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Folding of cytochrome c initiated by submillisecond mixing
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of special interest. In this and the study by Yeh et al. [24], a different, ultrarapid continuous mixing design was used to study the folding of cytochrome c. Instead of a freely flowing jet, the liquids that emerge from the mixer flow through a cuvette, which permits spectroscopic measurements with improved signal-to-noise. Resonance Raman spectroscopy shows that misligation of the heme by the histidine residues produces trapping of misfolded structures and complex kinetics, even at acid pH, in which protonation was previously thought to more completely prevent histidine binding.
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Proc Natl Acad Sci USA
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Laser temperature jump for the study of early events in protein folding
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°. Monitoring the fluorescence of a probe attached to the N terminus of the same peptide used in the infrared studies in [25] shows ~ 10-fold shorter relaxation time than is observed for the average helical content. The shorter relaxation time is attributed to fraying of the helix
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°. Monitoring the fluorescence of a probe attached to the N terminus of the same peptide used in the infrared studies in [25] shows ~ 10-fold shorter relaxation time than is observed for the average helical content. The shorter relaxation time is attributed to fraying of the helix.
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of special interest. Carbon monoxide destabilizes reduced cytochrome c by binding to the heme, thereby removing a covalent heme-methionine link that stabilizes the native state. Nanosecond photodissociation of the carbon monoxide in the unfolded state could therefore be used to trigger folding as in [28]. As the denaturant concentration is high and held fixed in these experiments, collapse of the protein is not observed prior to folding, as determined by fluorescence measurements of energy transfer between the tryptophan and heme.
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Diffusion-limited contact formation in unfolded cytochrome c: Estimating the maximum rate of protein folding
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of special interest. In two papers [30,31], nanosecond photodissociation experiments are used to measure the bimolecular binding rate of free methionine and histidine to the heme attached to an 11-residue peptide of cytochrome c. Analysis of these results show that the intramolecular binding of methionine residues to the heme of the intact protein (measured in [28]) is diffusion-limited, whereas the binding of histidine is reaction-limited.
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Direct observation of fast protein folding: The initial collapse of apomyoglobin
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of special interest. 196; In two papers [39,40], the folding of apomyoglobin from the cold-denatured state is investigated by nanosecond laser temperature jump. A decrease in tryptophan fluorescence at ~ 5 μs is attributed to contact quenching of Trp14 on the A helix by Met131 on the H helix. This process has little or no temperature dependence and a significant viscosity dependence, thereby supporting the interpretation of global collapse.
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Observation of distinct nanosecond and microsecond protein folding events
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The folding pathway of a protein at high resolution from microseconds to seconds
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of special interest. In this paper, which is an extension of [41], the results for 26 mutants were used to probe the structures of an intermediate state and the two transition states connecting it to the unfolded and folded states. This study allows a detailed structural characterization of these states by comparing the rates and equilibrium constants of the mutants and wild-type protein (Fersht φ analysis; see this issue [pp 3-9]).
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