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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.
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PMID:Ultrarapid mixing experiments shed new light on the characteristics of the initial conformational ensemble during the folding of ribonuclease A. 1557 90

Rubredoxins are the simplest type of iron-sulphur proteins and in recent years they have been used as model systems in protein folding and stability studies, especially the proteins from thermophilic sources. Here, we report our studies on the rubredoxin from the hyperthermophile Methanococcus jannaschii (T opt = 85 degrees C), which was investigated in respect to its thermal unfolding kinetics by temperature jump experiments. Different spectroscopic probes were used to monitor distinct structural protein features during the thermal transition: the integrity of the iron-sulphur centre was monitored by visible absorption spectroscopy, whereas tertiary structure was followed by intrinsic tryptophan fluorescence and exposure of protein hydrophobic patches was sensed by 1-anilinonaphthalene-8-sulphonate fluorescence. The studies were performed at acidic pH conditions in which any stabilising contributions from salt bridges are annulled due to protonation of protein side chain groups. In these conditions, M. jannaschii rubredoxin assumes a native-like, albeit more flexible and open conformation, as indicated by a red shift in the tryptophan emission maximum and 1-anilinonaphthalene-8-sulphonate binding. Temperature jumps were monitored by the three distinct techniques and showed that the protein undergoes thermal denaturation via a simple two step mechanism, as loss of tertiary structure, hydrophobic collapse, and disintegration of the iron-sulphur centre are concomitant processes. The proposed mechanism is framed with the multiphasic one proposed for Pyrococcus furiosus rubredoxin, showing that a common thermal unfolding mechanism is not observed between these two closely related thermophilic rubredoxins.
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PMID:Probing the mechanism of rubredoxin thermal unfolding in the absence of salt bridges by temperature jump experiments. 1597 57

Two-site fluorescence resonance energy transfer (FRET) measurements have been made to determine how two intra-molecular distances contract in the sub-millisecond collapse reaction that occurs initially during the refolding of the small protein barstar. FRET measurements were made on two, single-Cys and single-Trp-containing mutant forms of barstar, Cys25 and Cys62, in each of which a thionitrobenzoate (TNB) adduct was attached to the cysteine thiol. In each protein, the core tryptophan, Trp53, acted as the FRET donor, and the TNB adduct, located either at C25 or at C62, acted as the FRET acceptor. The stabilities as well as observable folding kinetics of the Cys25 and Cys62 mutant proteins were found to be identical. The presence of the TNB adduct on the cysteine did not alter the stability or folding kinetics of either protein. Thus, the FRET-monitored changes in the two labeled mutant proteins, Cys25-TNB and Cys62-TNB, could be compared directly. Refolding was commenced from unfolded protein in 8M urea, and both the Trp53 to C25-TNB distance and the Trp53 to C62-TNB distance were found to contract upon dilution of urea. The extent of contraction of each distance, which was measured at a few milliseconds of refolding, was dependent continuously on the concentration of urea present during refolding, and was different for the two distances. For either FRET pair, the gradual contraction of distance with a decrease in the concentration of urea in which refolding occurs, was continuous with the contraction of the polypeptide chain that is seen with a decrease in the concentration of urea in the range in which the protein remains completely unfolded. It therefore appears that the products of the initial sub-millisecond refolding reaction of barstar are collapsed forms, whose dimensions do not change cooperatively in an all-or-none manner, but instead, change gradually with a change in concentration of urea. Thus, the sub-millisecond polypeptide chain collapse reaction of barstar upon denaturant dilution, appears to be a continuous structural transition.
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PMID:Dependence of the size of the initially collapsed form during the refolding of barstar on denaturant concentration: evidence for a continuous transition. 1618 74

