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

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

doi:10.1016/j.jmb.2012.08.001

Cited by 22 publications

(54 citation statements)

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“…3) (Orevi et al 2009). Stopped flow mixing was used for the initiation of refolding (Ben Ishay et al 2012b). Upon transition to folding conditions, the initial transient collapsed ensemble of AK conformers appeared disordered and refolded to a native structure through an apparent two-state mechanism with a rate constant of 0.3 s −1 .…”

Section: Direct and Indirect Findings Consistent With The Closed Longmentioning

confidence: 99%

“…Upon transition to folding conditions, the initial transient collapsed ensemble of AK conformers appeared disordered and refolded to a native structure through an apparent two-state mechanism with a rate constant of 0.3 s −1 . At the end of the dead-time of the stopped flow device (5 ms), the distributions of several intramolecular distances were broad, in particular those that are far apart along the primary sequence of the chain, with a mean distance characteristic of the collapsed globule (e.g., the distance between residues 18 and 203, 28 and 203, 58 and 86, and 73 and 203) (Fig 3) (Ben Ishay et al 2012b). Several secondary structure elements that were monitored showed slow transition to the native mean end-to-end distance at the same rate as the cooperative folding transition (0.3 s −1 ).…”

Section: Direct and Indirect Findings Consistent With The Closed Longmentioning

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%

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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: Direct and Indirect Findings Consistent With The Closed Longmentioning

confidence: 99%

Section: Direct and Indirect Findings Consistent With The Closed Longmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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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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“…by the double kinetics experiment (Figures 8 and 9). The micromilli second kinetics of change of < E(t) > between the ends of the three N terminal loops, I, II, and III, were native-like within the dead time of the stopped flow device [111,201]. In sharp contrast, the FRET efficiency between the ends of loops V, VI, and VII increased at a much slower rate, comparable to the rate of the global folding transition, (192)(193)(194)(195)(196)(197)(198) 188 and 203 …”

Section: Microsecond Kineticsmentioning

confidence: 94%

“…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%

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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How cooperative are protein folding and unfolding transitions?

2016

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A thermodynamically and kinetically simple picture of protein folding envisages only two states, native (N) and unfolded (U), separated by a single activation free energy barrier, and interconverting by cooperative two-state transitions. The folding/unfolding transitions of many proteins occur, however, in multiple discrete steps associated with the formation of intermediates, which is indicative of reduced cooperativity. Furthermore, much advancement in experimental and computational approaches has demonstrated entirely non-cooperative (gradual) transitions via a continuum of states and a multitude of small energetic barriers between the N and U states of some proteins. These findings have been instrumental towards providing a structural rationale for cooperative versus noncooperative transitions, based on the coupling between interaction networks in proteins. The cooperativity inherent in a folding/unfolding reaction appears to be context dependent, and can be tuned via experimental conditions which change the stabilities of N and U. The evolution of cooperativity in protein folding transitions is linked closely to the evolution of function as well as the aggregation propensity of the protein. A large activation energy barrier in a fully cooperative transition can provide the kinetic control required to prevent the accumulation of partially unfolded forms, which may promote aggregation. Nevertheless, increasing evidence for barrier-less "downhill" folding, as well as for continuous "uphill" unfolding transitions, indicate that gradual non-cooperative processes may be ubiquitous features on the free energy landscape of protein folding.

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

Cited by 22 publications

References 50 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 closure of long loops at the initiation of the folding transition of globular proteins studied by time-resolved FRET-based methods

How cooperative are protein folding and unfolding transitions?

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