An instrument for fast acquisition of fluorescence decay curves at picosecond resolution designed for “double kinetics” experiments: Application to fluorescence resonance excitation energy transfer study of protein folding

Ishay, Eldad Ben; Hazan, Gershon; Rahamim, Gil; Amir, Dan; Haas, Elisha

doi:10.1063/1.4737632

Cited by 15 publications

(23 citation statements)

References 30 publications

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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%

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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Section: The Loop Hypothesismentioning

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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“…This is enabled by time-resolved dynamic non-radiative excitation energy transfer [time-resolved fluorescence resonance energy transfer (trFRET)] measurements in the "double kinetics" mode. [17][18][19] In the double kinetics experiment, the fluorescence decay curves of the probes (donor and/or acceptor), occurring on a nanosecond timescale, are determined at selected time points during the fast refolding transition of the protein, occurring on a microsecond-to-second timescales. This experiment can monitor the time-dependent status of ensembles of selected subdomain structural elements in the whole protein molecule, in situ in real time.…”

Section: Introductionmentioning

confidence: 99%

Fast Subdomain Folding Prior to the Global Refolding Transition of E. coli Adenylate Kinase: A Double Kinetics Study

Ishay¹,

Rahamim²,

Orevi³

et al. 2012

Journal of Molecular Biology

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“…Preparation of a series of labeled mutants of one protein enables monitoring of the conformational changes in each sub-domain structure. Very fast data collection enables determination of a series of sequential distance distributions along the folding pathway [99,[109][110][111][112][113][114][115][116][117]. Such experiments yield meaningful information describing specific conformational changes and the order of their occurrence along the folding pathway.…”

Section: Experimental Strategymentioning

confidence: 99%

“…The microsecond folding kinetics detected by FRET and other probes was used to characterize the ensemble of fast collapsed disordered molecules, which include a few specific interactions [105,113,117,151,166,184]. Barrier-limited chain contraction and specific sub-domain interactions, in particular by isoleucine, leucine, and valine (ILV) clusters, were observed upon transfer of the GndHCl denatured state ensemble to native-like conditions [166,185,189,190].…”

Section: The Ensemble Of Collapsed Moleculesmentioning

confidence: 99%

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Fast closure of long loops at the initiation of the folding transition of globular proteins studied by time-resolved FRET-based methods

Orevi

Rahamim

Shemesh

et al. 2014

Bio-Algorithms and Med-Systems

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The protein folding problem would be considered "solved" when it will be possible to "read genes", i.e., to predict the native fold of proteins, their dynamics, and the mechanism of fast folding based solely on sequence data. The long-term goal should be the creation of an algorithm that would simulate the stepwise mechanism of folding, which constrains the conformational space and in which random search for stable interactions is possible. Here, we focus attention on the initial phases of the folding transition starting with the compact disordered collapsed ensemble, in search of the initial sub-domain structural biases that direct the otherwise stochastic dynamics of the backbone. Our studies are designed to test the "loop hypothesis", which suggests that fast closure of long loop structures by non-local interactions between clusters of mainly non-polar residues is an essential conformational step at the initiation of the folding transition of globular proteins. We developed and applied experimental methods based on time-resolved resonance excitation energy transfer (trFRET) measurements combined with fast mixing methods and studied the initial phases of the folding of Escherichia coli adenylate kinase (AK). A series of AK mutants were prepared, in which the ends of selected backbone segments that form long closed loops or secondary structure elements were labeled by donors and acceptors of excitation energy. The end-to-end distance distributions of such segments were determined under equilibrium and during the fast folding transitions. These experiments show that three out of seven long loops that were labeled in the AK molecule are closed very early in the transition. The N terminal 26-residue loop (loop I) is closed in < 200 μs after the initiation of folding, while the β strand included in loop I is still disordered. The closure of the second 44-residue loop (loop II, which starts at the end of loop I) is also complete within < 300 μs. Four other loops as well as five secondary structures of the CORE domain of AK (an α helix and four β strands) are formed at a late step, at a rate of 0.5 ± 0.3 s -1 , the rate of the cooperative folding of the molecule. These experiments reveal a hierarchically ordered pathway of folding of the AK molecule, ranging from microseconds to seconds. The results reviewed here, obtained mainly from studying a small number of model proteins, support the counterintuitive mechanism whereby non-local interactions are effective in the initiation of the folding pathways. The experiments presented demonstrate the importance of mapping the rates of sub-domain structural transitions along the folding transition, in situ, in the context of the other sections of the chain, whether folded or disordered. These experiments also show the power of the time-resolved FRET measurements in achieving this goal. A large body of data obtained by theoretical and experimental studies that support, or can accommodate, the loop hypothesis is reviewed. We suggest that mapping multiple sub-domain structural tr...

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An instrument for fast acquisition of fluorescence decay curves at picosecond resolution designed for “double kinetics” experiments: Application to fluorescence resonance excitation energy transfer study of protein folding

Cited by 15 publications

References 30 publications

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

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

Fast Subdomain Folding Prior to the Global Refolding Transition of E. coli Adenylate Kinase: A Double Kinetics Study

Fast closure of long loops at the initiation of the folding transition of globular proteins studied by time-resolved FRET-based methods

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