Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0344329 (collapse)
 28,634 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Specific three- and two-disulfide intermediates that accumulate transiently during reduction of the disulfide bonds of Ca(2+)-bound bovine alpha-lactalbumin have been trapped, isolated, and characterized. The three-disulfide intermediate was shown to lack the Cys6-120 disulfide bond, confirming the observations of others. The newly-recognized two-disulfide form has been shown to lack the Cys6-120 and Cys28-111 native disulfide bonds. The remaining native disulfide bonds in the two partially reduced derivatives of alpha-lactalbumin are stable only when the proteins are in a Ca(2+)-bound state. Otherwise, they adopt an equilibrium between molten globule and unfolded conformations, and rapid thiol-disulfide interchange occurs, at a rate as high as when the proteins are fully unfolded in 8 M urea, to generate distinct mixtures of rearranged products. Urea gradient electrophoresis, circular dichroism, fluorescence, and ANS binding have been combined to give a detailed structural picture of alpha-lactalbumin, its derivatives with native and with nonnative disulfide bonds, and the fully reduced protein. The native structure of alpha-lactalbumin appears to be split by selective disulfide bond cleavage into at least one subdomain, which retains the Ca(2+)-binding site. The alpha-lactalbumin molten globule state is shown largely to result from nonspecific hydrophobic collapse, to be devoid of cooperative or specific tertiary interactions, and not to be stabilized substantially by the native or rearranged disulfide bonds.
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PMID:Structural characterization of the disulfide folding intermediates of bovine alpha-lactalbumin. 846 9

The folding-unfolding kinetics of human alpha 1-antitrypsin (alpha 1-AT) were examined by monitoring intrinsic Trp fluorescence and extrinsic ANS fluorescence. While the unfolding of alpha 1-AT followed a single exponential phase, refolding exhibited three exponential phases. The fast phase (tau 1r < 40 sec). which was independent of urea concentration, appears to be hydrophobic collapse that may be limited by cis-trans isomerization of prolyl residue. The medium phase (tau 2s = 200 sec) yielded an intermediate (IN), which is capable of elastase binding. The slowest (tau 3r = 1000 sec) phase completes refolding to the native protein, which intersects with the unfolding kinetics at the same urea concentration (1.9 M) as the equilibrium midpoint. Concentration-dependence of the amplitude of major refolding phases indicated that IN is prone to kinetic competition between the on-pathway to native protein and aggregation.
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PMID:Folding pathway of human alpha 1-antitrypsin: characterization of an intermediate that is active but prone to aggregation. 880 43

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.
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PMID:Hexafluoroacetone hydrate as a structure modifier in proteins: characterization of a molten globule state of hen egg-white lysozyme. 914 78

Thermal denaturation of equinatoxin II (EqTxII) in glycine buffer solutions (pH 1.1, 2.0, 3.0, and 3.5) and in triple distilled water (pH 5.5-6.0) was examined by differential scanning calorimetry, UV and CD spectroscopy and fluorescence emission spectroscopy of the added hydrophobic fluorescent probe ANS. At pH 5.5-6.0 and at temperatures below 60 degrees C, the protein exists in a native state characterized by a pronounced tertiary structure, a beta-rich secondary structure and a low degree of ANS-binding. At higher temperatures, it undergoes a two-state conformational transition, (delta H degree)VH = (delta H degree)DSC, into an unfolded state, which is characterized by a complete collapse of its tertiary structure and an incomplete denaturation of its secondary structure. At acidic pH, the EqTxII temperature-induced conformational transition appears at lower temperatures as non-two-state transition accompanied by the formation of an intermediate state which shows characteristics of molten globules, i.e., absence of defined tertiary structure, increase in alpha-rich secondary structure, and high affinity for ANS. At pH 2.0, the low-temperature initial state of EqTxII is already partially denatured; the tertiary structure is partially disrupted, and a pronounced inequality (delta H degree)VH > (delta H degree)DSC is observed. At pH value of 1.1 and below 60 degrees C, EqTxII exists in a stable acid-denatured compact state which shows all the characteristics of a molten globule, which even at 95 degrees C is not completely denatured. According to numerous studies on the pore forming toxins, such acid-denatured compact states may contribute to the protein's ability to penetrate into biological membranes.
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PMID:pH and temperature-induced molten globule-like denatured states of equinatoxin II: a study by UV-melting, DSC, far- and near-UV CD spectroscopy, and ANS fluorescence. 939 52

