Mesophilic Counterparts Research Articles

Microbial Community Ability to Adapt to Altered Temperature Conditions Influences Operating Stability in Anaerobic Digestion

Abstract This study examined the influence of altered operating temperature on microbial community composition and how this affects the ability of anaerobic degradation (AD) processes to cope with increasing loading rate. Four digesters fed household and slaughterhouse waste, mimicking two large-scale processes, were operated in sets of two at 37°C or 52°C, followed by a gradual increase or decrease in temperature in one digester in each set. All digesters were then subjected to step-wise increases in organic loading rate (OLR) from 3 to 7g VS/L/day, concurrently with decreased hydraulic retention time (HRT). Temporal microbial changes were monitored by Illumina MiSeq analysis of bacterial and archaeal 16S rRNA gene sequences. The digester transformed from thermophilic to mesophilic conditions failed at 6g VS/L/day, whereas the reference digesters and the mesophilic-to-thermophilic digester remained stable at all OLR investigated. The bacterial community was characterised by relatively diversified structure dominated by Bacteriodetes and Firmicutes in mesophilic conditions. Increasing temperature caused loss of complexity at phyla level and enhanced the relative abundance of Firmicutes and Thermotogae. Temperature adaptation in the thermophilic-to-mesophilic digester resulted in a bacterial community structure reflecting the communities observed in digesters with initial temperature of 37°C. However, certain populations found in the mesophilic digesters did not appear at detectable levels during operation at 37°C in the thermophilic-to-mesophilic digester. Overall, the results showed that anaerobic process stability was highly dependent on the innate resilience of the microbial community. The mesophilic microbiota formed from a thermophilic community had a slightly different structure than its mesophilic counterpart and was considerably less resistant to increased OLR in digesters examined within this study.

PHYSIOLOGICAL AND BIOCHEMICAL CHANGES IN ALGAL CULTURES OF CHLORELLA VULGARIS AND SYNECHOCYSTIS SALINA (MESOPHILIC AND ANTARCTIC ISOLATES) OCCURING AFTER TREATMENT WITH UV-B RADIATION

Determination of biomass production and viability of algal cells of Chlorella vulgaris and Synechocystis salina exposed to UV-B radiation were carried out in this study together with comparison of the mesophilic and antarctic isolates of both investigated strains. Estimation of the content of the pigments: chlorophyll a, chlorophyll b, β-carotene, C-phycocyanin and allo-phycocyanin in algal cells exposed to UV-B radiation was also accomplished. The obtained results showed that the antarctic algae are more resistant to oxidative stress than their mesophilic counterparts. The antarctic isolates of Ch. vulgaris and S. salina compared with the mesophilic ones - up to 72 h showed tolerance to low exposures of radiation, expressed in a slight stimulation of growth and viability of the cells. Antarctic isolates also showed greater resistance to low doses of UV-B radiation manifested by stimulation of the synthesis of chlorophyll a and β-carotene. The registered increase in the amount of C- and allo-phycocyanin in antarctic isolates of S. salina showed that they had developed protective strategies against UV-B radiation by increasing the concentration of the phycobiliproteins. As a result of increased UV-B background, in antarctic isolates, stronger antioxidant defence mechanisms are triggered, which proved the possibility of using them as markers of oxidative stress.

Structural and functional insights into thermally stable cytochrome c' from a thermophile.

Thermophilic Hydrogenophilus thermoluteolus cytochrome c′ (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c′ (AVCP), which has a homo‐dimeric structure and ligand‐binding ability. To understand the thermal stability mechanism and ligand‐binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo‐dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 Å. In the PHCP structure, six specific residues appeared to strengthen the heme‐related and subunit–subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit–subunit interactions were more severely destabilized than ones having altered heme‐related interactions. The PHCP structure further revealed a ligand‐binding channel and a penta‐coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme‐related and subunit–subunit interactions with conservation of the ligand‐binding ability.Brief SummaryWe report the X‐ray crystal structure of cytochrome c′ (PHCP) from thermophilic Hydrogenophilus thermoluteolus. The high thermal stability of PHCP was attributed to heme‐related and subunit–subunit interactions, which were confirmed by a mutagenesis study. The ligand‐binding ability of PHCP was examined by spectrophotometry. PHCP acquired the thermal stability with conservation of the ligand‐binding ability. This study furthers the understanding of the stability and function of cytochromes c.

