Direct evidence for a dry molten globule intermediate during the unfolding of a small protein.
ABSTRACT: Little is known about how proteins begin to unfold. In particular, how and when water molecules penetrate into the protein interior during unfolding, thereby enabling the dissolution of specific structure, is poorly understood. The hypothesis that the native state expands initially into a dry molten globule, in which tight packing interactions are broken, but whose hydrophobic core has not expanded sufficiently to be able to absorb water molecules, has very little experimental support. Here, we report our analysis of the earliest observable events during the unfolding of single chain monellin (MNEI), a small plant protein. Far- and near-UV circular dichroism measurements of GdnHCl-induced unfolding indicate that a molten globule intermediate forms initially, before the major slow unfolding reaction commences. Steady-state fluorescence resonance energy transfer measurements show that the C-terminal end of the single helix of MNEI initially moves rapidly away from the single tryptophan residue that is close to the N-terminal end of the helix. The average end-to-end distance of the protein also expands during unfolding to the molten globule intermediate. At this time, water has yet to penetrate the protein core, according to the evidence from intrinsic tryptophan fluorescence and 8-anilino-1-naphthalenesulfonic acid fluorescence-monitored kinetic unfolding measurements. Our results therefore provide direct evidence for a dry molten globule intermediate at the initial stage of unfolding. Our results further suggest that the structural transition between the native and dry molten globule states could be an all-or-none transition, whereas further swelling of the globule appears to occur gradually.
Project description:Dissecting a protein unfolding process into individual steps can provide valuable information on the forces that maintain the integrity of the folded structure. Solvation of the protein core determines stability, but it is not clear when such solvation occurs during unfolding. In this study, far-UV circular dichroism measurements suggest a simplistic two-state view of the unfolding of barstar, but the use of multiple other probes brings out the complexity of the unfolding reaction. Near-UV circular dichroism measurements show that unfolding commences with the loosening of tertiary interactions in a native-like intermediate, N(∗). Fluorescence resonance energy transfer measurements show that N(∗) then expands rapidly but partially to form an early unfolding intermediate IE. Fluorescence spectral measurements indicate that both N(∗) and IE have retained native-like solvent accessibility of the core, suggesting that they are dry molten globules. Dynamic quenching measurements at the single tryptophan buried in the core suggest that the core becomes solvated only later in a late wet molten globule, IL, which precedes the unfolded form. Fluorescence anisotropy decay measurements show that tight packing around the core tryptophan is lost when IL forms. Of importance, the slowest step is unfolding of the wet molten globule and involves a solvated transition state.
Project description:We investigated the pathway for pressure unfolding of metmyoglobin using molecular dynamics (MD) for a range of pressures (0.1 MPa to 1.2 GPa) and a temperature of 300 K. We find that the unfolding of metmyoglobin proceeds via a two-step mechanism native --> molten globule intermediate --> unfolded, where the molten globule forms at 700 MPa. The simulation describes qualitatively the experimental behavior of metmyoglobin under pressure. We find that unfolding of the alpha-helices follows the sequence of migrating hydrogen bonds (i,i + 4) --> (i,i + 2).
Project description:?-Cardiotoxin is a novel member of the snake venom three-finger toxin (3FTX) family. This is the first exogenous protein to antagonize ?-adrenergic receptors and thereby causing reduction in heart rates (bradycardia) when administered into animals, unlike the conventional cardiotoxins as reported earlier. 3FTXs are stable all ?-sheet peptides with 60-80 amino acid residues. Here, we describe the three-dimensional crystal structure of ?-cardiotoxin together with the identification of a molten globule intermediate in the unfolding pathway of this protein. In spite of the overall structural similarity of this protein with conventional cardiotoxins, there are notable differences observed at the loop region and in the charge distribution on the surface, which are known to be critical for cytolytic activity of cardiotoxins. The molten globule intermediate state present in the thermal unfolding pathway of ?-cardiotoxin was however not observed during the chemical denaturation of the protein. Interestingly, circular dichroism (CD) and NMR studies revealed the presence of ?-helical secondary structure in the molten globule intermediate. These results point to substantial conformational plasticity of ?-cardiotoxin, which might aid the protein in responding to the sometimes conflicting demands of structure, stability, and function during its biological lifetime.