The kinetics of refolding of TEM-1 beta-lactamase from solution in guanidine hydrochloride have been investigated on the manual and stopped-flow mixing time scales. The kinetics of change of far-UV circular dichroism and of intrinsic and ANS fluorescence have been compared with changes in the quenching of fluorescence by acrylamide as a probe of the accessibility of solvent to tryptophan. The binding of ANS points to hydrophobic collapse in the very early stages of folding which take place in the burst phase. This is accompanied by regain of 60-65% of native ellipticity, indicating formation of a significant proportion of secondary structure. Also in the burst phase, the tryptophan residues, which are largely exposed to solvent in the native protein, become less accessible to acrylamide, and the intrinsic fluorescence increases markedly. An early intermediate is thus formed in which tryptophan is more buried than in the native protein. Further intermediates are formed over the next 20 s. Quenching by acrylamide increases during this period, as the transient nonnative state is disrupted and the tryptophan residue(s) become(s) reexposed to solvent. The two slowest phases are determined by the isomerization of incorrect prolyl isomers, but double jump tryptophan fluorescence and acrylamide quenching experiments show little, if any, effect of proline isomerization on the earlier phases. Hydrophobic collapse thus occurs to a folding intermediate in which there is a nonnative element of structure which has to rearrange in the later steps of folding, resulting in a nonhierarchical folding pathway. The C-terminal W290 is suggested as being involved in the nonnative intermediate. beta-Lactamase provides further evidence for the occurrence of nonnative intermediates in protein folding.
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PMID:A collapsed intermediate with nonnative packing of hydrophobic residues in the folding of TEM-1 beta-lactamase. 948 21

Prothymosin alpha has previously been shown to be unfolded at neutral pH, thus belonging to a growing family of "natively unfolded" proteins. The structural properties and conformational stability of recombinant human prothymosin alpha were characterized at neutral and acidic pH by gel filtration, SAXS, circular dichroism, ANS fluorescence, (1)H NMR, and resistance to urea-induced unfolding. Interestingly, prothymosin alpha underwent a cooperative transition from the unfolded state into a partially folded conformation on lowering the pH. This conformation of prothymosin alpha is a compact denatured state, with structural properties different from those of the molten globule. The formation of alpha-helical structure by the glutamic acid-rich elements of the protein accompanied by the partial hydrophobic collapse is expected at lower pH due to the neutralization of the negatively charged residues. It is possible that such conformational changes may be associated with the protein function.
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PMID:Natively unfolded human prothymosin alpha adopts partially folded collapsed conformation at acidic pH. 1055 83

Although molten globules have been widely accepted as a general intermediate in protein folding, there is no clear evidence to show their presence during nascent peptide folding. This paper concentrates on whether the molten globule state occurs, and if it does, when does it form during nascent peptide folding, by comparing the changes in conformation during peptide chain extension of staphylococcal nuclease R. The results show that a large N-terminal fragment of staphylococcal nuclease, SNR121, which already contains more than 80% amino acid sequence of the nuclease, is found to fulfill all the criteria for the molten globule state, suggesting that the molten globule should occur at a later stage of peptide elongation. At this stage the hydrophobic collapse of the polypeptide chain occurs driven by the hydrophobic force, which leads to the formation of a solvent-accessible non-polar core, characterized by the high ANS-binding fluorescence. The nascent peptide folding of the nuclease is a hierarchical process that at the very least includes the following steps: secondary structure accumulation, pre-molten globule state, molten globule state, post-molten globule state and finally the native state. Constant conformation adjustment is necessary for correct folding and active expression of the protein.
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PMID:An in vitro peptide folding model suggests the presence of the molten globule state during nascent peptide folding. 1067 28

