Bharat Madan

In silico Study on the Effect of Surface Lysines and Arginines on the Electrostatic Interactions and Protein Stability

Charged amino acids are mostly exposed on a protein surface, thereby forming a network of interactions with the surrounding amino acids as well as with water. In particular, positively charged arginine and lysine have different side chain geometries and provide a different number of potential electrostatic interactions. This study reports a comparative analysis of the difference in the number of two representative electrostatic interactions, such as salt-bridges and hydrogen bonds, contributed by surface arginine and lysine, as well as their effect on protein stability using molecular modeling and dynamics simulation techniques. Two in silico variants, the R variant with all arginines and the K variant with all lysines on the protein surface, were modeled by mutating all the surface lysines to arginines and the surface arginines to lysines, respectively, for each of the 10 model proteins. A structural comparison of the respective two variants showed that the majority of R variants possessed more salt-bridges and hydrogen bond interactions than the K variants, indicating that arginine provides a higher probability of electrostatic interactions than lysine owing to its side chain geometry. Molecular dynamics simulations of these variants revealed the R variants to be more stable than the K variants at room temperature but this effect was not prominent under protein denaturating conditions, such as 353 and 333 K with 8 M urea. These results suggest that the arginine residues on a protein surface contribute to the protein stability slightly more than lysine by enhancing the electrostatic interactions.

Structural and sequence features of two residue turns in beta-hairpins.

Beta‐turns in beta‐hairpins have been implicated as important sites in protein folding. In particular, two residue β‐turns, the most abundant connecting elements in beta‐hairpins, have been a major target for engineering protein stability and folding. In this study, we attempted to investigate and update the structural and sequence properties of two residue turns in beta‐hairpins with a large data set. For this, 3977 beta‐turns were extracted from 2394 nonhomologous protein chains and analyzed. First, the distribution, dihedral angles and twists of two residue turn types were determined, and compared with previous data. The trend of turn type occurrence and most structural features of the turn types were similar to previous results, but for the first time Type II turns in beta‐hairpins were identified. Second, sequence motifs for the turn types were devised based on amino acid positional potentials of two‐residue turns, and their distributions were examined. From this study, we could identify code‐like sequence motifs for the two residue beta‐turn types. Finally, structural and sequence properties of beta‐strands in the beta‐hairpins were analyzed, which revealed that the beta‐strands showed no specific sequence and structural patterns for turn types. The analytical results in this study are expected to be a reference in the engineering or design of beta‐hairpin turn structures and sequences. Proteins 2014; 82:1721–1733.

A comparative study on the stability and structure of two different green fluorescent proteins in organic co-solvent systems

Green fluorescent protein (GFP) has been used as a reporter marker in a wide range of biological and bioengineering studies. The expanded use of GFP in the field of biosensors, biochips and bio-conjugations requires the stability of GFP in organic co-solvent systems. This prompted us to examine the kinetic stability of two different GFP sequences, n-GFP and s-GFP, showing different folding robustness and thermodynamic stability, under a range of organic co-solvent systems. n-GFP and s-GFP are variants whose biophysical properties are comparable to wild type and super folder GFPs, respectively. The stability of n-GFP and s-GFP in 50% water-miscible organic solvents showed that s-GFP with higher thermodynamic stability exhibited much higher stability against organic solvents than n-GFP, which has lower thermodynamic stability. s-GFP was quite stable even in 90% organic solvents. Circular dichroism analysis confirmed that s-GFP maintained its native structure in organic co-solvent systems, whereas n-GFP showed structural variations under these conditions. Four highly fluctuating loop regions were identified from molecular dynamic simulations under the organic cosolvent conditions. A structural comparison of n-GFP and s-GFP suggested that the improved kinetic stability of s-GFP was due to its larger number of hydrogen bonds and salt-bridges that were present in four loop regions. This study suggests that thermodynamically stable s-GFP can be a good choice for use under harsh organic co-solvent conditions.

NADH-dependent Lactate Dehydrogenase from Alcaligenes eutrophus H16 Reduces 2-oxoadipate to 2-hydroxyadipate

Adipic acid is an important monomer for the production of nylon-6,6 polyamide. One novel biological route for the synthesis of adipic acid, which combines the lysine synthetic pathway and glutaconic acid production pathway, has been suggested, but this route has suffered from the lack of an efficient 2-oxoadipate reductase connecting the two pathways or converting 2-oxoadipate to 2-hydroxyadipate. In this study, we report that the lactate dehydrogenase of Alcaligenes eutrophus H16 is a promising catalyst for this reaction. The lactate dehydrogenase gene (Ae-ldhO) was cloned, expressed in Escherichia coli, purified, and characterized. The recombinant enzyme, having a molecular weight of 36.7 kDa, exhibited broad substrate specificity for various 2-oxoacids. NADH was the preferred coenzyme over NADPH for all 2-oxoacids tested. The maximum specific activity of Ae-LdhO on 2-oxoadipate was 454.5 ± 20.1 U/mg protein at pH 7.0 and 30℃. The Km values for 2-oxoadipic acid and NADH were 0.32 ± 0.02 and 0.09 ± 0.002 mM, respectively. The activity of Ae-LdhO was enhanced in the presence of some metal ions, such as Mg2+, Co2+ or Ni2+, whereas it was completely inhibited by Hg2+, Ag+, Cu2+ and DTT.

