UMLS:C0344329 - FACTA Search

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This review traces some of the key features of the folding of beta-lactamases and their relevance to the way proteins fold in general. Studies on the enzymes have highlighted the nature and role of equilibrium and transient condensed states. The kinetics of folding are multiphasic, and when monitored by acrylamide quenching of the tryptophan fluorescence, an early phase provides evidence for the transient accumulation of a nonnative intermediate involving burial of tryptophan in a nonpolar environment. Intermediate phases can be understood in terms of progressive folding of different parts of the molecule. The later, slow phases are associated with proline isomerization in the TEM-1 enzyme and, in its P167T mutant form, with isomerization from trans to cis of the E166 T167 peptide bond. Coupled with kinetic and X-ray crystallographic studies of the beta-lactamase from Staphylococcus aureus and its D179Q mutant, it appears that the final stage of folding is that of collapse and packing of the omega-loop on to the main body of the protein.
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PMID:Beta-lactamases as models for protein-folding studies. 961 75

Early conformational states in the refolding of hen lysozyme from guanidine hydrochloride have been characterized by measuring both the fluorescence and the solvent exchange properties of tryptophan side chains. The indole proton occupancies indicate that at pH 5.5, 25 degrees C, half the protection against pulse labeling occurs in the dead time (4 ms) of the experiment, with the remaining protection developing with a time constant of 55 ms. Comparison of these data with the protection kinetics of backbone amides and with the fluorescence data provides evidence for hydrophobic collapse involving incorporation of tryptophan residues in a solvent-excluded state in advance of stable secondary structure formation. Analysis of the pH dependence of the indole hydrogen exchange protection is consistent with two or more structurally distinct collapsed states, and indicates that the generation of a correctly folded compact hydrophobic core is a key precursor to the formation of persistent native-like structure during refolding.
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PMID:Characterization of collapsed states in the early stages of the refolding of hen lysozyme. 962 99

The mechanism of unfolding of ferricytochrome c induced by the surfactant sodium dodecyl sulfate has been studied by heme absorption, tryptophan fluorescence, circular dichroism, resonance Raman scattering, stopped-flow and time-resolved resonance energy transfer to obtain a comprehensive view of the whole process. Unfolding occurred at an almost specific molecular ratio of SDS/cytochrome c in the concentration range (20-50 microM) studied here. However there appears to be a point at approximately 0.6 mM SDS where unfolding begins to occur for lower cytochrome c concentrations. The kinetics of unfolding revealed only a single transition with a rate constant of 33 s(-1) (at 298 K, [SDS] = 8.7 mM) and activation energy barrier of approximately 16 kJ/mol, indicating that other associated steps, if any, are too fast to be significantly populated. The free energy change (deltaG(o)) involved with the unfolding transition was estimated to be about 16.8 kJ/mol. The CD spectrum at 220 nm of SDS-unfolded cytochrome c shows only a partial decrease (25%), indicating that a significant amount of helical structure remains folded in contrast to a complete loss of helical structure in GdnHCl-denatured cytochrome c. The heme structure in SDS-unfolded cytochrome c, as deduced from heme absorption and resonance Raman spectra, shows a major population (approximately 95%) of mis-ligated histidine to the heme which acts as a kinetic trap in the folding process. The structural changes associated with cytochrome c unfolding were also monitored by time-resolved resonance energy transfer which shows a drastic increase in tryptophan fluorescence lifetime from 12 ps in the native protein to 0.63 ns in the unfolded one, associated with a movement of Trp59 by 10 A away from heme. The maximum entropy method analysis of fluorescence decay indicated the growth of various conformational substates in SDS-unfolded cytochrome c in contrast to narrowly distributed conformations in the native protein. The refolding was comprised of three kinetic steps; the first was significantly fast (approximately 8 ms) and was assigned to the dissociation of His26 that paves the protein towards correct folding pathway. The other two slower steps probably arise from chain misorganization and prolyl isomerization. The absence of a burst-phase amplitude supports the idea that the burst phase observed in the folding from completely unfolded cytochrome c corresponds to a molecular collapse that produces significant secondary structure. The partially unfolded state represents a unique intermediate state in the folding pathway.
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PMID:Characterization of a partially unfolded structure of cytochrome c induced by sodium dodecyl sulphate and the kinetics of its refolding. 968 80

