Protein structure and function at low temperatures

Proteins represent the major components in the living cell that provide the whole repertoire of constituents of cellular organization and metabolism. In the process of evolution, adaptation to extreme conditions mainly referred to temperature, pH and low water activity. With respect to life at low temperatures, effects on protein structure, protein stability and protein folding need consideration. The sequences and topologies of proteins from psychrophilic, mesophilic and thermophilic organisms are found to be highly homologous. Commonly, adaptive changes refer to multiple alterations of the amino acid sequence, which presently cannot be correlated with specific changes of structure and stability; so far it has not been possible to attribute specific increments in the free energy of stabilization to well-defined amino-acid exchanges in an unambiguous way. The stability of proteins is limited at high and low temperatures. Their expression and self-organization may be accomplished under conditions strongly deviating from optimum growth conditions. Molecular adaptation to extremes of temperature seems to be accompanied by a flattening of the temperature profile of the free energy of stabilization. In principle, the free energy of stabilization of proteins is small compared to the total molecular energy. As a consequence, molecular adaptation to extremes of physical conditions only requires marginal alterations of the intermolecular interactions and packing density. Careful statistical and structural analyses indicate that altering the number of ion pairs and hydrophobic interactions allows the flexibility of proteins to be adjusted so that full catalytic function is maintained at varying temperatures.     

Protein stability and molecular adaptation to extreme conditions.

Proteins, due to the delicate balance of stabilizing and destabilizing interactions, are only marginally stable. Adaptation to extreme environments tends to shift the 'mesophilic' characteristics of proteins to the respective extremes of temperature, hydrostatic pressure, pH and salinity, such that, under the mutual physiological conditions, the molecular properties are similar regarding overall topology, flexibility and solvation. Enhanced intrinsic stability requires only minute local structural changes so that general strategies of stabilization cannot be established. Apart from mutative changes of amino-acid sequences, extrinsic factors (or cellular components) may be involved in 'extremophilic adaptation'. The molecular basis of acidophilic, alkalophilic and barophilic adaptation is still obscure. Mechanisms of enhanced thermal stability involve improved packing density, as well as specific local interactions. In halophiles, water and salt binding of the intrinsically stable protein inventory is accomplished by favoring acidic over basic amino acid residues and decreased hydrophobicity. General limits of viability are: (a) the susceptibility of the covalent structure of the polypeptide chain toward hydrolysis or hydrothermal degradation; (b) the competition of extreme solvent parameters with the weak electrostatic and hydrophobic interactions involved in protein stabilization; (c) perturbations of the folding and assembly of proteins; and (d) 'dislocation' of biochemical pathways due to effects of extreme conditions on the intricate network of metabolic reactions.     

Helix capping in the GCN4 leucine zipper.

Capping interactions associated with specific sequences at or near the ends of alpha-helices are important determinants of the stability of protein secondary and tertiary structure. We investigate here the role of the helix-capping motif Ser-X-X-Glu, a sequence that occurs frequently at the N termini of alpha helices in proteins, on the conformation and stability of the GCN4 leucine zipper. The 1.8 A resolution crystal structure of the capped molecule reveals distinct conformations, packing geometries and hydrogen-bonding networks at the amino terminus of the two helices in the leucine zipper dimer. The free energy of helix stabilization associated with the hydrogen-bonding and hydrophobic interactions in this capping structure is -1.2 kcal/mol, evaluated from thermal unfolding experiments. A single cap thus contributes appreciably to stabilizing the terminated helix and thereby the native state. These results suggest that helix capping plays a further role in protein folding, providing a sensitive connector linking alpha-helix formation to the developing tertiary structure of a protein.     