The enzyme rhodanese contains two globular domains connected by a tether region and associated by strong hydrophobic interactions. The protein has proven to be very difficult to refold without assistance to prevent oxidation and aggregation. For this study, the active site cysteine 247, near the interdomain region, was modified with the environmentally sensitive fluorescent probe, 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS), to yield a derivative that reversibly unfolds. Structural transitions during urea unfolding/refolding were complex and multiphasic. Increasing urea concentrations increased the IAANS fluorescence intensity and polarization. Both values reached maxima at approximately 4 m urea, where there is a concomitant large exposure of hydrophobic sites as reported by both IAANS and the noncovalent fluorescent probe, bis-ANS. The exposure of the hydrophobic sites arises from the decrease in strong interaction between the domain interfaces, which lead to their partial separation. This correlates with the loss of activity of the unlabeled enzyme. Above 4.5 m urea, there is progressive loss of rigid, hydrophobic surfaces, and both fluorescence and polarization of IAANS decrease, with accompanying loss of secondary structure. These results are consistent with a folding model in which there is an initial, rapid hydrophobic collapse of the denatured form to an intermediate with native like secondary structure, with exposed interdomain, hydrophobic surfaces. This step is followed by adjustment of the domain-domain interactions and the proper positioning of reduced cysteine 247 at the active site.
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PMID:Alteration around the active site of rhodanese during urea-induced denaturation and its implications for folding. 1080 29

Sac7d unfolds at low pH in the absence of salt, with the greatest extent of unfolding obtained at pH 2. We have previously shown that the acid unfolded protein is induced to refold by decreasing the pH to 0 or by addition of salt (McCrary BS, Bedell J. Edmondson SP, Shriver JW, 1998, J Mol Biol 276:203-224). Both near-ultraviolet circular dichroism spectra and ANS fluorescence enhancements indicate that the acid- and salt-induced folded states have a native fold and are not molten globular. 1H,15N heteronuclear single quantum coherence NMR spectra confirm that the native, acid-, and salt-induced folded states are essentially identical. The most significant differences in amide 1H and 15N chemical shifts are attributed to hydrogen bonding to titrating carboxyl side chains and through-bond inductive effects. The 1H NMR chemical shifts of protons affected by ring currents in the hydrophobic core of the acid- and salt-induced folded states are identical to those observed in the native. The radius of gyration of the acid-induced folded state at pH 0 is shown to be identical to that of the native state at pH 7 by small angle X-ray scattering. We conclude that acid-induced collapse of Sac7d does not lead to a molten globule but proceeds directly to the native state. The folding of Sac7d as a function of pH and anion concentration is summarized with a phase diagram that is similar to those observed for other proteins that undergo acid-induced folding except that the A-state is encompassed by the native state. These results demonstrate that formation of a molten globule is not a general property of proteins that are refolded by acid.
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PMID:The acid-induced folded state of Sac7d is the native state. 1110 60

The unfolding of shikimate kinase (SK) from Erwinia chrysanthemi by urea and its subsequent refolding on dilution of the denaturing agent has been studied in detail [Eur. J. Biochem. 269 (2002) 2124]. Comparison of the effects of urea on the enzyme with those of guanidinium chloride (GdmCl) and NaCl indicated that chloride ions significantly weakened the binding of shikimate. This finding prompted a more detailed examination of the effects of salts on the structure, function and stability of the enzyme; the effects of NaCl and Na(2)SO(4) were investigated in detail. These salts have very small effects on the secondary structure as judged by far UV CD circular dichroism (CD), although the near UV CD spectra suggest that some limited changes in the environment of aromatic amino acids may occur. Both salts inhibit SK activity and analysis of the kinetic and substrate binding parameters point to a complex mechanism for the inhibition. Inclusion of salts leads to a marked stabilisation against unfolding of the enzyme by urea. When the enzyme is unfolded by incubation in 4 M urea, addition of NaCl or Na(2)SO(4) leads to a relatively slow refolding of the enzyme as judged by the regain of native-like CD and fluorescence. In addition, the refolded enzyme can bind shikimate, though more weakly than the native enzyme. However, the refolded enzyme does not appear to be capable of binding nucleotides, nor does it possess detectable catalytic activity. The refolding process brought about by addition of salt in the presence of 4 M urea is not associated with any change in the fluorescence of the probe 8-anilino-1-naphthalenesulfonic acid (ANS), indicating that an intermediate formed by hydrophobic collapse is unlikely to be significantly populated. The results point to both specific and general effects of salts on SK. These are discussed in the light of the structural information available on the enzyme.
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PMID:Effects of salts on the function and conformational stability of shikimate kinase. 1275 46


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