Tryptophan residues in chitosanase from Streptomyces sp. N174 (Trp28, Trp101, and Trp227) were mutated to phenylalanine, and thermal unfolding experiments of the proteins were done in order to investigate the role of tryptophan residues in thermal stability. Four types of mutants (W28F, W101F, W227F and W28F/W101F) were produced in sufficient quantity in our expression system using Streptomyces lividans TK24. Each unfolding curve obtained by CD at 222 nm did not exhibit a two-state transition profile, but exhibited a biphasic profile: a first cooperative phase and a second phase that is less cooperative. The single tryptophan mutation decreased the midpoint temperature (Tm) of the first transition phase by about 7 degrees C, and the double mutation by about 11 degrees C. The second transition phase in each mutant chitosanase was more distinct and extended than that in the wild-type. On the other hand, each unfolding curve obtained by tryptophan fluorescence exhibited a typical two-state profile and agreed with the first phase of transition curves obtained by CD. Differential scanning calorimetry profiles of the proteins were consistent with the data obtained by CD. These data suggested that the mutation of individual tryptophan residues would partly collapse the side chain interactions, consequently decreasing Tm and enhancing the formation of a molten globule-like intermediate in the thermal unfolding process. The tryptophan side chains are most likely to play important roles in cooperative stabilization of the protein.
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PMID:Thermal unfolding of chitosanase from Streptomyces sp. N174: role of tryptophan residues in the protein structure stabilization. 998 21

The refolding kinetics of cobrotoxin (CBTX), a small-molecular-weight ( approximately 7 kDa) all beta-sheet protein, has been monitored using a variety of biophysical techniques. The secondary structure formation and hydrophobic collapse occur as distinct events during the refolding of the protein. Complete secondary structure formation occurs prior to the clustering of the hydrophobic residues. The late stage(s) of the refolding pathway of CBTX is characterized by change(s) in the local environment and optical asymmetry of the indole ring of the sole tryptophan residue. The results obtained in the present study, to our knowledge, represent the first unambiguous experimental support for the framework model of protein folding.
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PMID:Secondary structure formation is the earliest structural event in the refolding of an all beta-sheet protein. 1038 80

Obstructive sleep apnea hypopnea syndrome (OSAHS) is a prevalent disorder, for which there are no universally effective pharmacotherapeutics. We hypothesized that in OSAHS, excitatory serotoninergic influences are important for maintaining patency of the upper airway in waking, and that in sleep, reduced serotoninergic drive plays a significant role in upper airway collapse and OSAHS. The previously reported small responses in humans with OSAHS to serotoninergics may relate, in part, to study design and the drugs/doses selected. We therefore performed multitrials/dose, multidose, randomized sleep studies testing the effectiveness of a combination of serotoninergics, trazodone, and L-tryptophan, in our animal model of OSAHS, the English bulldog. Trazodone/L-tryptophan caused dose-dependent reductions in respiratory events in non-rapid-eye-movement sleep (NREMS) and rapid-eye-movement sleep (REMS). During NREMS, the respiratory disturbance index (RDI) +/- standard error was 6.3 +/- 1.4 events/h (placebo) and 0.9 +/- 0.3 (highest dose), p < 0.01. During REMS, the RDI was 31.4 +/- 6.1 events/h (placebo) and 11.5 +/- 4.3 (highest dose), p = 0.002. Trazodone/ L-tryptophan dose-dependently reduced sleep fragmentation, p = 0.03, increased sleep efficiency, p = 0.005, enhanced slow-wave sleep, p = 0.0004, and minimized sleep-related suppression of upper airway dilator activity, p < 0.02. Trazodone with L-tryptophan can treat sleep-disordered breathing (SDB) in an animal model of OSAHS; the effectiveness of this therapy may be related to increased upper airway dilator activity in sleep and/or enhanced slow-wave sleep.
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PMID:The effects of trazodone with L-tryptophan on sleep-disordered breathing in the English bulldog. 1055 37

Fluorescence resonance energy transfer (FRET) is one of the few methods available to measure the rate at which a folding protein collapses. Using staphylococcal nuclease in which a cysteine residue was engineered in place of Lys64, permitted FRET measurements of the distance between the donor tryptophan 140 and 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid-labeled Cys64. These measurements were undertaken on both equilibrium partially folded intermediates at low pH (A states), as well as transient intermediates during stopped-flow refolding. The results indicate that there is an initial collapse of the protein in the deadtime of the stopped-flow instrument, corresponding to a regain of approximately 60% of the native signal, followed by three slower transients. This is in contrast to circular dichroism measurements which show only 20-25% regain of the native secondary structure in the burst phase. Thus hydrophobic collapse precedes the formation of substantial secondary structure. The first two detected transient intermediate species have FRET properties essentially identical with those of the previously characterized equilibrium A state intermediates, suggesting similar structures between the equilibrium and transient intermediates. The effects of anions on the folding of acid-unfolded staphylococcal nuclease, and urea on the unfolding of the resulting A states, indicates that in folding the protein becomes compact prior to formation of major secondary structure, whereas in unfolding the protein expands prior to major loss of secondary structure. Comparison of the kinetics of refolding of staphylococcal nuclease, monitored by FRET, and for a proline-free variant, indicate that folding occurs via two partially folded intermediates leading to a native-like species with one (or more) proline residues in a non-native conformation. For the A states an excellent correlation between compactness measured by FRET, and compactness determined from small-angle X-ray scattering, was observed. Further, a linear relationship between compactness and free energy of unfolding was noted. Formation of soluble aggregates of the A states led to dramatic enhancement of the FRET, consistent with intermolecular fluorescence energy transfer.
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PMID:Fluorescence energy transfer indicates similar transient and equilibrium intermediates in staphylococcal nuclease folding. 1084 64