Electric charge balance mechanism of extended soluble proteins

Extended proteins such as calmodulin and troponin C have two globular terminal domains linked by a central region that is exposed to water and often acts as a function-regulating element. The mechanisms that stabilize the tertiary structure of extended proteins appear to differ greatly from those of globular proteins. Identifying such differences in physical properties of amino acid sequences between extended proteins and globular proteins can provide clues useful for identification of extended proteins from complete genomes including orphan sequences. In the present study, we examined the structure and amino acid sequence of extended proteins. We found that extended proteins have a large net electric charge, high charge density, and an even balance of charge between the terminal domains, indicating that electrostatic interaction is a dominant factor in stabilization of extended proteins. Additionally, the central domain exposed to water contained many amphiphilic residues. Extended proteins can be identified from these physical properties of the tertiary structure, which can be deduced from the amino acid sequence. Analysis of physical properties of amino acid sequences can provide clues to the mechanism of protein folding. Also, structural changes in extended proteins may be caused by formation of molecular complexes. Long-range effects of electrostatic interactions also appear to play important roles in structural changes of extended proteins.     

Specific interactions for ab initio folding of protein terminal regions with secondary structures

Proteins fold into unique three-dimensional structures by specific, orientation-dependent interactions between amino acid residues. Here, we extract orientation-dependent interactions from protein structures by treating each polar atom as a dipole with a direction. The resulting statistical energy function successfully refolds 13 out of 16 fully unfolded secondary-structure terminal regions of 10-23 amino acid residues in 15 small proteins. Dissecting the orientation-dependent energy function reveals that the orientation preference between hydrogen-bonded atoms is not enough to account for the structural specificity of proteins. The result has significant implications on the theoretical and experimental searches for specific interactions involved in protein folding and molecular recognition between proteins and other biologically active molecules.     

Stable submolecular folding units in a non-compact form of cytochrome c.

Studies of structure, dynamics, and stability of cytochrome c (cyt c) at low pH in a non-compact pre-molten globule state indicate that the protein contains submolecular folding units that are independently stable. In high salt, acid cyt c (pD 2.2; where D is deuterium) is nearly as compact as the native form. Nuclear magnetic resonance (n.m.r.) line broadening typical of the molten globule form is seen, indicating loosened packing and increased mobility not only for side-chains but also for the main chain. As NaCl concentration is decreased below 0.05 M, cyt c expands due to the deshielding of electrostatic repulsions, attaining a linear extent perhaps double that of the native protein (viscosity, fluorescence). In the extended form, tertiary structural hydrogen bonds are largely broken (hydrogen exchange rate), some normally buried parts of the protein are exposed to water (fluorescence), and many of the native side-chain contacts must be lost. Nevertheless, almost all of the helical content is retained (circular dichroism). The helices involve the same amino acid residues that are helical in the native state (hydrogen exchange labeling monitored by 2-dimensional n.m.r.). The equilibrium constant for helix formation at 20 degrees C (0.02 M-NaCl, pD 2.2) is about 10 (hydrogen exchange rate), even though the individual helical segments when isolated have little or no structure. Additional experiments were done to check assumptions and calibrate parameters that underlie the hydrogen exchange analysis of protein folding. These results indicate that the native-like helical segments in the expanded non-globular form of cyt c exist as part of somewhat larger submolecular folding units that possess significant equilibrium stability. Results from equilibrium and kinetic studies of protein folding support the generality of this conclusion. This view is contrary to the two-state paradigm for equilibrium folding and inconsistent with the idea that side-chain packing constraints determine folding motifs. The result suggests an extension of the thermodynamic hypothesis for protein structure to kinetic folding processes, so that the amino acid code for equilibrium and kinetic folding may be the same, and also seems pertinent to the biological evolution of contemporary protein structures.     