The role of polypeptide collapse and formation of intermediates in protein folding is still under debate. Miniproteins, small globular peptide structures, serve as ideal model systems to study the basic principles that govern folding. Experimental investigations of folding dynamics of such small systems, however, turn out to be challenging, because requirements for high temporal and spatial resolution have to be met simultaneously. Here, we demonstrate how selective quenching of an extrinsic fluorescent label by the amino acid tryptophan (Trp) can be used to probe folding dynamics of Trp-cage (TC), the smallest protein known to date. Using fluorescence correlation spectroscopy, we monitor folding transitions as well as conformational flexibility in the denatured state of the 20-residue protein under thermodynamic equilibrium conditions with nanosecond time resolution. Besides microsecond folding kinetics, we reveal hierarchical folding of TC, hidden to previous experimental studies. We show that specific collapse of the peptide to a molten globule-like intermediate enhances folding efficiency considerably. A single point mutation destabilizes the intermediate, switching the protein to two-state folding behavior and slowing down the folding process. Our results underscore the importance of preformed structure in the denatured state for folding of even the smallest globular structures. A unique method emerges for monitoring conformational dynamics and ultrafast folding events of polypeptides at the nanometer scale.
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PMID:A microscopic view of miniprotein folding: enhanced folding efficiency through formation of an intermediate. 1626 42

We have examined the architecture of a protein complex in the absence of bulk water. By determining collision cross sections of assemblies of the trp RNA binding protein, TRAP, we established that the 11-membered ring topology of the complex can be maintained within a mass spectrometer. We also found that the binding of tryptophan enhances the stability of the ring structure and that addition of a specific RNA molecule increases the size of the complex and prevents structural collapse. These results provide definitive evidence that protein quaternary structure can be maintained in the absence of bulk water and highlight the potential of ion mobility separation for defining shapes of heterogeneous macromolecular assemblies.
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PMID:Evidence for macromolecular protein rings in the absence of bulk water. 1629 22

The uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, into vacuoles isolated from Catharanthus roseus cells has been studied by silicone layer floatation filtering. The transport across the tonoplast of MACC is stimulated fourfold by 5 millimolar MgATP, has a K(m) of about 2 millimolar, an optimum pH around 7, and an optimum temperature at 30 degrees C. Several effectors known to inhibit ATPase (N,N'-dicyclohexylcarbodiimide) and to collapse the transtonoplastic H(+) electrochemical gradient (carbonylcyanide m-chlorophenylhydrazone, gramicidin, and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with SCN(-) and valinomycin also greatly inhibited MACC transport. Our data demonstrate that MACC accumulates in the vacuole against a concentration gradient by means of a proton motive force generated by a tonoplastic ATPase. The involvement of a protein carrier is suggested by the strong inhibition of uptake by compounds known to block SH-, OH-, and NH(2)- groups. MACC uptake is antagonized competitively by malonyl-d-tryptophan, indicating that the carrier also accepts malonyl-d-amino acids. Neither the moities of these compounds taken separately [1-aminocyclopropane-1-carboxylic acid, malonate, d-tryptophan or d-phenylalanine] nor malate act as inhibitors of MACC transport. The absence of inhibition of malate uptake by MACC suggests that MACC and malate are taken up by two different carriers. We propose that the carrier identified here plays an important physiological role in withdrawing from the cytosol MACC and malonyl-d-amino acids generated under stress conditions.
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PMID:Carrier-Mediated Uptake of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Vacuoles Isolated from Catharanthus roseus Cells. 1666 82

Melittin, an amphipathic peptide from honeybee venom, consists of 26 amino acid residues and adopts different conformations from a random coil, to an alpha-helix, and to a self-assembled tetramer under certain aqueous environments. We report here our systematic studies of the hydration dynamics in these conformations using single intrinsic tryptophan (W19) as a molecular probe. With femtosecond resolution, we observed the solvation dynamics occurring in 0.62 and 14.7 ps in a random-coiled primary structure. The former represents bulklike water motion, and the latter reflects surface-type hydration dynamics of proteins. As a comparison, a model tripeptide (KWK) was also studied. At a membrane-water interface, melittin folds into a secondary alpha-helical structure, and the interfacial water motion was found to take as long as 114 ps, indicating a well-ordered water structure along the membrane surface. In high-salt aqueous solution, the dielectric screening and ionic solvation promote the hydrophobic core collapse in melittin aggregation and facilitate the tetramer formation. This self-assembled tertiary structure is also stabilized by the strong hydrophilic interactions of charged C-terminal residues and associated ions with water molecules in the two assembled regions. The hydration dynamics was observed to occur in 87 ps, significantly slower than typical water relaxation at protein surfaces but similar to water motion at membrane interfaces. Thus, the observed time scale of approximately 100 ps probably implies appropriate water mobility for mediating the formation of high-order structures of melittin in an alpha-helix and a self-assembled tetramer. These results elucidate the critical role of hydration dynamics in peptide conformational transitions and protein structural stability and integrity.
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PMID:Ultrafast hydration dynamics in melittin folding and aggregation: helix formation and tetramer self-assembly. 1685 51