Neocarzinostatin is a potent enediyne antitumor antibiotic complex in which a chromophore is noncovalently bound to a carrier protein. The protein regulates availability of the drug by proper release of the biologically active chromophore. To understand the physiological mechanism of the drug delivery system, we have examined the trifluoroethanol (TFE)-induced conformational changes of the protein with special emphasis on their relation to the release of the chromophore from holoneocarzinostatin. The effect of the alpha helix-inducing agent, TFE, on all the beta-sheet neocarzinostatin proteins was studied by circular dichroism, fluorescence, and (1)H NMR studies. By using binding of anilinonaphthalene sulfonic acid as a probe, we observed that the protein exists in a stable, partially structured intermediate state around 45-50% TFE, which is consistent with the results from tryptophan fluorescence and circular dichroism studies. The native state is stable until 20% TFE and is half-converted into the intermediate state at 30% TFE, which starts to collapse beyond 50%. High pressure liquid chromatographic analysis of the release of the chromophore caused by TFE treatment at 0 degrees C suggests that the release process, which occurs below 20% TFE, does not result from an observable conformational change in the protein. Kinetic measurements of the release of chromophore at 25 degrees C reveal that TFE does stimulate the rate of release, which increases sharply at 15% and reaches a maximum at 20% TFE, although no major secondary or tertiary structural change of the carrier protein is observed under these same conditions. Our data suggest that chromophore release results from a fluctuation of the protein structure that is stimulated by TFE. Complete release of the chromophore occurs at TFE concentrations where no overall observable unfolding of the apoprotein is seen. Thus, the results suggest that denaturation of the protein by TFE is not a necessary step for release of the tightly bound chromophore.
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PMID:Release of the neocarzinostatin chromophore from the holoprotein does not require major conformational change of the tertiary and secondary structures induced by trifluoroethanol. 1098 12

Unfolding of the immunoglobulin binding domain B1 of streptococcal protein G (GB1) was induced by guanidine hydrochloride (GdnHCl) and studied by circular dichroism, steady-state, and time-resolved fluorescence spectroscopy. The fluorescence methods employed the single tryptophan residue of GB1 as an intrinsic reporter. While the transitions monitored by circular dichroism and steady-state fluorescence coincided with each other, the transitions followed by dynamic fluorescence were markedly different. Specifically, fluorescence anisotropy data showed that a relaxation spectrum of tryptophan contained a slow motion with relaxation times of 9 ns in the native state and 4 ns in the unfolded state in 6 M GdnHCl. At intermediate GdnHCl concentrations of 3.8-4.2 M, however, the slow relaxation time increased to 18 ns. The fast nanosecond motion had an average time of 0.8 ns and showed no dependence on the formation of native structure. Overall, dynamic fluorescence revealed two preliminary stages in GB1 folding, which are equated with the formation of local structure in the beta(3)-strand hairpin and the initial collapse. Both stages exist without alpha-helix formation, i. e., before the appearance of any ordered secondary structure detectable by circular dichroism. Another stage in GB1 folding might exist at very low ( approximately 1 M) GdnHCl concentrations.
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PMID:Nanosecond dynamics of the single tryptophan reveals multi-state equilibrium unfolding of protein GB1. 1098 67

Flavoproteins can function as hydrophobic sites for vitamin B(2) (riboflavin) or, in other structures, with cofactors for catalytic reactions such as glucose oxidation. In this contribution, we report direct observation of charge separation and recombination in two flavoproteins: riboflavin-binding protein and glucose oxidase. With femtosecond resolution, we observed the ultrafast electron transfer from tryptophan(s) to riboflavin in the riboflavin-binding protein, with two reaction times: approximately 100 fs (86% component) and 700 fs (14%). The charge recombination was observed to take place in 8 ps, as probed by the decay of the charge-separated state and the recovery of the ground state. The time scale for charge separation and recombination indicates the local structural tightness for the dynamics to occur that fast and with efficiency of more than 99%. In contrast, in glucose oxidase, electron transfer between flavin-adenine-dinucleotide and tryptophan(s)/tyrosine(s) takes much longer times, 1.8 ps (75%) and 10 ps (25%); the corresponding charge recombination occurs on two time scales, 30 ps and nanoseconds, and the efficiency is still more than 97%. The contrast in time scales for the two structurally different proteins (of the same family) correlates with the distinction in function: hydrophobic recognition of the vitamin in the former requires a tightly bound structure (ultrafast dynamics), and oxidation-reduction reactions in the latter prefer the formation of a charge-separated state that lives long enough for chemistry to occur efficiently. Finally, we also studied the influence on the dynamics of protein conformations at different ionic strengths and denaturant concentrations and observed the sharp collapse of the hydrophobic cleft and, in contrast, the gradual change of glucose oxidase.
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PMID:Femtosecond dynamics of flavoproteins: charge separation and recombination in riboflavine (vitamin B2)-binding protein and in glucose oxidase enzyme. 1159 97

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