Fine structure analysis of a protein folding transition state; distinguishing between hydrophobic stabilization and specific packing

Developing a detailed understanding of the structure and energetics of protein folding transition states is a key step in describing the folding process. The phi-value analysis approach allows the energetic contribution of side-chains to be mapped out by comparing wild-type with individual mutants where conservative changes are introduced. Studies where multiple substitutions are made at individual sites are much rarer but are potentially very useful for understanding the contribution of each element of a side-chain to transition state formation, and for distinguishing the relative importance of specific packing versus hydrophobic interactions. We have made a series of conservative mutations at multiple buried sites in the N-terminal domain of L9 in order to assess the relative importance of specific side-chain packing versus less specific hydrophobic stabilization of the transition state. A total of 28 variants were prepared using both naturally occurring and non-naturally occurring amino acids at six sites. Analysis of the mutants by NMR and CD showed no perturbation of the structure. There is no correlation between changes in hydrophobicity and changes in stability. In contrast, there is excellent linear correlation between the hydrophobicity of a side-chain and the log of the folding rate, ln(k(f)). The correlation between ln(k(f)) and the change in hydrophobicity holds even for substitutions that change the shape and/or size of a side-chain significantly. For most sites, the correlation with the logarithm of the unfolding rate, ln(k(u)), is much worse. Mutants with more hydrophobic amino acid substitutions fold faster, and those with less hydrophobic amino acid substitutions fold slower. The results show that hydrophobic interactions amongst core residues are an important driving force for forming the transition state, and are more important than specific tight packing interactions. Finally, a number of substitutions lead to negative phi-values and the origin of these effects are described.     

Protein structure and neutral theory of evolution

The neutral theory of evolution is extended to the origin of protein molecules. Arguments are presented which suggest that the amino acid sequences of many globular proteins mainly represent "memorized" random sequences while biological evolution reduces to the "editing" these random sequences. Physical requirements for a functional globular protein are formulated and it is shown that many of these requirement do not involve strategical selection of amino acid sequences during biological evolution but are inherent also for typical random sequences. In particular, it is shown that random sequences of polar and amino acid residues can form alpha-helices and beta-strand with lengths and arrangement along the chain similar to those in real globular proteins. These alpha- and beta-regions in random sequences can form three-dimensional folding patterns also similar to those in proteins. The arguments are presented suggesting that even the tight packing of side groups inside protein core do not require very strong biological selection of amino acid sequences either. Thus many structural features of real proteins can exist also in random sequences and the biological selection is needed mainly for the creation of active site of protein and for their stability under physiological conditions.     

The relationship between conservation, thermodynamic stability, and function in the SH3 domain hydrophobic core

To investigate the relationships between sequence conservation, protein stability, and protein function, we have measured the thermodynamic stability, folding kinetics, and in vitro peptide-binding activity of a large number of single-site substitutions in the hydrophobic core of the Fyn SH3 domain. Comparison of these data to that derived from an analysis of a large alignment of SH3 domain sequences revealed a very good correlation between the distinct pattern of conservation observed at each core position and the thermodynamic stability of mutants. Conservation was also found to correlate well with the unfolding rates of mutants, but not to the folding rates, suggesting that evolution selects more strongly for optimal native state packing interactions than for maximal folding rates. Structural analysis suggests that residue-residue core packing interactions are very similar in all SH3 domains, which provides an explanation for the correlation between conservation and mutant stability effects studied in a single SH3 domain. We also demonstrate a correlation between stability and the in vivo activity of mutants, and between conservation and activity. However, the relationship between conservation and activity was very strong only for the three most conserved hydrophobic core positions. The weaker correlation between activity and conservation seen at the other seven core positions indicates that maintenance of protein stability is the dominant selective pressure at these positions. In general, the pattern of conservation at hydrophobic core positions appears to arise from conserved packing constraints, and can be effectively utilized to predict the destabilizing effects of amino acid substitutions.     

Protein Structures and Neutral Theory of Evolution

Abstract

The neutral theory of evolution is extended to the origin of protein molecules. Arguments are presented which suggest that the amino acid sequences of many globular proteins mainly represent “memorized” random sequences while biological evolution reduces to the “editing” these random sequences. Physical requirements for a functional globular protein are formulated and it is shown that many of these requirements do not involve strategical selection of amino acid sequences during biological evolution but are inherent also for typical random sequences. In particular, it is shown that random sequences of polar and unpolar amino acid residues can form α-helices and β-strands with lengths and arrangement along the chain similar to those in real globular proteins. These α- and β-regions in random sequences can form three-dimensional folding patterns also similar to those in real proteins. The arguments are presented suggesting that even the tight packing of side groups inside protein core do not require very strong biological selection of amino acid sequences either. Thus many structural features of real proteins can exist also in random sequences and the biological selection is needed mainly for the creation of active sites of proteins and for their stability under physiological conditions.     

