA binding domain of interferon regulatory factor 1 (IRF1 DBD) for its DNA binding site depends strongly on salt concentration and salt type. These dependencies are consistent with IRF1 DBD binding to DNA, resulting in the release of cations from the DNA and both release of anions from the protein and uptake of a cation by the protein. We demonstrated this by utilizing the fact that the release of fluoride from protein upon complex formation does not contribute to the salt concentration dependence of binding and by studying mutants in which charged residues in IRF1 DBD that form salt bridges with DNA phosphates are changed to alanine. The salt concentration dependencies of the dissociation constants of wild-type IRF1 DBD and the mutants R64A, D73A, K75A, and D73A/K75A were measured in buffer containing NaF, NaCl, or NaBr. The salt concentration and type dependencies of the mutants relative to wild-type IRF1 DBD provide evidence of charge neutralization by solution ions for R64 and by a salt bridge between D73 and K75 in buffer containing chloride or bromide salts. These data also allowed us to determine the number, type, and localization of condensed ions around both IRF1 DBD and its DNA binding site.     

Effects of engineered salt bridges on the stability of subtilisin BPN'

Variants designed using PROTEUS have been produced in an attempt to engineer stabilizing salt bridges into subtilisin BPN'. All the mutants constructed by site-directed mutagenesis were secreted by Bacillus subtilis, except L75K. Q19E, expressed as a single variant and also in a double variant, Q19E/Q271E, appears to form a stabilizing salt bridge based on X-ray crystal structure determination and differential scanning calorimeter measurements. Although the double mutant was found to be less thermodynamically stable than the wild-type, it did exhibit an autolytic stability about two-fold greater under hydrophobic conditions. Four variants, A98K, S89E, V26R and L235R, were found to be nearly identical to wild-type in thermal stability, indicative of stable structures without evidence of salt bridge formation. Variants Q271E, V51K and T164R led to structures that resulted in varying degrees of thermodynamic and autolytic instability. A computer-modeling analysis of the PROTEUS predictions reveals that the low percentage of salt bridge formation is probably due to an overly simplistic electrostatic model, which does not account for the geometry of the pairwise interactions.     

Comparison studies of the structural stability of rabbit prion protein with human and mouse prion proteins

Background: Prion diseases are fatal and infectious neurodegenerative diseases affecting humans and animals. Rabbits are one of the few mammalian species reported to be resistant to infection from prion diseases isolated from other species (I. Vorberg et al., Journal of Virology 77 (3) (2003) 2003-2009). Thus the study of rabbit prion protein structure to obtain insight into the immunity of rabbits to prion diseases is very important.Findings: The paper is a straight forward molecular dynamics simulation study of wild-type rabbit prion protein (monomer cellular form) which apparently resists the formation of the scrapie form. The comparison analyses with human and mouse prion proteins done so far show that the rabbit prion protein has a stable structure. The main point is that the enhanced stability of the C-terminal ordered region especially helix 2 through the D177-R163 salt-bridge formation renders the rabbit prion protein stable. The salt bridge D201-R155 linking helixes 3 and 1 also contributes to the structural stability of rabbit prion protein. The hydrogen bond H186-R155 partially contributes to the structural stability of rabbit prion protein.Conclusions: Rabbit prion protein was found to own the structural stability, the salt bridges D177-R163, D201-R155 greatly contribute and the hydrogen bond H186-R155 partially contributes to this structural stability. The comparison of the structural stability of prion proteins from the three species rabbit, human and mouse showed that the human and mouse prion protein structures were not affected by the removing these two salt bridges. Dima et al. (Biophysical Journal 83 (2002) 1268-1280 and Proceedings of the National Academy of Sciences of the United States of America 101 (2004) 15335-15340) also confirmed this point and pointed out that “correlated mutations that reduce the frustration in the second half of helix 2 in mammalian prion proteins could inhibit the formation of PrP^Sc”.     

Contribution of surface salt bridges to protein stability: guidelines for protein engineering

The small globular protein, ubiquitin, contains a pair of oppositely charged residues, K11 and E34, that according to the three-dimensional structure are located on the surface of this protein with a spatial orientation characteristic of a salt bridge. We investigated the strength of this salt bridge and its contribution to the global stability of the ubiquitin molecule. Using the "double mutant cycle" analysis, the strength of the pairwise interactions between K11 and E34 was estimated to be favorable by 3.6kJ/mol. Further, the salt bridge of the reverse orientation, i.e. E11/K34, can be formed and is found to have a strength (3.8kJ/mol) similar to that of the K11/E34 pair. However, the global stability of the K11/E34 variant of ubiquitin is 2.2kJ/mol higher than that of the E11/K34 variant. The difference in the contribution of the opposing salt bridge orientations to the overall stability of the ubiquitin molecule is attributed to the difference in the charge-charge interactions between residues forming the salt bridge and the rest of the ionizable groups in this protein. On the basis of these results, we concluded that surface salt bridges are stabilizing, but their contribution to the overall protein stability is strongly context-dependent, with charge-charge interactions being the largest determinant. Analysis of 16 salt bridges from six different proteins, for which detailed experimental data on energetics have been reported, support the conclusions made from the analysis of the salt bridge in ubiquitin. Implications of these findings for engineering proteins with enhanced thermostability are discussed.     

