Biomedical subjects

Rajiv Bhat

Publications and source records attributed to Rajiv Bhat.

10 recordsLinked to original sources

PubMed2006

Stabilization of yeast hexokinase A by polyol osmolytes: correlation with the physicochemical properties of aqueous solutions.

Osmolytes of the polyol series are known to accumulate in biological systems under stress and stabilize the structures of a wide variety of proteins. While increased surface tension of aqueous solutions has been considered an important factor in protein stabilization effect, glycerol is an exception, lowering the surface tension of water. To clarify this anomalous effect, the effect of a series of polyols on the thermal stability of a highly thermolabile two domain protein yeast hexokinase A has been investigated by differential scanning calorimetry and by monitoring loss in the biological activity of the enzyme as a function of time. A larger increase in the T(m) of domain 1 compared with that of domain 2, varying linearly with the number of hydroxyl groups in polyols, has been observed, sorbitol being the best stabilizer against both thermal as well as urea denaturation. Polyols help retain the activity of the enzyme considerably and a good correlation of the increase in T(m) (DeltaT(m)) and the retention of activity with the increase in the surface tension of polyol solutions, except glycerol, which breaks this trend, has been observed. However, the DeltaT(m) values show a linear correlation with apparent molal heat capacity and volume of aqueous polyol solutions including glycerol. These results suggest that while bulk solution properties contribute significantly to protein stabilization, interfacial properties are not always a good indicator of the stabilizing effect. A subtle balance of various weak binding and exclusion effects of the osmolytes mediated by water further regulates the stabilizing effect. Understanding these aspects is critical in the rational design of stable protein formulations.

Circular Dichroism↗

PubMed2006

Bose-Einstein condensates in rotating lattices.

Strongly interacting bosons in a two-dimensional rotating square lattice are investigated via a modified Bose-Hubbard Hamiltonian. Such a system corresponds to a rotating lattice potential imprinted on a trapped Bose-Einstein condensate. Second-order quantum phase transitions between states of different symmetries are observed at discrete rotation rates. For the square lattice we study, there are four possible ground-state symmetries.

Journal Article↗

PubMed2005

Efficient refolding of aggregation-prone citrate synthase by polyol osmolytes: how well are protein folding and stability aspects coupled?

Efficient refolding of proteins and prevention of their aggregation during folding are of vital importance in recombinant protein production and in finding cures for several diseases. We have used citrate synthase (CS) as a model to understand the mechanism of aggregation during refolding and its prevention using several known structure-stabilizing cosolvent additives of the polyol series. Interestingly, no parallel correlation between the folding effect and the general stabilizing effect exerted by polyols was observed. Although increasing concentrations of polyols increased protein stability in general, the refolding yields for CS decreased at higher polyol concentrations, with erythritol reducing the folding yields at all concentrations tested. Among the various polyols used, glycerol was the most effective in enhancing the CS refolding yield, and a complete recovery of enzymatic activity was obtained at 7 m glycerol and 10 mug/ml protein, a result superior to the action of the molecular chaperones GroEL and GroES in vitro. A good correlation between the refolding yields and the suppression of protein aggregation by glycerol was observed, with no aggregation detected at 7 m. The polyols prevented the aggregation of CS depending on the number of hydroxyl groups in them. Stopped-flow fluorescence kinetics experiments suggested that polyols, including glycerol, act very early in the refolding process, as no fast and slow phases were detectable. The results conclusively demonstrate that both the thermodynamic and kinetic aspects are critical in the folding process and that all structure-stabilizing molecules need not always help in productive folding to the native state. These findings are important for the rational design of small molecules for efficient refolding of various aggregation-prone proteins of commercial and medical relevance.

Alcohols↗

PubMed2004

Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A.

The earliest folding events in single-tryptophan mutants of RNase A were investigated by fluorescence measurements by using a combination of stopped-flow and continuous-flow mixing experiments covering the time range from 70 micros to 10 s. An ultrarapid double-jump mixing protocol was used to study refolding from an unfolded ensemble containing only native proline isomers. The continuous-flow measurements revealed a series of kinetic events on the submillisecond time scale that account for the burst-phase signal observed in previous stopped-flow experiments. An initial increase in fluorescence within the 70-micros dead time of the continuous-flow experiment is consistent with a relatively nonspecific collapse of the polypeptide chain whereas a subsequent decrease in fluorescence with a time constant of approximately 80 micros is indicative of a more specific structural event. These rapid conformational changes are not observed if RNase A is allowed to equilibrate under denaturing conditions, resulting in formation of nonnative proline isomers. Thus, contrary to previous expectations, the isomerization state of proline peptide bonds can have a major impact on the structural events during early stages of folding.