Structural insights into the cold adaptation of the photosynthetic pigment-protein C-phycocyanin from an Arctic cyanobacterium

The cold adaptation mechanism of phycobiliproteins, the major photosynthetic pigment-proteins in cyanobacteria and red algae, has rarely been studied. Here we reported the biochemical, structural, and molecular dynamics simulation study of the C-phycocyanin from Arctic cyanobacterial strain Pseudanabaena sp. LW0831. We characterized the phycobilisome components of LW0831 and obtained their gene sequences. Compared to the mesophilic counterpart from Arthrospira platensis (Ar-C-PC), LW0831 C-phycocyanin (Ps-C-PC) has a decreased thermostability (∆Tm of −16°C), one of the typical features of cold-adapted enzymes. To uncover its structural basis, we resolved the crystal structure of Ps-C-PC 1 at 2.04Å. Consistent with the decrease in thermostability, comparative structural analyses revealed decreased intra-trimer and inter-trimer interactions in Ps-C-PC 1, compared to Ar-C-PC. However, comparative molecular dynamics simulations indicated that Ps-C-PC 1 shows similar flexibilities to Ar-C-PC for both the (αβ)3 trimer and (αβ)6 hexamer. Therefore, the optimization mode is clearly different from cold-adapted enzymes, which usually have increased flexibilities. Detailed analyses demonstrated different optimization modes for the α and β subunits and it was revealed that hydrophobic interactions are key to this difference, though salt bridges, hydrogen bonds, and surface hydrophobicity are also involved. This study is the first report of the structure of cold-adapted phycobiliproteins and provides insights into the cold-adaptation strategies of non-enzyme proteins.

Cloning, Expression, and Characterization of a Cold-Adapted Shikimate Kinase from the Psychrophilic Bacterium Colwellia psychrerythraea 34H.

Most cold-adapted enzymes possess higher Km and kcat values than those of their mesophilic counterparts to maximize the reaction rate. This characteristic is often ascribed to a high structural flexibility and improved dynamics in the active site. However, this may be less convincing to cold-adapted metabolic enzymes, which work at substrate concentrations near Km. In this respect, cold adaptation of a shikimate kinase (SK) in the shikimate pathway from psychrophilic Colwellia psychrerythraea (CpSK) was characterized by comparing it with a mesophilic Escherichia coli homolog (EcSK). The optimum temperatures for CpSK and EcSK activity were approximately 30°C and 40°C, respectively. The melting points were 33°C and 45°C for CpSK and EcSK, respectively. The ΔGH2O (denaturation in the absence of denaturing agent) values were 3.94 and 5.74 kcal/mol for CpSK and EcSK, respectively. These results indicated that CpSK was a cold-adapted enzyme. However, contrary to typical kinetic data, CpSK had a lower Km for its substrate shikimate than most mesophilic SKs, and the kcat was not increased. This observation suggested that CpSK may have evolved to exhibit increased substrate affinity at low intracellular concentrations of shikimate in the cold environment. Sequence analysis and homology modeling also showed that some important salt bridges were lost in CpSK, and higher Arg residues around critical Arg 140 seemed to increase flexibility for catalysis. Taken together, these data demonstrate that CpSK exhibits characteristics of cold adaptation with unusual kinetic parameters, which may provide important insights into the cold adaptation of metabolic enzymes.

In Silico Designing of an Industrially Sustainable Carbonic Anhydrase Using Molecular Dynamics Simulation.

Carbonic anhydrase (CA) is a family of metalloenzymes that has the potential to sequestrate carbon dioxide (CO2) from the environment and reduce pollution. The goal of this study is to apply protein engineering to develop a modified CA enzyme that has both higher stability and activity and hence could be used for industrial purposes. In the current study, we have developed an in silico method to understand the molecular basis behind the stability of CA. We have performed comparative molecular dynamics simulation of two homologous α-CA, one of thermophilic origin (Sulfurihydrogenibium sp.) and its mesophilic counterpart (Neisseria gonorrhoeae), for 100 ns each at 300, 350, 400, and 500 K. Comparing the trajectories of two proteins using different stability-determining factors, we have designed a highly thermostable version of mesophilic α-CA by introducing three mutations (S44R, S139E, and K168R). The designed mutant α-CA maintains conformational stability at high temperatures. This study shows the potential to develop industrially stable variants of enzymes while maintaining high activity.