Project description:Although the intrinsic tryptophan fluorescence of proteins offers a convenient probe of protein folding, interpretation of the fluorescence spectrum is often difficult because it is sensitive to both global and local changes. Infrared (IR) spectroscopy offers a complementary measure of structural changes involved in protein folding, because it probes changes in the secondary structure of the protein backbone. Here we demonstrate the advantages of using multiple probes, infrared and fluorescence spectroscopy, to study the folding of the FBP28 WW domain. Laser-induced temperature jumps coupled with fluorescence or infrared spectroscopy have been used to probe changes in the peptide backbone on the submillisecond time scale. The relaxation dynamics of the ?-sheets and ?-turn were measured independently by probing the corresponding IR bands assigned in the amide I region. Using these wavelength-dependent measurements, we observe three kinetics phases, with the fastest process corresponding to the relaxation kinetics of the turns. In contrast, fluorescence measurements of the wild-type WW domain and tryptophan mutants exhibit single-exponential kinetics with a lifetime that corresponds to the slowest phase observed by infrared spectroscopy. Mutant sequences provide evidence of an intermediate dry molten globule state. The slowest step in the folding of this WW domain is the tight packing of the side chains in the transition from the dry molten globule intermediate to the native structure. This study demonstrates that using multiple complementary probes enhances the interpretation of protein folding dynamics.
Project description:Neocarzinostatin is a potent antitumor drug consisting of an enediyne chromophore and a protein carrier.We characterized an intermediate in the equilibrium unfolding pathway of aponeocarzinostatin, using a variety of biophysical techniques including 1-anilino-8-napthalene sulfonate binding studies, size-exclusion fast protein liquid chromatography, intrinsic tryptophan fluorescence, circular dichroism, and 1H-15N heteronuclear single quantum coherence spectroscopy.The partially unfolded protein is in molten globule-like state, in which approximately 60% and approximately 20% tertiary and secondary structure is disrupted respectively. Despite lacking a fully coordinated tertiary structure for assembling a functional binding cleft, the protein in molten globule-like state is still able to fully protect the labile chromophore. Titration of chromophore leads the partially denatured apoprotein to fold into its native state.These findings bring insight into conserving mechanism of neocarzinostatin under harsh environment, where even the partially denatured apoprotein exhibits protective effect, confirming the superiority of the drug carrier.
Project description:Kinetic intermediates that appear early during protein folding often resemble the relatively stable molten globule intermediates formed by several proteins under mildly denaturing conditions. Molten globules have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule that is observed during folding of alpha-beta parallel flavodoxin from Azotobacter vinelandii is a remarkably non-native species. This folding intermediate is helical and contains no beta-sheet and is kinetically off-pathway to the native state. It can be trapped under native-like conditions by substituting residue Phe(44) for Tyr(44). To characterize this species at the residue level, in this study, use is made of interrupted hydrogen/deuterium exchange detected by NMR spectroscopy. In the molten globule of flavodoxin, the helical region comprising residues Leu(110)-Val(125) is shown to be better protected against exchange than the other ordered parts of the folding intermediate. This helical region is better buried than the other helices, causing its context-dependent stabilization against unfolding. Residues Leu(110)-Val(125) thus form the stable core of the helical molten globule of alpha-beta parallel flavodoxin, which is almost entirely structured. Non-native docking of helices in the molten globule of flavodoxin prevents formation of the parallel beta-sheet of native flavodoxin. Hence, to produce native alpha-beta parallel protein molecules, the off-pathway species needs to unfold.