Kinetics↗

PubMed2004

Thermal inactivation of protective antigen of Bacillus anthracis and its prevention by polyol osmolytes.

Protective antigen (PA) of Bacillus anthracis is the main immunogen of all anthrax vaccines. It is a highly thermolabile molecule and loses its activity rapidly when exposed to higher temperatures. Earlier some cosolvents had been used to stabilize PA with variable success but no study has been done to find out the primary cause of PA thermal inactivation. This study aims at elucidating the predominant cause of thermal inactivation of PA in order to develop more effective strategies for its thermostabilization. The prime cause for the loss of biological activity of PA at high temperature was its aggregation and an inverse correlation between PA activity and its aggregation on heating was observed. Inactivation of the protein by autolysis did not occur. This paper reports the use of a series of polyol osmolytes to stabilize PA. Different polyols stabilized PA to a different extent against thermal inactivation in a concentration dependent manner, with glycerol stabilizing to the maximum extent. Addition of NaCl to glycerol solution further enhanced the thermal stability of PA. An increase in the T(1/2) value, the temperature at which 50% of the activity is retained during short-term incubation, of more than 20 degrees C was observed. The half-life (t(1/2)) of PA thermal inactivation at 40 degrees C increased by more than 6 times in the presence of the mixture of glycerol and NaCl as compared to control. This study demonstrates for the first time that aggregation of the PA molecule is the predominant cause of its thermal inactivation, and can be very effectively prevented by the use of glycerol and other polyols to increase the shelf life of the recombinant vaccine against anthrax.

Animals↗

PubMed2004

Effect of pH on the stability and structure of yeast hexokinase A. Acidic amino acid residues in the cleft region are critical for the opening and the closing of the structure.

pH and salts have a marked effect on the stability, structure, and function of many globular proteins due to their ability to influence the electrostatic interactions. In this work, calorimetry, CD, and fluorescence studies have been carried out to understand the pH-dependent conformational changes of the two-domain protein yeast hexokinase A. In conjunction with the crystal structural data available, the present results have enabled the complete characterization and analysis of the pH-dependent conformational changes of the enzyme that have strong implications in understanding its structure-function relationship. The calorimetric profiles show a single thermal transition in the acidic pH range, whereas two independent transitions were observed in the alkaline pH range, suggesting the structural merger of the domains at the acidic pH. Comparison of the thermal transitions at pH 8.5 studied by different techniques suggests that the first transition corresponds to the smaller domain, and the second transition corresponds to the larger domain. The acid-denatured state of hexokinase A has high secondary structure content with little or no tertiary interactions and binds to the hydrophobic dye 8-anilinonaphthalene-1-sulfonic acid, suggesting that it is a molten globule-like state, whereas the alkali-denatured state is less structured than the acid-denatured state but more structured than the urea-denatured state, suggestive of a premolten globule-like state. Structural analysis using the published hexokinase B structure as well as the hexokinase A structure with the revised amino acid sequence in conjunction with the results obtained by us suggests that the ionization state of the acidic residues at the active site could regulate domain movements that are responsible for the opening and the closure of the cleft between the two domains and in turn affect the structure and function of the enzyme.

Acids↗

PubMed2004

The osmoprotectants glycine and its methyl derivatives prevent the thermal inactivation of protective antigen of Bacillus anthracis.

Protective antigen (PA) is the main immunogenic constituent of all vaccines against anthrax. It is known to lose its biological activity even at 37 degrees C. Its thermolabile nature has, thus, remained a cause of concern as even transient exposure of the vaccine to higher temperature could compromise its efficacy. Various types of cosolvent excipients have been used to stabilize a number of proteins with variable success. However, no comprehensive and systematic study to stabilize anthrax PA molecule using this approach has ever been undertaken. We have carried out a systematic study on the effect of osmoprotectants like glycine and its methyl derivatives, sarcosine, dimethylglycine, and betaine, on the thermostability of PA. The thermal stability of PA was found to be highly sensitive to pH with maxima at pH 7.9. All the cosolvent additives used were able to enhance the thermal stability of PA as inferred from an increase in T(1/2) values, the temperature at which 50% activity was retained during short-term incubation. Glycine was found to be the best stabilizer, while the ability of its methyl derivatives to stabilize PA decreased with an increase in the number of substituted methyl groups suggesting perturbation of hydrophobic interactions. On extended incubation at 40 degrees C the half-life of PA thermal inactivation increased more than four times in the presence of glycine. Thus, glycine could be used as an effective stabilizer to enhance the shelf life of recombinant vaccine against anthrax.