Specific amino acids responsible for the cold adaptedness of Micrococcus antarcticus β-glucosidase BglU.

Psychrophilic enzymes display efficient activity at moderate or low temperatures (4-25°C) and are therefore of great interest in biotechnological industries. We previously examined the crystal structure of BglU, a psychrophilic β-glucosidase from the bacterium Micrococcus antarcticus, at 2.2Å resolution. In structural comparison and sequence alignment with mesophilic (BglB) and thermophilic (GlyTn) counterpart enzymes, BglU showed much lower contents of Pro residue and of charged amino acids (particularly positively charged) on the accessible surface area. In the present study, we investigated the roles of specific amino acid residues in the cold adaptedness of BglU. Mutagenesis assays showed that the mutations G261R and Q448P increased optimal temperature (from 25 to 40-45°C) at the expense of low-temperature activity, but had no notable effects on maximal activity or heat lability. Mutations A368P, T383P, and A389E significantly increased optimal temperature (from 25 to 35-40°C) and maximal activity (~1.5-fold relative to BglU). Thermostability of A368P and A389E increased slightly at 30°C. Mutations K163P, N228P, and H301A greatly reduced enzymatic activity-almost completely in the case of H301A. Low contents of Pro, Arg, and Glu are important factors contributing to BglU's psychrophilic properties. Our findings will be useful in structure-based engineering of psychrophilic enzymes and in production of mutants suitable for a variety of industrial processes (e.g., food production, sewage treatment) at cold or moderate temperatures.

Characteristics of Cold Adapted Enzyme and Its Comparison withMesophilic and Thermophilic Counterpart

The large proportion of earth’s biosphere (>70%) has cold environments in the form of ocean depths, glaciers, polar and alpine regions; and are dominated by psychrophiles. The ability of psychrophiles to proliferate in cold habitat is due to its unique capacity to transcribe, translate and synthesize cold-adapted enzymes which catalyses biochemical reactions at low temperature. The most of psychrophilic enzymes optimize their high activity at low temperature at expense of substrate affinity and reduction in free energy barrier of the transition state. Psychrophilic enzyme has optimum activity relatively at lower temperature than mesophilic and thermophilic counterpart. We have observed that there is no definite relation between Km value of psychrophilic, mesophilic and thermophilic enzymes. It has also been observed that psychrophilic enzymes denatured at relatively faster and at lower temperature than mesophilic and thermophilic counterpart. In this review, attempts have been made to compile up to date advances in the field of psychrophilic enzymes and compare its characteristics with mesophilic and thermophilic counterpart.

Structure features of GH10 xylanase from <italic>Caldicellulosiruptor bescii</italic>: implication for its thermophilic adaption and substrate binding preference

Caldicellulosiruptor bescii is the most thermophilic cellulolytic species of organisms known to date. In our previous study, GH10 xylanase CbXyn10B from C. bescii displayed outstanding hydrolytic activity toward various xylans at high temperatures. To understand the structural basis for this protein's catalysis and thermostability, we solved the crystal structures of CbXyn10B and its complexes with xylooligosaccharides. These structural models were used to guide comparison with its mesophilic counterpart PbXyn10B. A distinctive structural feature is that thermophilic CbXyn10B presents a relatively stable interaction between the extended loops L7 and L8 in the catalytic cleft by an extensive hydrogen bonding network, which is mediated by Lys306, Arg314 and three well-ordered water molecules. Moreover, a unique aromatic cluster consisting of Try17, Phe20, Phe21, and Phe337 may enhance the interaction between the N- and C- terminus. Targeted mutagenesis demonstrated that these interactions substantially contribute to enzyme stabilization, as indicated by a considerable decrease in the melting temperature (Tm) of CbXyn10B by substituting critical residues with Ala. Therefore, it was shown that not only the aromatic interaction connecting protein termini but also the extensive hydrogen bonding network formed between surface loops could restrict the local structural flexibility and contribute significantly to the overall stability of enzymes. Furthermore, the xylooligosaccharides were found to tightly bind to the glycone subsites of xylanase, indicating higher affinities at these subsites and reflecting its substrate binding preference. Our results suggest that CbXyn10B is stabilized with distinct rigidity at the catalytic cleft as well as the terminal regions, which provides insights into the evolutionary strategy for accommodating the functional needs of GH10 enzymes to high temperature.