Project description:Solution pH plays an important role in protein dynamics, stability, and folding; however, detailed mechanisms remain poorly understood. Here we use continuous constant pH molecular dynamics in explicit solvent with pH replica exchange to describe the pH profile of the folding cooperativity of a miniprotein BBL, which has drawn intense debate in the past. Our data reconciled the two opposing hypotheses (downhill vs. two-state) and uncovered a sparsely populated unfolding intermediate. As pH is lowered from 7 to 5, the folding barrier vanishes. As pH continues to decrease, the unfolding barrier lowers and denaturation is triggered by the protonation of Asp162, consistent with experimental evidence. Interestingly, unfolding proceeded via an intermediate, with intact secondary structure and a compact, unlocked hydrophobic core shielded from solvent, lending support to the recent hypothesis of a universal dry molten globule in protein folding. Our work demonstrates that constant pH molecular dynamics is a unique tool for testing this and other hypotheses to advance the knowledge in protein dynamics, stability, and folding.
Project description:Recently, the role of force in cellular processes has become more evident, and now with advances in force spectroscopy, the response of proteins to force can be directly studied. Such studies have found that native proteins are brittle, and thus not very deformable. Here, we examine the mechanical properties of a class of intermediates referred to as the molten globule state. Using optical trap force spectroscopy, we investigated the response to force of the native and molten globule states of apomyoglobin along different pulling axes. Unlike natively folded proteins, the molten globule state of apomyoglobin is compliant (large distance to the transition state); this large compliance means that the molten globule is more deformable and the unfolding rate is more sensitive to force (the application of force or tension will have a more dramatic effect on the unfolding rate). Our studies suggest that these are general properties of molten globules and could have important implications for mechanical processes in the cell.
Project description:The molten globule (MG) state of proteins is widely detected through binding with 1-anilino-8-naphthalene sulphonate (ANS), a fluorescent dye. This strategy is based upon the assumption that when in molten globule state, the exposed hydrophobic clusters of protein are readily bound by the nonpolar anilino-naphthalene moiety of ANS molecules which then produce brilliant fluorescence. In this work, we explored the acid-induced unfolding pathway of chymopapain, a cysteine proteases from Carica papaya, by monitoring the conformational changes over a pH range 1.0-7.4 by circular dichroism, intrinsic fluorescence, ANS binding, acrylamide quenching, isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The spectroscopic measurements showed that although maximum ANS fluorescence intensity was observed at pH 1.0, however protein exhibited ?80% loss of secondary structure which does not comply with the characteristics of a typical MG-state. In contrast at pH 1.5, chymopapain retains substantial amount of secondary structure, disrupted side chain interactions, increased hydrodynamic radii and nearly 30-fold increase in ANS fluorescence with respect to the native state, indicating that MG-state exists at pH 1.5 and not at pH 1.0. ITC measurements revealed that ANS molecules bound to chymopapain via hydrophobic interaction were more at pH 1.5 than at pH 1.0. However, a large number of ANS molecules were also involved in electrostatic interaction with protein at pH 1.0 which, together with hydrophobically interacted molecules, may be responsible for maximum ANS fluorescence. We conclude that maximum ANS-fluorescence alone may not be the criteria for determining the MG of chymopapain. Hence a comprehensive structural analysis of the intermediate is essentially required.
Project description:RsbW, an anti-sigma factor possessing kinase activity, is expressed by many Gram-positive bacteria including Staphylococcus aureus. To obtain clues about the domain structure and the folding-unfolding mechanism of RsbW, we have elaborately studied rRsbW, a recombinant S. aureus RsbW. Sequence analysis of the protein fragments, generated by the limited proteolysis of rRsbW, has proposed it to be a single-domain protein. The unfolding of rRsbW in the presence of GdnCl or urea was completely reversible in nature and occurred through the formation of at least two intermediates. The structure, shape, and the surface hydrophobicity of no intermediate completely matches with those of other intermediates or the native rRsbW. Interestingly, one of the intermediates, formed in the presence of less GdnCl concentrations, has a molten globule-like structure. Conversely, all of the intermediates, like native rRsbW, exist as dimers in aqueous solution. The putative molten globule and the urea-generated intermediates also have retained some kinase activity. Additionally, the putative ATP binding site/catalytic site of rRsbW shows higher denaturant sensitivity than the tentative dimerization region of this enzyme.