Influence of hydration on the conformational stability and formation of bends in terminally blocked dipeptides

The conformations of 23 terminally blocked dipeptide sequences were examined by conformational energy calculations that included the effects of the aqueous solvent. Starting structures were derived from combinations of minimum-energy conformations of hydrated single residues. Their conformational energies were then minimized using the ECEPP potential (Empirical Conformational Energy Program for Peptides) with hydration included. Short-range interactions dominate in stabilizing the conformations of the hydrated dipeptides. Differences between conformational stabilities of hydrated and unhydrated dipeptides in many cases are due to the competition of solute–water and intramolecular hydrogen bonds. In other cases, perturbation of the hydration shell of the solute by close approach of solute atoms alters conformational preferences. Probabilities of formation of bends were calculated and compared to the corresponding quantities for unhydrated dipeptides and to those calculated from x-ray structures. For bends in dipeptides containing two nonpolar amino acids, computations omitting hydration yield better results. However, better agreement with experimental (x-ray) bend probabilities for dipeptides containing glycine or polar amino acids is obtained only in some sequences when hydration is included. The results are rationalized by the observation that, in proteins, bends containing nonpolar sequences occur on the inside, shielded from the solvent. Bends containing glycine or polar amino acids occur frequently on the surface of the protein, but they are not completely hydrated.     

Amino acid conformational preferences and solvation of polar backbone atoms in peptides and proteins

Amino acids in peptides and proteins display distinct preferences for alpha-helical, beta-strand, and other conformational states. Various physicochemical reasons for these preferences have been suggested: conformational entropy, steric factors, hydrophobic effect, and backbone electrostatics; however, the issue remains controversial. It has been proposed recently that the side-chain-dependent solvent screening of the local and non-local backbone electrostatic interactions primarily determines the preferences not only for the alpha-helical but also for all other main-chain conformational states. Side-chains modulate the electrostatic screening of backbone interactions by excluding the solvent from the vicinity of main-chain polar atoms. The deficiency of this electrostatic screening model of amino acid preferences is that the relationships between the main-chain electrostatics and the amino acid preferences have been demonstrated for a limited set of six non-polar amino acid types in proteins only. Here, these relationships are determined for all amino acid types in tripeptides, dekapeptides, and proteins. The solvation free energies of polar backbone atoms are approximated by the electrostatic contributions calculated by the finite difference Poisson-Boltzmann and the Langevin dipoles methods. The results show that the average solvation free energy of main-chain polar atoms depends strongly on backbone conformation, shape of side-chains, and exposure to solvent. The equilibrium between the low-energy beta-strand conformation of an amino acid (anti-parallel alignment of backbone dipole moments) and the high-energy alpha conformation (parallel alignment of backbone dipole moments) is strongly influenced by the solvation of backbone polar atoms. The free energy cost of reaching the alpha conformation is by approximately 1.5 kcal/mol smaller for residues with short side-chains than it is for the large beta-branched amino acid residues. This free energy difference is comparable to those obtained experimentally by mutation studies and is thus large enough to account for the distinct preferences of amino acid residues. The screening coefficients gamma(local)(r) and gamma(non-local)(r) correlate with the solvation effects for 19 amino acid types with the coefficients between 0.698 to 0.851, depending on the type of calculation and on the set of point atomic charges used. The screening coefficients gamma(local)(r) increase with the level of burial of amino acids in proteins, converging to 1.0 for the completely buried amino acid residues. The backbone solvation free energies of amino acid residues involved in strong hydrogen bonding (for example: in the middle of an alpha-helix) are small. The hydrogen bonded backbone is thus more hydrophobic than the peptide groups in random coil. The alpha-helix forming preference of alanine is attributed to the relatively small free energy cost of reaching the high-energy alpha-helix conformation. These results confirm that the side-chain-dependent solvent screening of the backbone electrostatic interactions is the dominant factor in determining amino acid conformational preferences.     