Role of interdomain salt bridges in the pore-forming ability of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac

The four salt bridges (Asp(222)-Arg(281), Arg(233)-Glu(288), Arg(234)-Glu(274), and Asp(242)-Arg(265)) linking domains I and II in Cry1Aa were abolished individually in alpha-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp(242)-Arg(265) bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.     

The effect of deleting a putative salt bridge on the properties of the thermostable subtilisin-like proteinase, aqualysin I

Aqualysin I, is a subtilisin-like serine proteinase, from the thermophilic bacterium Thermus aquaticus. It is predicted that the enzyme contains a salt bridge, D17-R259, connecting the N- and C-terminal regions of the enzyme. Previously we reported on the stabilizing effect of the incorporation of a salt bridge at a corresponding site in VPR, a related cold adapted enzyme from a marine Vibrio sp. Here we describe the effect of the reverse change, i.e. the elimination of the salt bridge on the thermal stability and kinetic properties of aqualysin I. Deletion of the putative salt bridge in the D17N mutant of the enzyme destabilized the enzyme by 8-9 °C in terms of T??%, determined by thermal inactivation and over 4 °C in T(m), as measured from melting curves of the inhibited enzyme. The mutation, however, had no significant effect on the kinetic parameters of the enzyme under standard assay conditions.     

Protein Thermal Stability Enhancement by Designing Salt Bridges: A Combined Computational and Experimental Study

Protein thermal stability is an important factor considered in medical and industrial applications. Many structural characteristics related to protein thermal stability have been elucidated, and increasing salt bridges is considered as one of the most efficient strategies to increase protein thermal stability. However, the accurate simulation of salt bridges remains difficult. In this study, a novel method for salt-bridge design was proposed based on the statistical analysis of 10,556 surface salt bridges on 6,493 X-ray protein structures. These salt bridges were first categorized based on pairing residues, secondary structure locations, and Cα–Cα distances. Pairing preferences generalized from statistical analysis were used to construct a salt-bridge pair index and utilized in a weighted electrostatic attraction model to find the effective pairings for designing salt bridges. The model was also coupled with B-factor, weighted contact number, relative solvent accessibility, and conservation prescreening to determine the residues appropriate for the thermal adaptive design of salt bridges. According to our method, eight putative salt-bridges were designed on a mesophilic β-glucosidase and 24 variants were constructed to verify the predictions. Six putative salt-bridges leaded to the increase of the enzyme thermal stability. A significant increase in melting temperature of 8.8, 4.8, 3.7, 1.3, 1.2, and 0.7°C of the putative salt-bridges N437K–D49, E96R–D28, E96K–D28, S440K–E70, T231K–D388, and Q277E–D282 was detected, respectively. Reversing the polarity of T231K–D388 to T231D–D388K resulted in a further increase in melting temperatures by 3.6°C, which may be caused by the transformation of an intra-subunit electrostatic interaction into an inter-subunit one depending on the local environment. The combination of the thermostable variants (N437K, E96R, T231D and D388K) generated a melting temperature increase of 15.7°C. Thus, this study demonstrated a novel method for the thermal adaptive design of salt bridges through inference of suitable positions and substitutions.     

Relationship between stability and flexibility in the most flexible region of Photinus pyralis luciferase

Firefly luciferase is a protein with a large N-terminal and a small C-terminal domain. B-factor analysis shows that its C-terminal is much more flexible than its N-terminal. Studies on hyperthermophile proteins have been shown that the increased thermal stability of hyperthermophile proteins is due to their enhanced conformational rigidity and the relationship between flexibility, stability and function in most of proteins is on debate. Two mutations (D474K and D476N) in the most flexible region of firefly luciferase were designed. Thermostability analysis shows that D476N mutation doesn't have any significant effect but D474K mutation destabilized protein. On the other hand, flexibility analysis using dynamic quenching and limited proteolysis demonstrates that D474K mutation became much more flexible than wild type although D476N doesn't have any significant difference. Intrinsic and ANS fluorescence studies demonstrate that D476N mutation is brought about by structural changes without significant effect on thermostability and flexibility. Molecular modeling reveals that disruption of a salt bridge between D(474) and K(445) accompanying with some H-bond deletion may be involved in destabilization of D474K mutant.     