A structural dissection of amino acid substitutions in helical transmembrane proteins

The evolution of protein folds is under strong constraints from their surrounding environment. Although folding in water‐soluble proteins is driven primarily by hydrophobic forces, the nature of the forces that determine the folding and stability of transmembrane proteins are still not fully understood. Furthermore, the chemically heterogeneous lipid bilayer has a non‐uniform effect on protein structure. In this article, we attempt to get an insight into the nature of this effect by examining the impact of various types of local structure environment on amino acid substitution, based on alignments of high‐resolution structures of polytopic helical transmembrane proteins combined with sequences of close homologs. Compared to globular proteins, burying amino acid sidechains, especially hydrophilic ones, led to a lower increase in conservation in both the lipid‐water interface region and the hydrocarbon core region. This observation is due to surface residues in HTM proteins especially in the HC region being relatively highly conserved, suggesting higher evolutionary constraints from their specific interactions with the surrounding lipid molecules. Polar and small residues, particularly Pro and Gly, show a noticeable increase in conservation as they are positioned more towards the centre of the membrane, which is consistent with their recognized key roles in structural stability. In addition, the examination of hydrogen bonds in the membrane environment identified some exposed hydrophilic residues being better conserved when not hydrogen‐bonded to other residues, supporting the importance of lipid‐protein sidechain interactions. The conclusions presented in this study highlight the distinct features of substitution matrices that take into account the membrane environment, and their potential role in improving sequence‐structure alignments of transmembrane proteins. Proteins 2010; © 2010 Wiley‐Liss, Inc.     

Comparison of helix interactions in membrane and soluble alpha-bundle proteins

Helix-helix interactions are important for the folding, stability, and function of membrane proteins. Here, two independent and complementary methods are used to investigate the nature and distribution of amino acids that mediate helix-helix interactions in membrane and soluble alpha-bundle proteins. The first method characterizes the packing density of individual amino acids in helical proteins based on the van der Waals surface area occluded by surrounding atoms. We have recently used this method to show that transmembrane helices pack more tightly, on average, than helices in soluble proteins. These studies are extended here to characterize the packing of interfacial and noninterfacial amino acids and the packing of amino acids in the interfaces of helices that have either right- or left-handed crossing angles, and either parallel or antiparallel orientations. We show that the most abundant tightly packed interfacial residues in membrane proteins are Gly, Ala, and Ser, and that helices with left-handed crossing angles are more tightly packed on average than helices with right-handed crossing angles. The second method used to characterize helix-helix interactions involves the use of helix contact plots. We find that helices in membrane proteins exhibit a broader distribution of interhelical contacts than helices in soluble proteins. Both helical membrane and soluble proteins make use of a general motif for helix interactions that relies mainly on four residues (Leu, Ala, Ile, Val) to mediate helix interactions in a fashion characteristic of left-handed helical coiled coils. However, a second motif for mediating helix interactions is revealed by the high occurrence and high average packing values of small and polar residues (Ala, Gly, Ser, Thr) in the helix interfaces of membrane proteins. Finally, we show that there is a strong linear correlation between the occurrence of residues in helix-helix interfaces and their packing values, and discuss these results with respect to membrane protein structure prediction and membrane protein stability.     