Protein design simulations suggest that side-chain conformational entropy is not a strong determinant of amino acid environmental preferences

Loss of side-chain conformational entropy is an important force opposing protein folding and the relative preferences of the amino acids for being buried or solvent exposed may be partially determined by which amino acids lose more side-chain entropy when placed in the core of a protein. To investigate these preferences, we have incorporated explicit modeling of side-chain entropy into the protein design algorithm, RosettaDesign. In the standard version of the program, the energy of a particular sequence for a fixed backbone depends only on the lowest energy side-chain conformations that can be identified for that sequence. In the new model, the free energy of a single amino acid sequence is calculated by evaluating the average energy and entropy of an ensemble of structures generated by Monte Carlo sampling of amino acid side-chain conformations. To evaluate the impact of including explicit side-chain entropy, sequences were designed for 110 native protein backbones with and without the entropy model. In general, the differences between the two sets of sequences are modest, with the largest changes being observed for the longer amino acids: methionine and arginine. Overall, the identity between the designed sequences and the native sequences does not increase with the addition of entropy, unlike what is observed when other key terms are added to the model (hydrogen bonding, Lennard-Jones energies, and solvation energies). These results suggest that side-chain conformational entropy has a relatively small role in determining the preferred amino acid at each residue position in a protein.     

Characteristic sequential residue environment of amino acids in proteins

The occurrence of all di- and tripeptide segments of proteins was counted in a large data base containing about 119 000 residues. It was found that the abundance of the amino acids does not determine the frequency of the various di- and tripeptide segments. In addition, the frequency of the various tripeptides cannot be predicted from the observed pair-frequency values. The pair-frequency distribution of amino acids is highly asymmetrical, pairs formed from identical residues are generally preferred and amino acids cannot be clustered on the basis of their first neighbour preferences. These data indicate the existence of general short range regularities in the primary structure of proteins. The consequences of these short range regularities were studied by comparing Chou-Fasman parameters with analogous parameters determined from the results of conformational energy calculations of single amino acids. This comparison shows that Chou-Fasman parameters carry significant information about the environment of each amino acid. The success of the Chou-Fasman's prediction and the properties of the pair and triplet distribution of the amino acid residues suggest that every amino acid has a characteristic sequential residue environment in proteins. The observed preferences could be invoked, for example, in protein design or in the study of the evolutionary relationship of proteins.     

Molecular orbital studies of the conformation around the 5 position in angiotensin II

Analogs of angiotensin are more potent when the side chain at position 5 is branched rather than unbranched. We have performed molecular orbital calculations of conformational preferences of l-valine (branched side chain) and l-a-aminobutyric acid (unbranched side chain) as amino acid residues. The results of these calculations illustrate the differing conformational preferences of the two classes of amino acids and support a stereochemical role for position 5 of angiotensin.     

Differential geometry and protein conformation. V. Medium-range conformational influence of the individual amino acids

A previous differential geometric analysis of the conformational properties of the various amino acids has been extended to study their influence on folding over a larger backbone interval. In addition, statistical effects associated with variation in the number of the individual amino acids in the database have been treated in greater detail, using a simulation method. It is found that the amino acids can be divided into three groups on the basis of their conformational influence over four-C^α units in the interval i ? 6 ? j ? i + 6. Group Ia is composed of seven amino acids (His, Leu, Ala, Met, Lys, Gln, Ile) that encourage the formation of A_R-helical structure. Group Ib (Glu, Phe, Trp, Val, Asp) is composed of amino acids with some helix-forming tendency but that also show positive extended-strand formation tendency. They therefore act as a bridge between group Ia and group II (Cys, Gly, Asn, Pro, Arg, Ser, Thr, Tyr) that contains amino acids that encourage the formation of extended structure and bends. The detailed four-C^α conformational properties of each of the amino acids are shown, and the ability of amino acids to exert conformational influence in both directions along the backbone is examined. It is shown that, in general, such influence extends farther in the N-terminal direction than in the C-terminal direction. A framework is briefly sketched for using the present data to investigate actual folding mechanisms.     

Analysis of the code relating sequence to conformation in globular proteins. The distribution of residue pairs in turns and kinks in the backbone chain

1. The residue pair is considered as the fundamental unit which differentiates alpha-helix, beta-pleated sheet and the various turns and kink structures of the protein backbone. 2. The HPLG alphabet (Robson & Pain, 1974) is used to group pairs of residues, giving 16 possible conformational pairs, all of which are found with differing frequencies in the nine proteins examined. 3. The frequencies of occurrence of the 16 different types of turn or kink are analysed in relation to the constituent amino acids. Those containing the L or G conformation are of low frequency and are grouped for purposes of this analysis. 4. The distribution of amino acids within all the conformational pairs is non-random, with distinct preferences shown by certain residues. 5. All pairs containing an L or G conformation require the presence of a glycine or a proton-donor side chain. 6. The results are discussed in terms of the determination of these ;random' structures by local interactions.     