Contributions of engineered surface salt bridges to the stability of T4 lysozyme determined by directed mutagenesis.

Six designed mutants of T4 lysozyme were created in an attempt to create putative salt bridges on the surface of the protein. The first three of the mutants, T115E (Thr 115 to Glu), Q123E, and N144E, were designed to introduce a new charged side chain close to one or more existing charged groups of the opposite sign on the surface of the protein. In each of these cases the putative electrostatic interactions introduced by the mutation include possible salt bridges between residues within consecutive turns of an alpha-helix. Effects of the mutations ranged from no change in stability to a 1.5 degrees C (0.5 kcal/mol) increase in melting temperature. In two cases, secondary (double) mutants were constructed as controls in which the charge partner was removed from the primary mutant structure. These controls proteins indicate that the contributions to stability from each of the engineered salt bridges is very small (about 0.1-0.25 kcal/mol in 0.15 M KCl). The structures of the three primary mutants were determined by X-ray crystallography and shown to be essentially the same as the wild-type structure except at the site of the mutation. Although the introduced charges in the T115E and Q123E structures are within 3-5 A of their intended partner, the introduced side chains and their intended partners were observed to be quite mobile. It has been shown that the salt bridge between His 31 and Asp 70 in T4 lysozyme stabilizes the protein by 3-5 kcal/mol [Anderson, D. E., Becktel, W. J., & Dahlquist, F. W. (1990) Biochemistry 29, 2403-2408]. To test the effectiveness of His...Asp interactions in general, three additional double mutants, K60H/L13D, K83H/A112D, and S90H/Q122D, were created in order to introduce histidine-aspartate charge pairs on the surface of the protein. Each of these mutants destabilizes the protein by 1-3 kcal/mol in 0.15 M KCl at pH values from 2 to 6.5. The X-ray crystallographic structure of the mutant K83H/A112D has been determined and shows that there are backbone conformational changes of 0.3-0.6 A extending over several residues. The introduction of the histidine and aspartate presumably introduces strain into the folded protein that destabilizes this variant. It is concluded that pairs of oppositely charged residues that are on the surface of a protein and have freedom to adopt different conformations do not tend to come together to form structurally localized salt bridges. Rather, such residues tend to remain mobile, interact weakly if at all, and do not contribute significantly to protein stability. It is argued that the entropic cost of localizing a pair of solvent-exposed charged groups on the surface of a protein largely offsets the interaction energy expected from the formation of a defined salt bridge. There are examples of strong salt bridges in proteins, but such interactions require that the folding of the protein provides the requisite driving energy to hold the interacting partners in the correct rigid alignment.     

Structures and free-energy landscapes of the wild type and mutants of the Abeta(21-30) peptide are determined by an interplay between intrapeptide electrostatic and hydrophobic interactions

The initial events in protein aggregation involve fluctuations that populate monomer conformations, which lead to oligomerization and fibril assembly. The highly populated structures, driven by a balance between hydrophobic and electrostatic interactions in the protease-resistant wild-type Aβ_21-30 peptide and mutants E22Q (Dutch), D23N (Iowa), and K28N, are analyzed using molecular dynamics simulations. Intrapeptide electrostatic interactions were connected to calculated pK_a values that compare well with the experimental estimates. The pK_a values of the titratable residues show that E22 and D23 side chains form salt bridges only infrequently with the K28 side chain. Contacts between E22-K28 are more probable in “dried” salt bridges, whereas D23-K28 contacts are more probable in solvated salt bridges. The strength of the intrapeptide hydrophobic interactions increases as D23N < WT < E22Q < K28A. Free-energy profiles and disconnectivity representation of the energy landscapes show that the monomer structures partition into four distinct basins. The hydrophobic interactions cluster the Aβ_21-30 peptide into two basins, differentiated by the relative position of the DVG(23-25) and GSN(25-27) fragments about the G25 residue. The E22Q mutation increases the population with intact VGSN turn compared to the wild-type (WT) peptide. The increase in the population of the structures in the aggregation-prone Basin I in E22Q, which occurs solely due to the difference in charge states between the Dutch mutant and the WT, gives a structural explanation of the somewhat larger aggregation rate in the mutant. The D23N mutation dramatically reduces the intrapeptide interactions. The K28A mutation increases the intrapeptide hydrophobic interactions that promote population of structures in Basin I and Basin II whose structures are characterized by hydrophobic interaction between V24 and K28 side chains but with well-separated ends of the backbone atoms in the VGSN turn. The intrapeptide electrostatic interactions in the WT and E22Q peptides roughen the free-energy surface compared to the K28A peptide. The D23N mutation has a flat free-energy surface, corresponding to an increased population of random coil-like structures with weak hydrophobic and electrostatic interactions. We propose that mutations or sequences that enhance the probability of occupying Basin I would promote aggregation of Aβ peptides.     