Helix packing moments reveal diversity and conservation in membrane protein structure

Helical membrane proteins are more tightly packed and the packing interactions are more diverse than those found in helical soluble proteins. Based on a linear correlation between amino acid packing values and interhelical propensity, we propose the concept of a helix packing moment to predict the orientation of helices in helical membrane proteins and membrane protein complexes. We show that the helix packing moment correlates with the helix interfaces of helix dimers of single pass membrane proteins of known structure. Helix packing moments are also shown to help identify the packing interfaces in membrane proteins with multiple transmembrane helices, where a single helix can have multiple contact surfaces. Analyses are described on class A G protein-coupled receptors (GPCRs) with seven transmembrane helices. We show that the helix packing moments are conserved across the class A family of GPCRs and correspond to key structural contacts in rhodopsin. These contacts are distinct from the highly conserved signature motifs of GPCRs and have not previously been recognized. The specific amino acid types involved in these contacts, however, are not necessarily conserved between subfamilies of GPCRs, indicating that the same protein architecture can be supported by a diverse set of interactions. In GPCRs, as well as membrane channels and transporters, amino acid residues with small side-chains (Gly, Ala, Ser, Cys) allow tight helix packing by mediating strong van der Waals interactions between helices. Closely packed helices, in turn, facilitate interhelical hydrogen bonding of both weakly polar (Ser, Thr, Cys) and strongly polar (Asn, Gln, Glu, Asp, His, Arg, Lys) amino acid residues. We propose the use of the helix packing moment as a complementary tool to the helical hydrophobic moment in the analysis of transmembrane sequences.     

Kinetic consequences of native state optimization of surface-exposed electrostatic interactions in the Fyn SH3 domain

Optimization of surface exposed charge-charge interactions in the native state has emerged as an effective means to enhance protein stability; but the effect of electrostatic interactions on the kinetics of protein folding is not well understood. To investigate the kinetic consequences of surface charge optimization, we characterized the folding kinetics of a Fyn SH3 domain variant containing five amino acid substitutions that was computationally designed to optimize surface charge-charge interactions. Our results demonstrate that this optimized Fyn SH3 domain is stabilized primarily through an eight-fold acceleration in the folding rate. Analyses of the constituent single amino acid substitutions indicate that the effects of optimization of charge-charge interactions on folding rate are additive. This is in contrast to the trend seen in folded state stability, and suggests that electrostatic interactions are less specific in the transition state compared to the folded state. Simulations of the transition state using a coarse-grained chain model show that native electrostatic contacts are weakly formed, thereby making the transition state conducive to nonspecific, or even nonnative, electrostatic interactions. Because folding from the unfolded state to the folding transition state for small proteins is accompanied by an increase in charge density, nonspecific electrostatic interactions, that is, generic charge density effects can have a significant contribution to the kinetics of protein folding. Thus, the interpretation of the effects of amino acid substitutions at surface charged positions may be complicated and consideration of only native-state interactions may fail to provide an adequate picture.     

Principles of protein folding--a perspective from simple exact models.

General principles of protein structure, stability, and folding kinetics have recently been explored in computer simulations of simple exact lattice models. These models represent protein chains at a rudimentary level, but they involve few parameters, approximations, or implicit biases, and they allow complete explorations of conformational and sequence spaces. Such simulations have resulted in testable predictions that are sometimes unanticipated: The folding code is mainly binary and delocalized throughout the amino acid sequence. The secondary and tertiary structures of a protein are specified mainly by the sequence of polar and nonpolar monomers. More specific interactions may refine the structure, rather than dominate the folding code. Simple exact models can account for the properties that characterize protein folding: two-state cooperativity, secondary and tertiary structures, and multistage folding kinetics--fast hydrophobic collapse followed by slower annealing. These studies suggest the possibility of creating "foldable" chain molecules other than proteins. The encoding of a unique compact chain conformation may not require amino acids; it may require only the ability to synthesize specific monomer sequences in which at least one monomer type is solvent-averse.     