Reconciling observations of sequence-specific conformational propensities with the generic polymeric behavior of denatured proteins

Radii of gyration of denatured proteins vary with chain length and are insensitive to details of amino acid sequence. Observations of sequence independence in polymeric properties conflict with results from spectroscopic experiments, which suggest the presence of sequence-specific residual structure in denatured states. Can we reconcile the two apparently conflicting sets of observations? To answer this question, we need knowledge of the ensemble of conformations accessible to proteins in good solvents. The excluded-volume limit provides an ideal mimic of polymers in good solvents. Therefore, we attempt to solve the "reconciliation problem" by simulating conformational ensembles accessible to peptides and proteins in the excluded-volume limit. Analysis of these ensembles for a variety of polypeptide sequences leads to results that are consistent with experimental observations of sequence-specific conformational preferences in short peptides and the scaling behavior of polymeric quantities for denatured proteins. Reconciliation in the excluded-volume limit comes about due to a tug of war between two factors, namely, minimization of steric overlap and the competing effects of conformational entropy. Minimization of steric overlap promotes chain stretching and leads to experimentally observed sequence-dependent preferences for locally extended segments such as polyproline II helices, beta-strands, and very short stretches of alpha-helix. Conformational entropy opposes chain stretching, and the calculated persistence length for sequence-dependent conformational preferences is less than five amino acids. This estimate does not vary with amino acid sequence. The short persistence lengths lead directly to experimental observations of generic sequence-independent behavior of radii of gyration for denatured proteins.     

Electronic properties of the amino acid side chains contribute to the structural preferences in protein folding.

A database of 118 non-redundant proteins was examined to determine the preferences of amino acids for secondary structures: alpha-helix, beta-strand and coil conformations. To better understand how the physicochemical properties of amino acid side chains might influence protein folding, several new scales have been suggested for quantifying the electronic effects of amino acids. These include the pKa at the amino group, localized effect substituent constants (esigma), and a composite of these two scales (epsilon). Amino acids were also classified into 5 categories on the basis of their electronic properties: O (strong electron donor), U (weak donor), Z (ambivalent), B (weak electron acceptor), and X (strong acceptor). Certain categories of amino acid appeared to be critical for particular conformations, e.g., O and U-type residues for alpha-helix formation. Pairwise analysis of the database according to these categories revealed significant context effects in the structural preferences. In general, the propensity of an amino acid for a particular conformation was related to the electronic features of the side chain. Linear regression analyses revealed that the electronic properties of amino acids contributed about as much to the folding preferences as hydrophobicity, which is a well-established determinant of protein folding. A theoretical model has been proposed to explain how the electronic properties of the side chain groups might influence folding along the peptide backbone.     

Alpha-helix stability in proteins. I. Empirical correlations concerning substitution of side-chains at the N and C-caps and the replacement of alanine by glycine or serine at solvent-exposed surfaces.

The importance of amino acid side-chains in helix stability has been investigated by making a series of mutations at the N-caps, C-caps and internal positions of the solvent-exposed faces of the two alpha-helices of barnase. There is a strong positional and context dependence of the effect of a particular amino acid on stability. Correlations have been found that provide insight into the physical basis of helix stabilization. The relative effects of Ala and Gly (or Ser) may be rationalized on the basis of solvent-accessible surface areas: burial of hydrophobic surface stabilizes the protein as does exposure to solvent of unpaired hydrogen bond donors or acceptors in the protein. There is a good correlation between the relative stabilizing effects of Ala and Gly at internal positions with the total change in solvent-accessible hydrophobic surface area of the folded protein on mutation of Ala----Gly. The relationship may be extended to the N and C-caps by including an extra term in hydrophilic surface area for the solvent exposure of the non-intramolecularly hydrogen-bonded main-chain CO, NH or protein side-chain hydrogen bonding groups. The requirement for solvent exposure of the C-cap main-chain CO groups may account for the strong preference for residues having positive phi and psi angles at this position, since this alpha L-conformation results in the largest solvent exposure of the C-terminal CO groups. Glycine in an alpha L-conformation results in the greatest exposure of these CO groups. Further, the side-chains of His, Asn, Arg and Lys may, with positive phi and psi-angles, form a hydrogen bond with the backbone CO of residue in position C -3 (residues are numbered relative to the C-cap). The preferences at the C-cap are Gly much greater than His greater than Asn greater than Arg greater than Lys greater than Ala approximately Ser approximately greater than Asp. The preferences at the N-cap are determined by hydrogen bonding of side-chains or solvent to the exposed backbone NH groups and are: Thr approximately Asp approximately Ser greater than Gly approximately Asn greater than Gln approximately Glu approximately His greater than Ala greater than Val much greater than Pro. These general trends may be obscured when mutation allows another side-chain to become a surrogate cap.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of hypermethylation on the functional properties of transfer ribonucleic acid. Formation of aminoacyl-transfer-ribonucleic acid