Animals↗

PubMed2003

Proline isomerization in bovine pancreatic ribonuclease A. 2. Folding conditions.

The kinetics of cis-trans isomerization of individual X-Pro peptide groups is used to study the backbone dynamics of bovine pancreatic ribonuclease A (RNase A). We previously developed and validated a fluorescence method for monitoring the cis-trans isomerization of the Tyr92-Pro93 and Asn113-Pro114 peptide groups of RNase A under unfolding conditions [Juminaga, D., Wedemeyer, W. J., and Scheraga, H. A. (1998) Biochemistry 37, 11614-11620]. The essence of this method is to introduce a fluorescent residue (Tyr or Trp) in a position adjacent to the isomerizing proline (if one is not already present) and to eliminate the fluorescence of other such residues adjacent to prolines by mutating them to phenylalanine. Here, we extend this method to observe the cis-trans isomerization of these peptide groups under folding conditions using two site-directed mutants (Y92F and Y115F) of RNase A. Both isomerizations decelerate with increasing concentrations of GdnHCl, with nearly identical m values (1.11 and 1.19 M(-1), respectively) and extrapolated zero-GdnHCl time constants (42 and 32 s, respectively); by contrast, under unfolding conditions, the cis-trans isomerizations of both Pro93 and Pro114 are independent of GdnHCl concentration. Remarkably, the isomerization rates under folding conditions at GdnHCl concentrations above 1 M are significantly slower than those measured under unfolding conditions. The temperature dependence of the Pro114 isomerization under folding conditions is also unusual; whereas Pro93 exhibits an activation energy typical of proline isomerization (19.4 kcal/mol), Pro114 exhibits a sharply reduced activation energy of 5.7 kcal/mol. A structurally plausible model accounts for these results and, in particular, shows that folding conditions strongly accelerate the cis-trans isomerization of both peptide groups to their native cis conformation, suggesting the presence of flickering local structure in their beta-hairpins.

Animals↗

PubMed2003

Why is trehalose an exceptional protein stabilizer? An analysis of the thermal stability of proteins in the presence of the compatible osmolyte trehalose.

Trehalose, a naturally occurring osmolyte, is known to be an exceptional stabilizer of proteins and helps retain the activity of enzymes in solution as well as in the freeze-dried state. To understand the mechanism of action of trehalose in detail, we have conducted a thorough investigation of its effect on the thermal stability in aqueous solutions of five well characterized proteins differing in their various physico-chemical properties. Among them, RNase A has been used as a model enzyme to investigate the effect of trehalose on the retention of enzymatic activity upon incubation at high temperatures. 2 m trehalose was observed to raise the transition temperature, Tm of RNase A by as much as 18 degrees C and Gibbs free energy by 4.8 kcal mol-1 at pH 2.5. There is a decrease in the heat capacity of protein denaturation (DeltaCp) in trehalose solutions for all the studied proteins. An increase in the DeltaG and a decrease in the DeltaCp values for all the proteins points toward a general mechanism of stabilization due to the elevation and broadening of the stability curve (DeltaG versus T). A direct correlation of the surface tension of trehalose solutions and the thermal stability of various proteins has been observed. Wyman linkage analysis indicates that at 1.5 m concentration 4-7 molecules of trehalose are excluded from the vicinity of protein molecules upon denaturation. We further show that an increase in the stability of proteins in the presence of trehalose depends upon the length of the polypeptide chain. The pH dependence data suggest that even though the charge status of a protein contributes significantly, trehalose can be expected to work as a universal stabilizer of protein conformation due to its exceptional effect on the structure and properties of solvent water compared with other sugars and polyols.

Animals↗

PubMed2002

Gln277 and Phe554 residues are involved in thermal inactivation of protective antigen of Bacillus anthracis.

Protective antigen (PA) is the main component of all the vaccines against anthrax. The currently available vaccines have traces of other proteins that contribute to its reactogenicity. Thus, purified PA is recommended for human vaccination. PA loses its biological activity within 48h at 37 degrees C and its thermolability has been a cause of concern as accidental exposure to higher temperatures during transportation or storage could decrease its efficacy. In the present study, we have used protein engineering approach to increase the thermostability of PA by mutating amino acid residues on the surface as well as the interior of the protein. After screening several mutants, the mutants Gln277Ala and Phe554Ala have been found to be more thermostable than the wild-type PA. Gln277Ala retains approximately 45% and Phe554Ala retains approximately 90% activity, even after incubation at 37 degrees C for 48h while in the same period wild-type PA loses its biological activity completely. It is the first report of increasing thermostability of PA using site-directed mutagenesis. Generation of such mutants could pave the way for better anthrax vaccines with longer shelf life.

Animals↗