Underlying features for the enhanced electrostatic strength of the extremophilic malate dehydrogenase interface salt-bridge compared to the mesophilic one.

Malate dehydrogenase (MDH) exists in multimeric form in normal and extreme solvent conditions where residues of the interface are involved in specific interactions. The interface salt-bridge (ISB) and its microenvironment (ME) residues may have a crucial role in the stability and specificity of the interface. To gain insight into this, we have analyzed 218 ISBs from 42 interfaces of 15 crystal structures along with their sequences. Comparative analyses demonstrate that the ISB strength is ∼30 times greater in extremophilic cases than that of the normal one. To this end, the interface residue propensity, ISB design and pair selection, and ME-residue's types, i.e., type-I and type-II, are seen to be intrinsically involved. Although Type-I is a common type, Type-II appears to be extremophile-specific, where the net ME-residue count is much lower with an excessive net ME-energy contribution, which seems to be a novel interface compaction strategy. Furthermore, the interface strength can be enhanced by selecting the desired mutant from the net-energy profile of all possible mutations of an unfavorable ME-residue. The study that applies to other similar systems finds applications in protein-protein interaction and protein engineering.Communicated by Ramaswamy H. Sarma.

Intrinsic basis of thermostability of prolyl oligopeptidase from Pyrococcus furiosus.

Salt-bridges play a key role in the thermostability of proteins adapted in stress environments whose intrinsic basis remains to be understood. We find that the higher hydrophilicity of PfP than that of HuP is due to the charged but not the polar residues. The primary role of these residues is to enhance the salt-bridges and their ME. Unlike HuP, PfP has made many changes in its intrinsic property to strengthen the salt-bridge. First, the desolvation energy is reduced by directing the salt-bridge towards the surface. Second, it has made bridge-energy more favorable by recruiting energetically advantageous partners with high helix-propensity among the six possible salt-bridge pairs. Third, ME-residues that perform intricate interactions have increased their energy contribution by making major changes in their binary properties. The use of salt-bridge partners as ME-residues, and ME-residues' overlapping usage, predominant in helices, and energetically favorable substitution are some of the favorable features of PfP compared to HuP. These changes in PfP reduce the unfavorable, increase the favorable ME-energy. Thus, the per salt-bridge stability of PfP is greater than that of HuP. Further, unfavorable target ME-residues can be identified whose mutation can increase the stability of salt-bridge. The study applies to other similar systems.

Insights from the salt bridge analysis of malate dehydrogenase from H. salinarum and E.coli.

Halophilic proteins have greater abundance of acidic over basic residues in sequence. In structure, the surface is decorated by negative charges, with lower content of Lysine. Using sequence BLOCKs and 3D model of malate dehydrogenase from halophilic archaea (Halobacterium salinarum; hsMDH) and X-ray structure from mesophilic bacteria (E. coli; ecMDH), we show that not only acidic and basic residues have higher mean relative abundance (MRA) and thus, impart higher polarity to the sequences, but also show their presence in the surface of the structure of hsMDH relative to its mesophilic counterpart. These observations may indicate that both the acidic and the basic residues have a concerted role in the stability of hsMDH. Analysis on salt bridges from hsMDH and ecMDH show that in the former, salt bridges are highly intricate, newly engineered and global in nature. Although, these salt bridges are abundant in hsMDH, in the active site the design remains unperturbed. In high salt where hydrophobic force is weak, these salt bridges seem to play a major role in the haloadaptation of the tertiary structure of hsMDH. This is the first report of such an observation.

Stability of buried and networked salt-bridges (BNSB)in thermophilic proteins.

Thermophilic proteins function at high temperature, unlike mesophilic proteins. Thermo-stability of these proteins is due to the unique buried and networked salt-bridge (BNSB). However, it is known that the desolvation cost of BNSB is too high compared to other favorable energy terms. Nonetheless, it is known that stability is provided generally by hydrophobic isosteres without the need for BNSB. We show in this analysis that the BNSB is the optimal evolutionary design of salt-bridge to offset desolvation cost efficiently. Hence, thermophilic proteins with a high level of BNSB provide thermo-stability.

Analysis of salt-bridges in prolyl oligopeptidase from Pyrococcus furiosus and Homo sapiens.