1. The ability of chemically hypermethylated Escherichia coli B transfer RNA to accept 19 amino acids was studied and the results were compared with those obtained with a control sample of E. coli B transfer RNA incubated under similar conditions in the absence of methylating agent. 2. There is a marked decrease in the ability of the modified transfer RNA to accept amino acids in almost all instances. 3. The acceptance of cysteine appears to be unique in that it is enhanced in the hypermethylated transfer RNA. 4. More detailed studies on the kinetics of acceptance for six amino acids is presented, emphasizing the variation in response of the individual amino acids. 5. Increasing hypermethylation causes a progressive decrease in the amino acid acceptance. 6. The results are discussed in terms of methylation at functional sites within the transfer RNA and possible conformational alterations to the structure of the macromolecule.     

Comparison of taste preferences in the three-spined Gasterosteus aculeatus and nine-spined Pungitius pungitius sticklebacks from the White Sea Basin

Similarity between the taste preferences of classical taste substances and free L-amino acids in adult three-spined stickleback Gasterosteus aculeatus (forma leiurus) and nine-spined stickleback Pungitius pungitius (forma laevis) was found. The strongest and the most significant responses in both species were evoked by citric acid and cysteine, asparatic, and glutamic acids. Sodium chloride, calcium chloride, sucrose, and the remaining 18 amino acids do not elicit a statistically significant effect on the consumption of agar-agar pellets by the fish or have weak taste attractiveness. Similarity of taste preferences in the three-spined and nine-spined sticklebacks are supported by correlation analysis. Absolute values of the consumption of pellets of the same types are also similar in the two species. There were, however, differences in the behavioral taste response, repeated snaps, and the duration of processing of the pellets. The taste response of the nine-spined stickleback is more similar to taste responses in fish of the limnophilic complex than to the response in the three-spined stickleback. It is hypothesized that taste spectra may be very similar in fish with similar ecology and feeding patterns not only in sticklebacks, but also in other related species.     

Cysteine-cysteine contact preference leads to target-focusing in protein folding

We perform a statistical analysis of amino-acid contacts to investigate possible preferences of amino-acid interactions. We include in the analysis only tertiary contacts, because they are less constrained--compared to secondary contacts--by proteins' backbone rigidity. Using proteins from the protein data bank, our analysis reveals an unusually high frequency of cysteine pairings relative to that expected from random. To elucidate the possible effects of cysteine interactions in folding, we perform molecular simulations on three cysteine-rich proteins. In particular, we investigate the difference in folding dynamics between a Gō-like model (where attraction only occurs between amino acids forming a native contact) and a variant model (where attraction between any two cysteines is introduced to mimic the formation/dissociation of native/nonnative disulfide bonds). We find that when attraction among cysteines is nonspecific and comparable to a solvent-averaged interaction, they produce a target-focusing effect that expedites folding of cysteine-rich proteins as a result of a reduction of conformational search space. In addition, the target-focusing effect also helps reduce glassiness by lowering activation energy barriers and kinetic frustration in the system. The concept of target-focusing also provides a qualitative understanding of a correlation between the rates of protein folding and parameters such as contact order and total contact distance.     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin.

In an attempt to delineate potential folding initiation sites for different protein structural motifs, we have synthesized series of peptides that span the entire length of the polypeptide chain of two proteins, and examined their conformational preferences in aqueous solution using proton nuclear magnetic resonance and circular dichroism spectroscopy. We describe here the behavior of peptides derived from a simple four-helix bundle protein, myohemerythrin. The peptides correspond to the sequences of the four long helices (the A, B, C and D helices), the N- and C-terminal loops and the connecting sequences between the helices. The peptides corresponding to the helices of the folded protein all exhibit preferences for helix-like conformations in solution. The conformational ensembles of the A- and D-helix peptides contain ordered helical forms, as shown by extensive series of medium-range nuclear Overhauser effect connectivities, while the B- and C-helix peptides exhibit conformational preferences for nascent helix. All four peptides adopt ordered helical conformations in mixtures of trifluoroethanol and water. The terminal and interconnecting loop peptides also appear to contain appreciable populations of conformers with backbone phi and psi angles in the alpha-region and include highly populated hydrophobic cluster and/or turn conformations in some cases. Trifluoroethanol is unable to drive these peptides towards helical conformations. Overall, the peptide fragments of myohemerythrin have a marked preference towards secondary structure formation in aqueous solution. In contrast, peptide fragments derived from the beta-sandwich protein plastocyanin are relatively devoid of secondary structure in aqueous solution (see accompanying paper). These results suggest that the two different protein structural motifs may require different propensities for formation of local elements of secondary structure to initiate folding, and that there is a prepartitioning of conformational space determined by the local amino acid sequence that is different for the helical and beta-sandwich structural motifs.     