Changes in the membrane fatty acid composition in Anoxybacillus flavithermus subsp. yunnanensis E13T as response to solvent stress.

Anoxybacillus flavithermus subsp. yunnanensis is currently the first species of strictly thermophilic bacteria that is able to tolerate a broad range of solvents. Unlike most of solvent-tolerant mesophilic bacteria, the bacterium does not synthesize unsaturated fatty acids. Our results revealed that in growing cells of A. flavithermus subsp. yunnanensis E13T, ethanol and toluene resulted in an increase in straight-chain fatty acids, mainly C16:0, leading to a more rigid membrane. Moreover, the increase in straight-chain fatty acids caused by ethanol was much higher than that of toluene. High temperature had little effect on the fatty acid composition by itself, whereas the combined conditions of high temperature and ethanol caused the dramatic increase in straight-chain fatty acids (mainly C16:0), that was balanced by decreasing branched fatty acids. The increase was also temperature dependent. The proportion of C16:0 further increased above 60°C. No similar evidence was found in four other species of Anoxybacillus. The results suggested that A. flavithermus subsp. yunnanesis seems to develop a different response to solvents compared to its mesophilic counterparts, which consist of an increase in the saturated straight/branched ratio.

Organic solvent-tolerant, cold-adapted lipases PML and LipS exhibit increased conformational flexibility in polar organic solvents

Organic solvent-tolerant enzymes are generally stable in organic solvents, but the conformational changes of the enzymes in organic solvents have not been well studied. As cold-adapted enzymes exhibit increased conformational flexibility as compared with their mesophilic counterparts, we investigated the effects of polar organic solvents (dimethyl sulfoxide, methanol, and ethanol) on the flexibility and stability relationship in the organic solvent-tolerant, cold-adapted lipases PML and LipS. To our surprise, both PML and LipS exhibited increased conformational flexibility as organic solvent concentration increased. The conformational flexibility required for maximal enzymatic activity differed between PML and LipS, suggesting distinct conformations of PML and LipS in each organic solvent for maximal enzymatic activity. However, greater structural flexibility of PML and LipS in organic solvents was associated with lower thermal denaturation midpoints. Our study suggests that the effects of organic solvents on conformational flexibility are specific to cold-adapted enzymes displaying intrinsic conformational flexibility at low temperatures. Organic solvent-tolerant, cold-adapted enzymes could be used to investigate the flexibility and stability relationship in enzymes in many different organic solvents.

Editorial: Enzymes from Extreme Environments.

Editorial: Enzymes from Extreme Environments.

A novel cold-adapted and highly salt-tolerant esterase from Alkalibacterium sp. SL3 from the sediment of a soda lake.

A novel esterase gene (estSL3) was cloned from the Alkalibacterium sp. SL3, which was isolated from the sediment of soda lake Dabusu. The 636-bp full-length gene encodes a polypeptide of 211 amino acid residues that is closely related with putative GDSL family lipases from Alkalibacterium and Enterococcus. The gene was successfully expressed in E. coli, and the recombinant protein (rEstSL3) was purified to electrophoretic homogeneity and characterized. rEstSL3 exhibited the highest activity towards pNP-acetate and had no activity towards pNP-esters with acyl chains longer than C8. The enzyme was highly cold-adapted, showing an apparent temperature optimum of 30 °C and remaining approximately 70% of the activity at 0 °C. It was active and stable over the pH range from 7 to 10, and highly salt-tolerant up to 5 M NaCl. Moreover, rEstSL3 was strongly resistant to most tested metal ions, chemical reagents, detergents and organic solvents. Amino acid composition analysis indicated that EstSL3 had fewer proline residues, hydrogen bonds and salt bridges than mesophilic and thermophilic counterparts, but more acidic amino acids and less hydrophobic amino acids when compared with other salt-tolerant esterases. The cold active, salt-tolerant and chemical-resistant properties make it a promising enzyme for basic research and industrial applications.

