Determination of intramolecular distance distribution during protein folding on the millisecond timescale

Ratner, Vladimir; Sinev, Michael A.; Haas, Elisha

doi:10.1006/jmbi.2000.3814

Cited by 53 publications

(62 citation statements)

References 28 publications

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“…The number of transitions seen in each trajectory varied widely. Many of the time traces showed no transitions, an expected result based on the time window of the experiment, which is limited by fluorophore photobleaching to 10-20 sec, compared with the ensemble folding time of Ϸ4 min [measured on the same mutant used here (20)]. …”

Section: Fret Distributions Of Ak In Vesicles Under Native and Midtramentioning

confidence: 86%

“…1, marked with arrows) in the mutant C77A of AK from E. coli (20). Fluorescence labeling was carried out in two steps following procedures described (20) and leading to molecules specifically labeled with the acceptor fluorophore (Texas red C2…”

Section: Methodsmentioning

confidence: 99%

“…AK is a 214-aa protein, the bulk folding reaction of which has been studied by several groups (18)(19)(20)(21). The equilibrium unfolding of this protein deviates from a simple two-state behavior, as evidenced by variations between equilibrium traces obtained with different methods (intrinsic fluorescence, circular dichroism, 8-anilino-1-naphthalene sulfonate binding, and FRET between different pairs of sites).…”

mentioning

confidence: 99%

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Watching proteins fold one molecule at a time

Rhoades

Gussakovsky²,

Haran³

2003

Proc. Natl. Acad. Sci. U.S.A.

335

304

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Recent theoretical work suggests that protein folding involves an ensemble of pathways on a rugged energy landscape. We provide direct evidence for heterogeneous folding pathways from single-molecule studies, facilitated by a recently developed immobilization technique. Individual fluorophore-labeled molecules of the protein adenylate kinase were trapped within surface-tethered lipid vesicles, thereby allowing spatial restriction without inducing any spurious interactions with the environment, which often occur when using direct surface-linking techniques. The conformational fluctuations of these protein molecules, prepared at the thermodynamic midtransition point, were studied by using fluorescence resonance energy transfer between two specifically attached labels. Folding and unfolding transitions appeared in experimental time traces as correlated steps in donor and acceptor fluorescence intensity. The size of the steps, in fluorescence resonance energy transfer efficiency units, shows a very broad distribution. This distribution peaks at a relatively low value, indicating a preference for small-step motion on the energy landscape. The time scale of the transitions is also distributed, and although many transitions are too fast to be time-resolved here, the slowest ones may take >1 sec to complete. These extremely slow changes during the folding of single molecules highlight the possible importance of correlated, non-Markovian conformational dynamics.

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Section: Fret Distributions Of Ak In Vesicles Under Native and Midtramentioning

confidence: 86%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Watching proteins fold one molecule at a time

Rhoades

Gussakovsky²,

Haran³

2003

Proc. Natl. Acad. Sci. U.S.A.

335

304

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“…trFRET experiments are ideal for detecting the formation of each closed long loop since it is possible to follow selected distances between two sites that are separated by large number of residues, their distributions, and fast fluctuations (Beechem and Haas 1989;Haas 2005). This led to our efforts towards the development of the trFRET-based "double kinetics" method for the detection of transient intramolecular distance distribution in the rapid collapsed state of globular proteins and during the full folding transition (Ratner et al 2000;Jacob et al 2005;Ben Ishay et al 2012a). …”

Section: The Loop Hypothesismentioning

confidence: 99%

“…Preparation of series of labeled mutants of one protein enables the monitoring of conformational changes of each sub-domain structure. Very fast data collection enables the determination of series of sequential distance distributions along the folding pathway (Ratner et al 2000(Ratner et al , 2005Ben Ishay et al 2012b). Such experiments yield meaningful information on specific conformational changes and the order of their occurrence along the folding pathway.…”

Section: Introductionmentioning

confidence: 99%

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

et al. 2013

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The extremely fast and efficient folding transition (in seconds) of globular proteins led to the search for some unifying principles embedded in the physics of the folding polypeptides. Most of the proposed mechanisms highlight the role of local interactions that stabilize secondary structure elements or a folding nucleus as the starting point of the folding pathways, i.e., a "bottom-up" mechanism. Nonlocal interactions were assumed either to stabilize the nucleus or lead to the later steps of coalescence of the secondary structure elements. An alternative mechanism was proposed, an "up-down" mechanism in which it was assumed that folding starts with the formation of very few non-local interactions which form closed long loops at the initiation of folding. The possible biological advantage of this mechanism, the "loop hypothesis", is that the hydrophobic collapse is associated with ordered compactization which reduces the chance for degradation and misfolding. In the present review the experiments, simulations and theoretical consideration that either directly or indirectly support this mechanism are summarized. It is argued that experiments monitoring the time-dependent development of the formation of specifically targeted early-formed sub-domain structural elements, either long loops or secondary structure elements, are necessary. This can be achieved by the timeresolved FRET-based "double kinetics" method in combination with mutational studies. Yet, attempts to improve the time resolution of the folding initiation should be extended down to the sub-microsecond time regime in order to design experiments that would resolve the classes of proteins which first fold by local or non-local interactions.

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Short‐Distance FRET Applied to the Polypeptide Chain

Jacob

Nau

2012

Protein and Peptide Folding, Misfolding, and Non‐Folding

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Determination of intramolecular distance distribution during protein folding on the millisecond timescale

Cited by 53 publications

References 28 publications

Watching proteins fold one molecule at a time

Watching proteins fold one molecule at a time

The loop hypothesis: contribution of early formed specific non-local interactions to the determination of protein folding pathways

Short‐Distance FRET Applied to the Polypeptide Chain

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