Influence of the hydrophilic face on the folding ability and stability of alpha-helix bundles: relevance to the peptide catalytic activity.

Although not the sole feature responsible, the packing of amino acid side chains in the interior of proteins is known to contribute to protein conformational specificity. While a number of amphipathic peptide sequences with optimized hydrophobic domains has been designed to fold into a desired aggregation state, the contribution of the amino acids located on the hydrophilic side of such peptides to the final packing has not been investigated thoroughly. A set of self-aggregating 18-mer peptides designed previously to adopt a high level of alpha-helical conformation in benign buffer is used here to evaluate the effect of the nature of the amino acids located on the hydrophilic face on the packing of a four alpha-helical bundle. These peptides differ from one another by only one to four amino acid mutations on the hydrophilic face of the helix and share the same hydrophobic core. The secondary and tertiary structures in the presence or absence of denaturants were determined by circular dichroism in the far- and near-UV regions, fluorescence and nuclear magnetic resonance spectroscopy. Significant differences in folding ability, as well as chemical and thermal stabilities, were found between the peptides studied. In particular, surface salt bridges may form which would increase both the stability and extent of the tertiary structure of the peptides. The structural behavior of the peptides may be related to their ability to catalyze the decarboxylation of oxaloacetate, with peptides that have a well-defined tertiary structure acting as true catalysts.     

Correlations of amino acids in proteins

A correlation analysis among 20 amino acids is performed for four protein structural classes (, β, /β, and +β) in a total of 204 proteins. The correlation relationships among amino acids can be classified into the following four types: (1) strong positive correlation, (2) strong negative correlation, (3) weak correlation, and (4) no correlation. The correlation relationships are different for different proteins and are correlated with the features of their structural classes. The amino acids with the weak correlation relationship can be treated as the independent basis functions for the space where proteins are defined. The amino acids with large correlation coefficients are linear correlative with each other and they are not independent. The strong correlation among amino acids reflects their mutual constrained relationship, as exhibited by their relevant structural features. The information obtained through the correlation analysis is used for predicting protein structural classes and a better prediction quality is obtained than that by the simple geometry distance methods without taking into account the correlation effects.     

Complete replacement set of amino acids at the C-terminus of thymidylate synthase: quantitative structure-activity relationship of mutants of an enzyme.

The C-terminal residue of thymidylate synthase (TS) is highly conserved and has been implicated in cofactor binding, catalysis, and a conformational change. The codon for the C-terminal valine of Lactobacillus casei TS has been replaced with those for 19 other amino acids and the amber stop codon. Fourteen of the resulting mutant proteins were active by genetic complementation using a Thy- strain of Escherichia coli, and 18 mutants were active by in vitro assay. Only the aspartate and amber mutations had undetectable activity. All of the mutants were expressed at high levels (5-30% of soluble protein) and were purified by phosphocellulose chromatography. In general, the alterations at position 316 led to little effect on the Km for dUMP, an increase in Km for the folate cofactor, and a decrease in kcat. The observations show that TS can tolerate the substitution of most amino acids for valine at the C-terminus without a complete loss of activity, that hydrophobic substitutions are preferred, and that the C-terminal side chain is involved in both cofactor binding and catalysis. There was an excellent correlation between log kcat and hydrophobicity of the side chain at position 316 and an inverse correlation between log Km and the hydrophobicity of this residue. Kinetic parameters of the cofactor-independent TS-catalyzed dehalogenation of BrdUMP showed no variation with the side chain at position 316. In context of the structure of TS, it is proposed that binding of the cofactor triggers a conformational change in which the C-terminal side chain undergoes hydrophobic interactions that stabilize a rate-limiting transition state of the TS reaction.     