Mechanical Aspects of Protein Thermostability

Single-molecule force-spectroscopies are remarkable tools for studying protein folding and unfolding. However it is legitimate to wonder whether the explored pathways differ from that traditionally explored via chemical or thermal denaturation, especially because the experimental reaction coordinates are very different. Here we address the following questions: how well do proteins that are very stable upon thermal denaturation resist under force? Are the same mechanisms involved for both types of stability? Our paradigm system is a ∼70-residue protein, cold-shock, that is present in thermophilic organisms and that has a very high melting temperature. We compare it to one of its mesophilic counterparts. Both thermal and mechanical stabilities are investigated with all-atom molecular dynamics simulations in explicit solvent. To verify the ability of our models to reproduce the experimental shift in the melting temperature of both protein homologues, we have implemented an enhanced-sampling algorithm (REST2) based on an Hamiltonian exchange scheme, into a publicly available simulation code (NAMD2). This allows us to explore the proteins’ free-energy landscape over a wide temperature range much faster than would be obtained using unperturbed simulations. The proteins mechanical stability is investigated using constant-force steered molecular-dynamics. In agreement with experimental data, we find that the typical unfolding force of the both variants are non-negligible but significantly lower than that of well-characterized mechanically stable proteins that are thermally stable as well. Our results suggest that thermal stability does not guarantee a mechanical one and that the associated mechanisms are very different in nature. Our simulations also aim at identifying the weak-points of the protein mechanical stability and how more stable mutants can be designed; such mutants are then expected to be more stable against thermal denaturation and could prove useful in numerous applications.

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures.

Genome-Scale Modeling of Thermophilic Microorganisms.

Thermophilic microorganisms are of increasing interest for many industries as their enzymes and metabolisms are highly efficient at elevated temperatures. However, their metabolic processes are often largely different from their mesophilic counterparts. These differences can lead to metabolic engineering strategies that are doomed to fail. Genome-scale metabolic modeling is an effective and highly utilized way to investigate cellular phenotypes and to test metabolic engineering strategies. In this review we chronicle a number of thermophilic organisms that have recently been studied with genome-scale models. The microorganisms spread across archaea and bacteria domains, and their study gives insights that can be applied in a broader context than just the species they describe. We end with a perspective on the future development and applications of genome-scale models of thermophilic organisms.

Cloning and In Silico Analysis of a High-Temperature Inducible Lipase from Brevibacillus

The genus Brevibacillus comprises diverse collection of gram-positive/gram-variable, endospore-forming, rod-shaped, aerobic/facultative anaerobic bacteria inhabitant of varied environmental habitats contributing many industrial enzymes. A thermophilic lipolytic bacterium was previously isolated from a hot spring of Orissa, India, and identified as Brevibacillus sp. AK-P2 via 16S rRNA gene technology. A 750-bp lipase gene from Brevibacillus sp. AK-P2 was isolated and sequenced, which encoded a deduced polypeptide of 250 amino acid residues. The comparative analysis of amino acid composition among thermostable and mesostable homologue of Brevibacillus lipases ascertains the role of neutral, charged, and aromatic amino acid residues in thermostability. Three amino acid residues Ser 94, His 242, and Asp 213 were identified from this putative lipase of Brevibacillus sp. AK-P2 as catalytic triad. The consensus ‘P-loop’ motif (-[AG]-X4-G-K-[ST]-) previously reported for Bacillus thermoalkalophilic lipases is modified to (-[AT]-[GE]-X5-G-[RQ]-[S]-) inBrevibacillus lipases. Multiple sequence alignment (MSA) revealed that there is a methionine residue (M) in the oxyanion hole consensus sequence of thermostable Brevibacillus lipases. The frequency of occurrence of AXXXA motif is more for thermophilic Brevibacillus lipases than their mesophilic counterparts ensures strong van der Waals interaction and stabilization of proteins. Higher percentage of Alanine (A) in thermophilic Brevibacillus lipases attributed toward thermostabilization of lipases. Codon usage analysis revealed that there was intermediate codon usage bias in lipase-coding genes, and the same supports our hypothesis that GC mutation pressure might determine codon usage bias.