Design of stable α‐helices using global sequence optimization

The rational design of peptide and protein helices is not only of practical importance for protein engineering but also is a useful approach in attempts to improve our understanding of protein folding. Recent modifications of theoretical models of helix‐coil transitions allow accurate predictions of the helix stability of monomeric peptides in water and provide new possibilities for protein design. We report here a new method for the design of α‐helices in peptides and proteins using AGADIR, the statistical mechanical theory for helix‐coil transitions in monomeric peptides and the tunneling algorithm of global optimization of multidimensional functions for optimization of amino acid sequences. CD measurements of helical content of peptides with optimized sequences indicate that the helical potential of protein amino acids is high enough to allow formation of stable α‐helices in peptides as short as of 10 residues in length. The results show the maximum achievable helix content (HC) of short peptides with fully optimized sequences at 5 °C is expected to be ～70–75%. Under certain conditions the method can be a powerful practical tool for protein engineering. Unlike traditional approaches that are often used to increase protein stability by adding a few favorable interactions to the protein structure, this method deals with all possible sequences of protein helices and selects the best one from them. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Universally conserved positions in protein folds: reading evolutionary signals about stability, folding kinetics and function.

Here, we provide an analysis of molecular evolution of five of the most populated protein folds: immunoglobulin fold, oligonucleotide-binding fold, Rossman fold, alpha/beta plait, and TIM barrels. In order to distinguish between "historic", functional and structural reasons for amino acid conservations, we consider proteins that acquire the same fold and have no evident sequence homology. For each fold we identify positions that are conserved within each individual family and coincide when non-homologous proteins are structurally superimposed. As a baseline for statistical assessment we use the conservatism expected based on the solvent accessibility. The analysis is based on a new concept of "conservatism-of-conservatism". This approach allows us to identify the structural features that are stabilized in all proteins having a given fold, despite the fact that actual interactions that provide such stabilization may vary from protein to protein. Comparison with experimental data on thermodynamics, folding kinetics and function of the proteins reveals that such universally conserved clusters correspond to either: (i) super-sites (common location of active site in proteins having common tertiary structures but not function) or (ii) folding nuclei whose stability is an important determinant of folding rate, or both (in the case of Rossman fold). The analysis also helps to clarify the relation between folding and function that is apparent for some folds.     

Characterization of the backbone geometry of protein native state structures

We present a simple method for analyzing the geometry of noncovalent residue-residue interactions stabilizing the protein structure, which takes into account the constraints on the local backbone geometry. We find that the principal geometrical constraints are amino acid aspecific and are associated with hydrogen bond formation in helices and sheets. In contrast, amino acid residues in nonhelical and nonextended conformations, which make noncovalent interactions stabilizing the protein tertiary structure, display greater flexibility. We apply the method to an analysis of the packing of helices in helical bundle proteins requiring an efficient packing of amino acid side-chains of the interacting helices.     

A concerted mechanism for the suppression of a folding defect through interactions with chaperones

Specific amino acid substitutions confer a temperature-sensitive-folding (tsf) phenotype to bacteriophage P22 coat protein. Additional amino acid substitutions, called suppressor substitutions (su), relieve the tsf phenotype. These su substitutions are proposed to increase the efficiency of procapsid assembly, favoring correct folding over improper aggregation. Our recent studies indicate that the molecular chaperones GroEL/ES are more effectively recruited in vivo for the folding of tsf:su coat proteins than their tsf parents. Here, the tsf:su coat proteins are studied with in vitro equilibrium and kinetic techniques to establish a molecular basis for suppression. The tsf:su coat proteins were monomeric, as determined by velocity sedimentation analytical ultracentrifugation. The stability of the tsf:su coat proteins was ascertained by equilibrium urea titrations, which were best described by a three-state folding model, N <--> I <--> U. The tsf:su coat proteins either had stabilized native or intermediate states as compared with their tsf coat protein parents. The kinetics of the I <--> U transition showed a decrease in the rate of unfolding and a small increase in the rate of refolding, thereby increasing the population of the intermediate state. The increased intermediate population may be the reason the tsf:su coat proteins are aggregation-prone and likely enhances GroEL-ES interactions. The N --> I unfolding rate was slower for the tsf:su proteins than their tsf coat parents, resulting in an increase in the native state population, which may allow more competent interactions with scaffolding protein, an assembly chaperone. Thus, the suppressor substitution likely improves folding in vivo through increased efficiency of coat protein-chaperone interactions.