Hyper thermophilic archaea not only tolerate high temperature but also operate its biochemical machineries, normally under these conditions. However, the structural signatures in proteins that answer for the hyper thermo-stability relative to its mesophilic homologue remains poorly understood. We present comparative analyses of sequences, structures and salt-bridges of prolyl-oligopeptidase from Pyrococcus furiosus (pfPOP - PDB ID: 5T88) and human (huPOP - PDB ID: 3DDU). A similar level of hydrophobic and hydrophilic residues in pfPOP and huPOP is observed. A low level of interactions between shell-waters and atom-types in pfPOP indicated hyper thermophilic features are negligible. Salt-bridge-forming-residues (sbfrs) are high in pfPOP's core and surface (pfPOP). Increased sbfrs largely indicate specific-electrostatic is important for thermo stability in pfPOP. Four classes of sbfrs are found namely positionally non-conservative (PNCS), conservative (PCS), unchanged (PU) and interchanged (PIC) type of substitutions. PNCS-sbfrs constitutes 28% and it is associated with the topology of pfPOP at high temperature. PCS helps to increase the salt-bridge population. It is also found that PU maintains similar salt-bridges at the active site and other binding sites while PIC abolishes mesophilic patterns.

PROPAB: Computation of Propensities and Other Properties from Segments of 3D structure of Proteins.

Residues in allelic positions, in the local segment of aligned sequences of proteins show wide variations. Here, we describe PROPAB that computes the propensity tables for helix, strand and coil types from multiple 3D structure files following ab initio statistical procedure. It also classifies them in range specific and chain specific manners. It further computes percentage composition and physicochemical properties along with residues propensities. It also prepares FASTA files for different segments (helix, strand and coil) in the exact order that they follow in the sequence. Representative analyses on orthologous (homologous across species) proteins demonstrate wide segmental variations of physicochemical properties. Such variations provide insights to relate the adaptation of these proteins in a given functional constraint under diverse environmental conditions. Thus, the program finds applications in the structural and evolutionary analysis of proteins. AVAILABILITY: PROPAB is freely available at http://sourceforge.net/projects/propab/for worldwide user.

POWAINDv1.0: A Program for Protein-Water Interactions Determination.

Protein is the most exposed biomolecule in the aqueous environment of the cell. Its structure maintains a delicate balance between the rigidity and the flexibility that imparts binding specificity to its substrate/ligand, etc. Intramolecular interactions of polar and non-polar groups of amino acid residues and intermolecular weak interactions between these groups and shell-waters may contribute to the overall stability of the tertiary structure. However, the question as to what are the dynamics of interactions of shell-water with respect to weak forces and atom-groups of protein (AGP), requires systematic investigations. In this end, we have developed a procedure POWAINDv1.0 that analyzes interactions of crystallographic shell-waters (CSH) in residues and AGP specific manner. The shell-water and AGP specific bridge-interactions are also extracted. Further, the program analyzes favorable and unfavorable nature of each interaction based on the actual and 75% of the sum of van der Waals (vdW) radii of interacting atoms. The EXCEL-outputs are useful in understanding the profile for AGP-CSH interactions and contribution of each component in AGP. Taken together, the program provides intricate details on CSHprotein interactions and finds application in the structural Bioinformatics.

Substitutional Analysis of Orthologous Protein Families Using BLOCKS.

Orthologous proteins, form due to divergence of parental sequence, perform similar function under different environmental and biological conditions. Amino acid changes at locus specific positions form hetero-pairs whose role in BLOCK evolution is yet to be understood. We involve eight protein BLOCKs of known divergence rate to gain insight into the role of hetero-pairs in evolution. Our procedure APBEST uses BLOCK-FASTA file to extract BLOCK specific evolutionary parameters such as dominantly used hetero-pair (D), usage of hetero-pairs (E), non-conservative to conservative substitution ratio (R), maximally-diverse residue (MDR), residue (RD) and class (CD) specific diversity. All these parameters show BLOCK specific variation. Conservative nature of D points towards restoration of function of BLOCK. While E sets the upper-limit of usage of hereto-pairs, strong correlation of R with divergence-rate indicates that the later is directly dependent on non-conservative substitutions. The observation that MDR, measure of positional diversity, occupy very limited positions in BLOCK indicates accommodation of diversity is positionally restricted. Overall, the study extract observed hetero-pair related quantitative and multi-parametric details of BLOCK, which finds application in evolutionary biology.

ADSBET2: Automated Determination of Salt-Bridge Energy-Terms version 2.