To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side-chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophan residues in an apolar environment. The kinetics of folding was observed by continuous and stopped-flow fluorescence measurements over refolding times ranging from 100 micros to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophan residues in the beta-barrel and the N-terminal part of the alpha-helical domain become partially shielded from the solvent at an early stage (<1 ms), indicating that this region undergoes a rapid collapse. For some variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophan residues are buried only during the late stages of folding. Other variants exhibit a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophan residues into close contact with intramolecular quenchers.
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PMID:Folding kinetics of staphylococcal nuclease studied by tryptophan engineering and rapid mixing methods. 1733 34

Aromatic amine dehydrogenase uses a tryptophan tryptophylquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. In the reductive half-reaction, a proton is transferred from the substrate C1 to betaAsp-128 O-2, in a reaction that proceeds by H-tunneling. Using solution studies, kinetic crystallography, and computational simulation we show that the mechanism of oxidation of aromatic carbinolamines is similar to amine oxidation, but that carbinolamine oxidation occurs at a substantially reduced rate. This has enabled us to determine for the first time the structure of the intermediate prior to the H-transfer/reduction step. The proton-betaAsp-128 O-2 distance is approximately 3.7A, in contrast to the distance of approximately 2.7A predicted for the intermediate formed with the corresponding primary amine substrate. This difference of approximately 1.0 A is due to an unexpected conformation of the substrate moiety, which is supported by molecular dynamic simulations and reflected in the approximately 10(7)-fold slower TTQ reduction rate with phenylaminoethanol compared with that with primary amines. A water molecule is observed near TTQ C-6 and is likely derived from the collapse of the preceding carbinolamine TTQ-adduct. We suggest this water molecule is involved in consecutive proton transfers following TTQ reduction, and is ultimately repositioned near the TTQ O-7 concomitant with protein rearrangement. For all carbinolamines tested, highly stable amide-TTQ adducts are formed following proton abstraction and TTQ reduction. Slow hydrolysis of the amide occurs after, rather than prior to, TTQ oxidation and leads ultimately to a carboxylic acid product.
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PMID:New insights into the reductive half-reaction mechanism of aromatic amine dehydrogenase revealed by reaction with carbinolamine substrates. 1747 20

Two antiparallel beta-strands connected by a turn make beta-hairpins an ideal model system to analyze the interactions and dynamics of beta-sheets. Site-specific conformational dynamics were studied by temperature-jump IR spectroscopy and isotopic labeling in a model based on the tryptophan zipper peptide, Trpzip2, developed by Cochran et al. (Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5578). The modified Trpzip2C peptides have nearly identical equilibrium spectral behavior as Trpzip2 showing that they also form well-characterized beta-hairpin conformations in aqueous solution. Selective introduction of 13C=O groups on opposite strands lead to distinguishable cross-strand coupling of the labeled residues as monitored in the amide I' band. These frequency patterns reflect theoretical predictions, and the coupled 13C=O band loses intensity with increase in temperature and unfolding of the hairpin. Thermal relaxation kinetics were analyzed for unlabeled and cross-strand isotopically labeled variants. T-jumps of approximately 10 degrees C induce relaxation times of a few microseconds that decrease with increase of the peptide temperature. Differences in kinetic behavior for the loss of beta-strand and gain of disordered structure can be used to distinguish localized structure dynamics by comparison of nonlabeled and labeled amide I' components. Analysis of the data supports multistate dynamic and equilibrium behavior, but because of this process it is not possible to clearly define a folding and unfolding rate. Nonetheless, site-specific relaxation kinetics could be seen to be consistent with a hydrophobic collapse hypothesis for hairpin folding.
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PMID:Site-specific relaxation kinetics of a tryptophan zipper hairpin peptide using temperature-jump IR spectroscopy and isotopic labeling. 1827 8


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