Conformational preference functions for predicting helices in membrane proteins

A suite of FORTRAN programs, PREF, is described for calculating preference functions from the data base of known protein structures and for comparing smoothed profiles of sequence-dependent preferences in proteins of unknown structure. Amino acid preferences for a secondary structure are considered as functions of a sequence environment. Sequence environment of amino acid residue in a protein is defined as an average over some physical, chemical, or statistical property of its primary structure neighbors. The frequency distribution of sequence environments in the data base of soluble protein structures is approximately normal for each amino acid type of known secondary conformation. An analytical expression for the dependence of preferences on sequence environment is obtained after each frequency distribution is replaced by corresponding Gaussian function. The preference for the α-helical conformation increases for each amino acid type with the increase of sequence environment of buried solvent-accessible surface areas. We show that a set of preference functions based on buried surface area is useful for predicting folding motifs in α-class proteins and in integral membrane proteins. The prediction accuracy for helical residues is 79% for 5 integral membrane proteins and 74% for 11 α-class soluble proteins. Most residues found in transmembrane segments of membrane proteins with known α-helical structure are predicted to be indeed in the helical conformation because of very high middle helix preferences. Both extramembrane and transmembrane helices in the photosynthetic reaction center M and L subunits are correctly predicted. We point out in the discussion that our method of conformational preference functions can identify what physical properties of the amino acids are important in the formation of particular secondary structure elements. © 1993 John Wiley & Sons, Inc.     

Ligand specificity and conformational stability of human fatty acid-binding proteins.

Fatty acid binding proteins (FABPs) are small cytosolic proteins with virtually identical backbone structures that facilitate the solubility and intracellular transport of fatty acids. At least eight different types of FABP occur, each with a specific tissue distribution and possibly with a distinct function. To define the functional characteristics of all eight human FABPs, viz. heart (H), brain (B), myelin (M), adipocyte (A), epidermal (E), intestinal (I), liver (L) and ileal lipid-binding protein (I-LBP), we studied their ligand specificity, their conformational stability and their immunological crossreactivity. Additionally, binding of bile acids to I-LBP was studied. The FABP types showed differences in fatty acid binding affinity. Generally, the affinity for palmitic acid was lower than for oleic and arachidonic acid. All FABP types, except E-FABP, I-FABP and I-LBP interacted with 1-anilinonaphtalene-8-sulphonic acid (ANS). Only L-FABP, I-FABP and M-FABP showed binding of 11-((5-dimethylaminonaphtalene-1-sulfonyl)amino)undecanoic acid (DAUDA). I-LBP showed increasing binding of bile acids in the order taurine-conjugated>glycine-conjugated>unconjugated bile acids. A hydroxylgroup of bile acids at position 7 decreased and at position 12 increased the binding affinity to I-LBP. The fatty acid-binding affinity and the conformation of FABP types were differentially affected in the presence of urea. Our results demonstrate significant differences in ligand binding, conformational stability and surface properties between different FABP types which may point to a specific function in certain cells and tissues. The preference of I-LBP (but not L-FABP) for conjugated bile acids is in accordance with a specific role in bile acid reabsorption in the ileum.     

A network representation of protein structures: implications for protein stability

This study views each protein structure as a network of noncovalent connections between amino acid side chains. Each amino acid in a protein structure is a node, and the strength of the noncovalent interactions between two amino acids is evaluated for edge determination. The protein structure graphs (PSGs) for 232 proteins have been constructed as a function of the cutoff of the amino acid interaction strength at a few carefully chosen values. Analysis of such PSGs constructed on the basis of edge weights has shown the following: 1), The PSGs exhibit a complex topological network behavior, which is dependent on the interaction cutoff chosen for PSG construction. 2), A transition is observed at a critical interaction cutoff, in all the proteins, as monitored by the size of the largest cluster (giant component) in the graph. Amazingly, this transition occurs within a narrow range of interaction cutoff for all the proteins, irrespective of the size or the fold topology. And 3), the amino acid preferences to be highly connected (hub frequency) have been evaluated as a function of the interaction cutoff. We observe that the aromatic residues along with arginine, histidine, and methionine act as strong hubs at high interaction cutoffs, whereas the hydrophobic leucine and isoleucine residues get added to these hubs at low interaction cutoffs, forming weak hubs. The hubs identified are found to play a role in bringing together different secondary structural elements in the tertiary structure of the proteins. They are also found to contribute to the additional stability of the thermophilic proteins when compared to their mesophilic counterparts and hence could be crucial for the folding and stability of the unique three-dimensional structure of proteins. Based on these results, we also predict a few residues in the thermophilic and mesophilic proteins that can be mutated to alter their thermal stability.