Thermal Adaptability of the Light-Harvesting Complex 2 from Thermochromatium tepidum: Temperature-Dependent Excitation Transfer Dynamics.

The photosynthetic purple bacterium Thermochromatium (Tch.) tepidum is a thermophile that grows at an optimal temperature of ∼50 °C. We have investigated, by means of steady-state and time-resolved optical spectroscopies, the effects of temperature on the near-infrared light absorption and the excitation energy transfer (EET) dynamics of its light-harvesting complex 2 (LH2), for which the mesophilic counterpart of Rhodobacter (Rba.) sphaeroides 2.4.1 (∼30 °C) was examined in comparison. In a limited range around the physiological temperature (10-55 °C), the B800-to-B850 EET process of the Tch. tepidum LH2, but not the Rba. sphaeroides LH2, was found to be characteristically temperature-dependent, mainly because of a temperature-tunable spectral overlap. At 55 °C, the LH2 complex from Tch. tepidum maintained efficient near-infrared light harvesting and B800-to-B850 EET dynamics, whereas this EET process was disrupted in the case of Rba. sphaeroides 2.4.1 owing to the structural distortion of the LH2 complex. Our results reveal a remarkable thermal adaptability of the light-harvesting function of Tch. tepidum, which could enhance our understanding of the survival strategy of this thermophile in response to environmental challenges.

Thermus thermophilus as a Source of Thermostable Lipolytic Enzymes.

Lipolytic enzymes, esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3), catalyze the hydrolysis of ester bonds between alcohols and carboxylic acids, and its formation in organic media. At present, they represent about 20% of commercialized enzymes for industrial use. Lipolytic enzymes from thermophilic microorganisms are preferred for industrial use to their mesophilic counterparts, mainly due to higher thermostability and resistance to several denaturing agents. However, the production at an industrial scale from the native organisms is technically complicated and expensive. The thermophilic bacterium Thermus thermophilus (T. thermophilus) has high levels of lipolytic activity, and its whole genome has been sequenced. One esterase from the T. thermophilus strain HB27 has been widely characterized, both in its native form and in recombinant forms, being expressed in mesophilic microorganisms. Other putative lipases/esterases annotated in the T. thermophilus genome have been explored and will also be reviewed in this paper.

A theoretical method to compute sequence dependent configurational properties in charged polymers and proteins.

A general formalism to compute configurational properties of proteins and other heteropolymers with an arbitrary sequence of charges and non-uniform excluded volume interaction is presented. A variational approach is utilized to predict average distance between any two monomers in the chain. The presented analytical model, for the first time, explicitly incorporates the role of sequence charge distribution to determine relative sizes between two sequences that vary not only in total charge composition but also in charge decoration (even when charge composition is fixed). Furthermore, the formalism is general enough to allow variation in excluded volume interactions between two monomers. Model predictions are benchmarked against the all-atom Monte Carlo studies of Das and Pappu [Proc. Natl. Acad. Sci. U. S. A. 110, 13392 (2013)] for 30 different synthetic sequences of polyampholytes. These sequences possess an equal number of glutamic acid (E) and lysine (K) residues but differ in the patterning within the sequence. Without any fit parameter, the model captures the strong sequence dependence of the simulated values of the radius of gyration with a correlation coefficient of R(2) = 0.9. The model is then applied to real proteins to compare the unfolded state dimensions of 540 orthologous pairs of thermophilic and mesophilic proteins. The excluded volume parameters are assumed similar under denatured conditions, and only electrostatic effects encoded in the sequence are accounted for. With these assumptions, thermophilic proteins are found-with high statistical significance-to have more compact disordered ensemble compared to their mesophilic counterparts. The method presented here, due to its analytical nature, is capable of making such high throughput analysis of multiple proteins and will have broad applications in proteomic studies as well as in other heteropolymeric systems.