UNLABELLED: Component (bridge: ΔΔGbrd , background: ΔΔGprot , desolvation: ΔΔGdsolv ) and net (ΔΔGnet ) energy-terms of salt-bridge-structure (SBS) are auto-generated by the program ADSBET that makes use of general purpose Adaptive Poison Boltzmann Solver (APBS) method. While the procedure reports gross energy terms (Kcal Mol(-1) ), report on bond-multiplicity corrected normalized energyterms (Kcal Mol(-1) Bond(-1) ) along with their accessibility (ASA) in monomer, isolated-SBS (ISBS) and networked-SBS (NSBS) format would be very useful for statistical comparison among SBSs and understanding their location in protein structure. In this end, ADSBET2 potentially incorporates these features along with additional model for side-chain. Gross and normalized energy-terms are redirected in monomer, ISBS and NSBS format along with their ASA informations. It works on any number of SBSs for any number of structure files present in a database. Taken together, ADSBET2 has been suitable for statistical analyses of SBSs energetics and finds applications in protein engineering and structural bioinformatics. AVAILABILITY: ADSBET2 is freely available at http://sourceforge.net/projects/ADSBET2/ for all users.

PHYSICO2: an UNIX based standalone procedure for computation of physicochemical, window-dependent and substitution based evolutionary properties of protein sequences along with automated block preparation tool, version 2.

UNLABELLED: Automated genome sequencing procedure is enriching the sequence database very fast. To achieve a balance between the entry of sequences in the database and their analyses, efficient software is required. In this end PHYSICO2, compare to earlier PHYSICO and other public domain tools, is most efficient in that it i] extracts physicochemical, window-dependent and homologousposition-based-substitution (PWS) properties including positional and BLOCK-specific diversity and conservation, ii] provides users with optional-flexibility in setting relevant input-parameters, iii] helps users to prepare BLOCK-FASTA-file by the use of Automated Block Preparation Tool of the program, iv] performs fast, accurate and user-friendly analyses and v] redirects itemized outputs in excel format along with detailed methodology. The program package contains documentation describing application of methods. Overall the program acts as efficient PWS-analyzer and finds application in sequence-bioinformatics. AVAILABILITY: PHYSICO2: is freely available at http://sourceforge.net/projects/physico2/ along with its documentation at https://sourceforge.net/projects/physico2/files/Documentation.pdf/download for all users.

SBION: A Program for Analyses of Salt-Bridges from Multiple Structure Files.

UNLABELLED: Salt-bridge and network salt-bridge are specific electrostatic interactions that contribute to the overall stability of proteins. In hierarchical protein folding model, these interactions play crucial role in nucleation process. The advent and growth of protein structure database and its availability in public domain made an urgent need for context dependent rapid analysis of salt-bridges. While these analyses on single protein is cumbersome and time-consuming, batch analyses need efficient software for rapid topological scan of a large number of protein for extracting details on (i) fraction of salt-bridge residues (acidic and basic). (ii) Chain specific intra-molecular salt-bridges, (iii) inter-molecular salt-bridges (protein-protein interactions) in all possible binary combinations (iv) network salt-bridges and (v) secondary structure distribution of salt-bridge residues. To the best of our knowledge, such efficient software is not available in public domain. At this juncture, we have developed a program i.e. SBION which can perform all the above mentioned computations for any number of protein with any number of chain at any given distance of ion-pair. It is highly efficient, fast, error-free and user friendly. Finally we would say that our SBION indeed possesses potential for applications in the field of structural and comparative bioinformatics studies. AVAILABILITY: SBION is freely available for non-commercial/academic institutions on formal request to the corresponding author (akbanerjee@biotech.buruniv.ac.in).

PHYSICO: An UNIX based Standalone Procedure for Computation of Individual and Group Properties of Protein Sequences.

UNLABELLED: In the genomic and proteomic era, efficient and automated analyses of sequence properties of protein have become an important task in bioinformatics. There are general public licensed (GPL) software tools to perform a part of the job. However, computations of mean properties of large number of orthologous sequences are not possible from the above mentioned GPL sets. Further, there is no GPL software or server which can calculate window dependent sequence properties for a large number of sequences in a single run. With a view to overcome above limitations, we have developed a standalone procedure i.e. PHYSICO, which performs various stages of computation in a single run based on the type of input provided either in RAW-FASTA or BLOCK-FASTA format and makes excel output for: a) Composition, Class composition, Mean molecular weight, Isoelectic point, Aliphatic index and GRAVY, b) column based compositions, variability and difference matrix, c) 25 kinds of window dependent sequence properties. The program is fast, efficient, error free and user friendly. Calculation of mean and standard deviation of homologous sequences sets, for comparison purpose when relevant, is another attribute of the program; a property seldom seen in existing GPL softwares. AVAILABILITY: PHYSICO is freely available for non-commercial/academic user in formal request to the corresponding author akbanerjee@biotech.buruniv.ac.in.