Surface expansion is independent of and occurs faster than core solvation during the unfolding of barstar

The denaturant-induced unfolding kinetics of the 89-residue protein, barstar, have been examined using fluorescence resonance energy transfer (FRET) at 25 degrees C and pH 8.0. The core tryptophan, Trp53, in barstar serves as a fluorescence donor, and a thionitrobenzoic acid moiety (TNB) attached to a cysteine residue acts as an acceptor to form an efficient FRET pair. Four different single-cysteine containing mutants of barstar with cysteine residues at positions 25, 40, 62, and 82 were studied. The unfolding kinetics of the four mutant forms of barstar were monitored by measurement of the changes in the fluorescence intensity of Trp53 in the unlabeled and TNB-labeled proteins. The rate of change of fluorescence of the single-tryptophan residue, Trp53, in the unlabeled protein, where no FRET occurs, yields the rate of solvation of the core. This rate is similar for all four unlabeled proteins. The rate of the increase in the fluorescence of Trp53 in the labeled protein, where FRET from the tryptophan to the TNB label occurs, yields the rate of decrease in FRET efficiency during unfolding. The decrease in FRET efficiency for proteins labeled at either of the two buried positions (Cys40 or Cys82) occurs at a rate similar to the rate of core solvation. The decrease in FRET efficiency for the acceptor at Cys40 is also shown to be sensitive to the isomerization of the Tyr47-Pro48 cis bond. For the proteins where the label is at a solvent-exposed position (Cys25 and Cys62), the decrease in FRET efficiency occurs in two kinetic phases; 15-25% of the FRET efficiency decreases in the faster phase, and the remaining FRET efficiency decreases in a slower phase, the rate of which is the same as the rate of core solvation. These results clearly indicate that, during unfolding, the protein surface expands faster than, and independently of, water intrusion into the core.     

The effect of mono‐amino acids on larval settlement of the barnacle,Balanus amphitrite Darwin

Settlement of barnacle larvae is believed to be induced by the chemical cues present in their surrounding environment. Here, an investigation was carried out on the effects of sixteen different mono‐amino acids with acidic, basic, uncharged polar and nonpolar side chains, and GABA on larval settlement of the barnacle, Balanus amphitrite. Settlement inducing activity by nine mono‐amino acids, viz. asparagine, glutamine, tyrosine, serine, glycine, tryptophan, leucine, isoleucine and valine (but not phenylalanine) with uncharged polar and nonpolar side chains was observed. Of these, the most active mono‐amino acids were serine, leucine and isoleucine, which were effective at a threshhold of 1.0 × 10^‐7 M. On the other hand, aspartic acid, glutamic acid, GABA, and the basic mono‐amino acids lysine, arginine and histidine did not have any inducing effect. These results suggest that uncharged polar and non‐polar end group of the amino acid chain play an important role in inducing the settlement process in cyprids.     

Characterization of the folding and unfolding reactions of single-chain monellin: evidence for multiple intermediates and competing pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.     

HX-ESI-MS and optical studies of the unfolding of thioredoxin indicate stabilization of a partially unfolded, aggregation-competent intermediate at low pH

Hydrogen exchange monitored by mass spectrometry (HX-MS), in conjunction with multiple optical probes, has been used to characterize the unfolding of thioredoxin. Equilibrium and kinetic studies have been carried out at pH 7 and 3. The HX-MS measurements are shown to be capable of distinguishing between native (N) and unfolded (U) protein molecules when both are present together, and their application in kinetic experiments allows the unfolding reaction to be delineated from the proline isomerization reaction to which it is coupled. At pH 7, equilibrium unfolding studies monitored by three optical probes, intrinsic fluorescence at 368 nm, ellipticity at 222 nm, and ellipticity at 270 nm, as well as by HX-MS, indicate that no intermediate is populated at pH 7, the unfolding reaction is slower than the proline isomerization reaction that follows it, and the three optical probes yield identical kinetics for unfolding, which occurs in a single kinetic phase. The fractional change in any of the three optical signals at any time of unfolding predicts the fraction of the molecules that have become U, as determined by HX-MS. Hence, unfolding at pH 7 appears to occur via a two-state N <==> U mechanism. In contrast at pH 3, HX-MS as well as optical measurements indicate that an unfolding intermediate is stabilized and hence accumulates in equilibrium with N and U, at concentrations of denaturant that define the transition zone of the equilibrium unfolding curve. The intermediate has lost the near-UV signal characteristic of N and possesses fewer amide hydrogen sites that are stable to exchange than does N. Kinetic experiments at pH 3, where unfolding is much faster than proline isomerization, show that more than one intermediate accumulates transiently during unfolding. Thus, the unfolding of thioredoxin occurs via an N <==> I <==> U mechanism, where I is a partially unfolded intermediate that is stabilized and hence populated at pH 3 but not at pH 7. It is shown that transient aggregation of this intermediate results in a deceleration of the kinetics of unfolding at high protein concentrations at pH 3 but not at pH 7.     

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods--spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.     