Deciphering the factors responsible for the stability of a GFP variant resistant to alkaline pH using molecular dynamics simulations

Charged amino acids having ionizable side chains play crucial roles in maintaining the solubility and stability of a protein. These charged amino acids are mostly exposed on protein surface and participate in electrostatic interactions with neighboring charged amino acids as well as with solvent. Therefore, the change in the solvent pH affects the protein stability in most cases. Previously, we reported a GFP variant, GFP14R having 14 surface lysines replaced with arginines, that showed enhanced stability under alkaline pH. Here, we analyzed the factors that contribute to the stability of the GFP14R under alkaline pH quantitatively using molecular dynamics simulations. Protonation state of the charged amino acids of GFP14R and control GFP under neutral pH and alkaline pH were modeled, and molecular dynamics simulations were performed. This comparative analysis revealed that the GFP14R with more arginine frequency on the surface maintained the stability under both pH conditions without much change in their salt-bridge interactions as well as the hydrogen bond interactions with solvent. On the other hand, these interactions were significantly reduced for the control GFP under alkaline pH due to the deprotonated lysine side chains. These results suggest that the advantageous property of arginine over lysine can be considered one of the parameter for the protein stability engineering under alkaline pH conditions.

Measuring the Conformational Distance of GPCR-related Proteins Using a Joint-based Descriptor

Joint-based descriptor is a new level of macroscopic descriptor for protein structure using joints of secondary structures as a basic element. Here, we propose how the joint-based descriptor can be applied to examine the conformational distances or differences of transmembrane (TM) proteins. Specifically, we performed three independent studies that measured the global and conformational distances between GPCR A family and its related structures. First, the conformational distances of GPCR A family and other 7TM proteins were evaluated. This provided the information on the distant and close families or superfamilies to GPCR A family and permitted the identification of conserved local conformations. Second, computational models of GPCR A family proteins were validated, which enabled us to estimate how much they reproduce the native conformation of GPCR A proteins at global and local conformational level. Finally, the conformational distances between active and inactive states of GPCR proteins were estimated, which identified the difference of local conformation. The proposed macroscopic joint-based approach is expected to allow us to investigate structural features, evolutionary relationships, computational models and conformational changes of TM proteins in a more simplistic manner.

Sequence and Structural Features of Subsite Residues in GH10 and GH11 Xylanases

Xylanases are the enzymes that breakdown complex plant cell wall polysaccharide xylan into xylose by hydrolysing the β-(1→4) glycosidic linkage between xylosides. They mainly belong to the families GH10 and GH11 of the glycoside hydrolase claβs of enzymes. GH10 xylanases have (α/β)8-barrel type of fold whereas GH11 xylanases have β-jelly roll type of fold. Both enzymes have several substrate binding subsites. This study analysed in detail the sequence and structural conservation of subsites residues by examining their 3D structures crystallized with homoxylan or its non-hydrolysable form as substrate. A total of 19 structures from GH10 and 6 structures from GH11 were analysed. It was found that in GH10 the subsites -3 to -1 consisted of conserved residues, whereas in GH11 subsites -1, -3 and +1 were found to be conserved. The substrate and subsite interaction analysed based on the presence of h-bonds and CH-π interactions showed that Face-to-Face or Edge-to-Face CH-π interactions are formed in the subsites of GH10, whereas such specific CH-π interactions were no at all observed in case of GH11 xylanases. The spatial conservation of subsite residues was also analysed using a distance matrix based approach. It was found that in GH10 xylanases conserved residues have conserved spatial position of those residues as opposed to GH11 enzymes where in subsites -2 and +2 conserved residues showed non-conservation in their spatial positions. The results presented in this study can be used in discovering new xylanases and in the engineering highly efficient xylanases.

Modulation of intracellular protein activity at level of protein folding by beta-turn engineering

Control of the intracellular protein activity is very important in various biological studies and biotechnology. This has generally been achieved at the transcription and translation levels. Although control of the intracellular activity at the protein folding level is conceptually possible, but there have been few studies. The present study examined this possibility by modulating the in vivo protein folding rate of green fluorescence protein (GFP) through beta-turn engineering. A type II’ two residue beta-turn in GFP was targeted to generate two sets of mutants. First, a switch-off mutant was designed to stop the protein activity completely. The modulation mutants were then constructed to change the rates of GFP folding. The design of mutants was based on the rationale that residues i+1 and i+2 of a beta-turn have defined residue preferences, and their perturbation affects the rate of protein folding. The in vivo fluorescence activity of the designed GFP variants was switched off and modulated as expected. The change in the in vivo folding patterns of the mutants was confirmed by SDS-PAGE and found to be similar to the intracellular fluorescence activities of the mutants. The in vitro refolding kinetics performed with purified variants showed correlations with the in vivo folding patterns. These results showed that the beta-turns in a protein can be a target for modulating the in vivo protein folding pattern and activity.