Entropic stabilization of proteins and its proteomic consequences

Abstract

Evolutionary traces of thermophilic adaptation are manifest, on the whole-genome level, in compositional biases toward certain types of amino acids. However, it is sometimes difficult to discern their causes without a clear understanding of underlying physical mechanisms of thermal stabilization of proteins. For example, it is well-known that hyperthermophiles feature a greater proportion of charged residues, but, surprisingly, the excess of positively charged residues is almost entirely due to lysines but not arginines in the majority of hyperthermophilic genomes. All-atom simulations show that lysines have a much greater number of accessible rotamers than arginines of similar degree of burial in folded states of proteins. This finding suggests that lysines would preferentially entropically stabilize the native state. Indeed, we show in computational experiments that arginine-to-lysine amino acid substitutions result in noticeable stabilization of proteins. We then hypothesize that if evolution uses this physical mechanism as a complement to electrostatic stabilization in its strategies of thermophilic adaptation, then hyperthermostable organisms would have much greater content of lysines in their proteomes than comparably sized and similarly charged arginines. Consistent with that, high-throughput comparative analysis of complete proteomes shows extremely strong bias toward arginine-to-lysine replacement in hyperthermophilic organisms and overall much greater content of lysines than arginines in hyperthermophiles. This finding cannot be explained by genomic GC compositional biases or by the universal trend of amino acid gain and loss in protein evolution. We discovered here a novel entropic mechanism of protein thermostability due to residual dynamics of rotamer isomerization in native state and demonstrated its immediate proteomic implications. Our study provides an example of how analysis of a fundamental physical mechanism of thermostability helps to resolve a puzzle in comparative genomics as to why amino acid compositions of hyperthermophilic proteomes are significantly biased toward lysines but not similarly charged arginines.

Figure 1. The Temperature Dependence of the Energy of Unfolding for Hydrolases, from E. coli (black rhombuses) and T. thermophilus (red squares)

Every simulation of unfolding started from the native structure and lasted for 2 × 10⁶ MC steps; absolute temperature increment is 0.2 and 0.1 in the vicinity of transition temperature. The error bars represent mean square fluctuations of energy at each temperature calculated within productive part of a run when trajectory reached equilibrium after temperature increment.

Figure 2. The Temperature Dependence of the Natural Logarithm of Number of Rotamers

(A) Arg (black rhombuses) versus lysine (red squares) rotamers of hydrolase H from E. coli; (B) Arg (black rhombuses) versus lysine (red squares) rotamers of hydrolase H from T. thermophilus; (C) Leu (dark blue rhombuses) versus Ile (light blue squares) rotamers of hydrolase H from E. coli; (C) Leu (dark blue rhombuses) versus Ile (light blue squares) rotamers of hydrolase H from T. thermophilus; (E) Thr (orange rhombuses) versus Ser (yellow squares) rotamers of hydrolase H from E. coli; (F) Thr (orange rhombuses) versus Ser (yellow squares) rotamers of hydrolase H from T. thermophilus; (G) Thr (orange rhombuses) versus Val (green-blue squares) rotamers of hydrolase H from E. coli; (H) Thr (orange rhombuses) versus Val (green-blue squares) rotamers of hydrolase H from T. thermophilus.

Figure 3. Distribution of the Ratios of the Number of Rotamers in Unfolded and Folded States in a Representative Set of Proteins

Completely unfolded state is achieved at absolute temperature T = 4, folded state at T = 1. (A) Lys versus Arg; (B) Ile versus Leu; (C) Phe versus Tyr; (D) Val versus Thr. Upper histogram in each panel corresponds to T = 4, lower histogram corresponds to T = 1.

Figure 4. The Temperature Dependence of the Energy of Unfolding for Mutated (Red Squares) versus Original Hydrolases H

(A) R171K mutant and wild-type of hydrolase H from T. thermophilus; (B) R171K mutant and wild-type of hydrolase H from E. coli; (C) R43,86,88,171K mutant and wild-type of hydrolase H from E. coli.

Figure 5. The Temperature Dependence of the Energy of Unfolding for R24,26,53,58,74,80,87,95K Mutant Compared with the Original Structure of Cytochrome C from R. sphaeroides

Figure 6. Histograms of the Content of Charged Amino Acid Residues in Hyperthermophilic Genomes Compared with Mesophilic Genomes

Top histogram shows percentage of each residue in mesophilic genomes; bottom histogram, in hyperthermophilic genomes. A total of 12 hyperthermophilic and 38 mesophilic genomes were analyzed (for the complete list, see Tables S1 and S2). (A) Arg; (B) Lys; (C) Asp; (D) Glu.