Equilibrium unfolding studies of monellin: the double-chain variant appears to be more stable than the single-chain variant

To improve our understanding of the contributions of different stabilizing interactions to protein stability, including that of residual structure in the unfolded state, the small sweet protein monellin has been studied in both its two variant forms, the naturally occurring double-chain variant (dcMN) and the artificially created single-chain variant (scMN). Equilibrium guanidine hydrochloride-induced unfolding studies at pH 7 show that the standard free energy of unfolding, ΔG°(U), of dcMN to unfolded chains A and B and its dependence on guanidine hydrochloride (GdnHCl) concentration are both independent of protein concentration, while the midpoint of unfolding has an exponential dependence on protein concentration. Hence, the unfolding of dcMN like that of scMN can be described as two-state unfolding. The free energy of dissociation, ΔG°(d), of the two free chains, A and B, from dcMN, as measured by equilibrium binding studies, is significantly lower than ΔG°(U), apparently because of the presence of residual structure in free chain B. The value of ΔG°(U), at the standard concentration of 1 M, is found to be ～5.5 kcal mol(-1) higher for dcMN than for scMN in the range from pH 4 to 9, over which unfolding appears to be two-state. Hence, dcMN appears to be more stable than scMN. It seems that unfolded scMN is stabilized by residual structure that is absent in unfolded dcMN and/or that native scMN is destabilized by strain that is relieved in native dcMN. The value of ΔG°(U) for both protein variants decreases with an increase in pH from 4 to 9, apparently because of the thermodynamic coupling of unfolding to the protonation of a buried carboxylate side chain whose pK(a) shifts from 4.5 in the unfolded state to 9 in the native state. Finally, it is shown that although the thermodynamic stabilities of dcMN and scMN are very different, their kinetic stabilities with respect to unfolding in GdnHCl are very similar.     

Fluoroalcohol-induced modulation of the pathway of amyloid protofibril formation by barstar

To understand how the conformational heterogeneity of protofibrils formed by any protein, as well as the mechanisms of their formation, are modulated by a change in aggregation conditions, we studied the formation of amyloid protofibrils by barstar at low pH by multiple structural probes in the presence of hexafluoroisopropanol (HFIP). In the presence of 10% HFIP, aggregation proceeds with the transient formation of spherical oligomers and leads to the formation of both protofibrils and fibrils. Curly short protofibrils and fibrils are seen to form early during the aggregation reaction, and both are seen to grow gradually in length during the course of the reaction. Atomic force microscopy images reveal that the HFIP-induced protofibrils are long (～300 nm in length), curly, and beaded and appear to be composed primarily of β-sheet bilayers, with heights of ～2.4 nm. The protofibrils formed in the presence of HFIP differ in both their structures and their stabilities from the protofibrils formed either in the absence of alcohol or in the presence of a related alcohol, trifluoroethanol (TFE). Aggregation appears to proceed via an isodesmic polymerization mechanism. Internal structure in the growing aggregates changes in two stages during protofibril formation. In the first stage, an α-helix-rich oligomeric intermediate is formed. In the second stage, the level of β-sheet structure increases at the expense of some α-helical structure. The second stage itself appears to occur in two distinct steps. The creation of thioflavin T binding sites occurs concomitantly with aggregate elongation and is seen to precede the change in secondary structure. The long straight fibrils with characteristic heights of 8-10 nm, which form in the course of the HFIP-induced aggregation reaction, have not been observed to form either in the absence of alcohol or in the presence of TFE.     

Induction,resuscitation and quantitative real-time polymerase chain reaction analyses of viable but nonculturable Vibrio vulnificus in artificial sea water

Vibrio vulnificus, an important food-borne pathogen, is known to enter viable but nonculturable (VBNC) state under low temperature and low nutrition stress conditions. Present study examined the time required for induction of VBNC state and temperature which induces resuscitation of V. vulnificus YJ016. The change in cell morphology and gene expression during VBNC state and in resuscitated cells was also examined. V. vulnificus incubated in artificial sea water at 4 °C entered VBNC state after considerably extended time (70 days). An increase in temperature by 6 °C from the VBNC induction temperature (4 °C) resulted in resuscitation of VBNC cells; however, maximum resuscitation was observed when VBNC cells were held at 23 °C for 24 h. VBNC cells changed their morphology from comma shape to coccoid shape. Two rounds of induction of VBNC and resuscitation were possible with V. vulnificus cells; however, there was progressive reduction in number of resuscitated cells and after 190 days cells failed to resuscitate. Significant up-regulation of genes related to membrane proteins [porinH (10.4-fold), ompU (2.9-fold)], regulatory proteins [envZ (5.6-fold), toxR (4.5-fold), toxS (4.8-fold)], oxidative stress related protein katG (2.3-fold), cell division/maintenance proteins [ftsZ (4.3), mreB (6.5-fold)] and resuscitating promoter factor yeaZ (fourfold) was observed during resuscitation with respect to VBNC state indicating that these genes play a role during resuscitation. Gene expression data presented here would enhance our understanding of resuscitation of V. vulnificus from VBNC state. The results also highlight the importance of maintenance of low temperature during storage of seafood.