Electrostatic contributions to T4 lysozyme stability: solvent-exposed charges versus semi-buried salt bridges

We carried our Poisson-Boltzmann (PB) calculations for the effects of charge reversal at five exposed sites (K16E, R119E, K135E, K147E, and R154E) and charge neutralization and proton titration of the H31-D70 semi-buried salt bridge on the stability of T4 lysozyme. Instead of the widely used solvent-exclusion (SE) surface, we used the van der Waals (vdW) surface as the boundary between the protein and solvent dielectrics (a protocol established in our earlier study on charge mutations in barnase). By including residual charge-charge interactions in the unfolded state, the five charge reversal mutations were found to have DeltaDeltaG(unfold) from -1.6 to 1.3 kcal/mol. This indicates that the variable effects of charge reversal observed by Matthews and co-workers are not unexpected. The H31N, D70N, and H31N/D70N mutations were found to destabilize the protein by 2.9, 1.3, and 1.6 kcal/mol, and the pK(a) values of H31 and D70 were shifted to 9.4 and 0.6, respectively. These results are in good accord with experimental data of Dahlquist and co-workers. In contrast, if the SE surface were used, the H31N/D70N mutant would be more stable than the wild-type protein by 1.3 kcal/mol. From these and additional results for 27 charge mutations on five other proteins, we conclude that 1) the popular view that electrostatic interactions are generally destabilizing may have been based on overestimated desolvation cost as a result of using the SE surface as the dielectric boundary; and 2) while solvent-exposed charges may not reliably contribute to protein stability, semi-buried salt bridges can provide significant stabilization.     

Formation of the death domain complex between FADD and RIP1 proteins in vitro

Fas-associated death domain (FADD) protein is an adapter molecule that bridges the interactions between membrane death receptors and initiator caspases. The death receptors contain an intracellular death domain (DD) which is essential to the transduction of the apoptotic signal. The kinase receptor-interacting protein 1 (RIP1) is crucial to programmed necrosis. The cell type interplay between FADD and RIP1, which mediates both necrosis and NF-κB activation, has been evaluated in other studies, but the mechanism of the interaction of the FADD and RIP1 proteins remain poorly understood. Here, we provided evidence indicating that the DD of human FADD binds to the DD of RIP1 in vitro. We developed a molecular docking model using homology modeling based on the structures of FADD and RIP1. In addition, we found that two structure-based mutants (G109A and R114A) of the FADD DD were able to bind to the RIP1 DD, and two mutations (Q169A and N171A) of FADD DD and four mutations (G595, K596, E620, and D622) of RIP1 DD disrupted the FADD–RIP1 interaction. Six mutations (Q169A, N171A, G595, K596, E620, and D622) lowered the stability of the FADD–RIP1 complex and induced aggregation that structurally destabilized the complex, thus disrupting the interaction.     

Relationship between stability and flexibility in the most flexible region of Photinus pyralis luciferase

Firefly luciferase is a protein with a large N-terminal and a small C-terminal domain. B-factor analysis shows that its C-terminal is much more flexible than its N-terminal. Studies on hyperthermophile proteins have been shown that the increased thermal stability of hyperthermophile proteins is due to their enhanced conformational rigidity and the relationship between flexibility, stability and function in most of proteins is on debate. Two mutations (D474K and D476N) in the most flexible region of firefly luciferase were designed. Thermostability analysis shows that D476N mutation doesn't have any significant effect but D474K mutation destabilized protein. On the other hand, flexibility analysis using dynamic quenching and limited proteolysis demonstrates that D474K mutation became much more flexible than wild type although D476N doesn't have any significant difference. Intrinsic and ANS fluorescence studies demonstrate that D476N mutation is brought about by structural changes without significant effect on thermostability and flexibility. Molecular modeling reveals that disruption of a salt bridge between D⁴⁷⁴ and K⁴⁴⁵ accompanying with some H-bond deletion may be involved in destabilization of D474K mutant.     

Structure of the 21-30 fragment of amyloid beta-protein

Folding and self-assembly of the 42-residue amyloid beta-protein (Abeta) are linked to Alzheimer's disease (AD). The 21-30 region of Abeta, Abeta(21-30), is resistant to proteolysis and is believed to nucleate the folding of full-length Abeta. The conformational space accessible to the Abeta(21-30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the "native" state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24-K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non-native states of this peptide are compact and retain a native-like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures.