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

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

doi:10.1007/s12551-013-0113-3

Cited by 16 publications

(20 citation statements)

References 117 publications

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“…A quantity of work has focused on the character of the unfolded state and its possible role in guiding subsequent folding (3,4,6,(40)(41)(42). Is the denatured state ensemble (DSE) compact at the start of the folding process?…”

Section: Discussionmentioning

confidence: 99%

Folding of a large protein at high structural resolution

Walters

Mayne²,

Hinshaw³

et al. 2013

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

107

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Kinetic folding of the large two-domain maltose binding protein (MBP; 370 residues) was studied at high structural resolution by an advanced hydrogen-exchange pulse-labeling mass-spectrometry method (HX MS). Dilution into folding conditions initiates a fast molecular collapse into a polyglobular conformation (<20 ms), determined by various methods including small angle X-ray scattering. The compaction produces a structurally heterogeneous state with widespread low-level HX protection and spectroscopic signals that match the equilibrium melting posttransition-state baseline. In a much slower step (7-s time constant), all of the MBP molecules, although initially heterogeneously structured, form the same distinct helix plus sheet folding intermediate with the same time constant. The intermediate is composed of segments that are distant in the MBP sequence but adjacent in the native protein where they close the longest residue-to-residue contact. Segments that are most HX protected in the early molecular collapse do not contribute to the initial intermediate, whereas the segments that do participate are among the less protected. The 7-s intermediate persists through the rest of the folding process. It contains the sites of three previously reported destabilizing mutations that greatly slow folding. These results indicate that the intermediate is an obligatory step on the MBP folding pathway. MBP then folds to the native state on a longer time scale (∼100 s), suggestively in more than one step, the first of which forms structure adjacent to the 7-s intermediate. These results add a large protein to the list of proteins known to fold through distinct native-like intermediates in distinct pathways.SAXS | HDX | protein collapse | denatured state ensemble F ifty years after Anfinsen's seminal demonstration that an unfolded protein can refold spontaneously when placed under native conditions, major questions concerning the folding process remain unanswered (1, 2). What is the unfolded state like, its degree of compaction, the reality and character of residual structure before folding begins, and its possible role in guiding the folding process (3-7)? Analogous questions relate to folding intermediates and the folding pathway itself. Do proteins fold through many alternative independent pathways as earlier theoretical investigations have suggested (8-12), or do they fold through necessary intermediates in a distinct pathway (13), as a growing list of experimental observations indicate (14, 15)? To answer these questions, it will be necessary to define experimentally the intermediate forms that proteins move through on their way to the native state. The problem has been that these transient states are beyond the reach of the usual high-resolution crystallographic and NMR structural methods. Most experimental folding studies have therefore relied on low-resolution optical methods that can follow folding in real time but rarely provide the structural information necessary to resolve the basic mechanistic questions.Recent work...

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Section: Discussionmentioning

confidence: 99%

Folding of a large protein at high structural resolution

Walters

Mayne²,

Hinshaw³

et al. 2013

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

107

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“…First, the experimental results obtained within the loop hypothesis (LH) context employing FRET techniques by Haas and collaborators (i.e., with an emphasis on recent results for the early kinetics of Escherichia coli adenylate kinase (AK) folding) will be shown to lend support to the main proposal of this Feature Article. 46,47 Then, the theoretical basis for the view of protein folding presented here will be seen to naturally emerge from the so-called sequential collapse model (SCM) of Bergasa-Caceres and Rabitz, 48 based on concepts first presented in Bergasa-Caceres' Ph.D. thesis. 49 A comparison also will be made between the SCM prediction and the LH experimental results for early nonlocal contact formation in adenylate kinase.…”

Section: ■ Introductionmentioning

confidence: 99%

Nature’s Shortcut to Protein Folding

2019

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This Feature Article presents a view of the protein folding transition based on the hypothesis that Nature has built features within the sequences that enable a Shortcut to efficient folding. Nature’s Shortcut is proposed to be the early establishment of a set of nonlocal weak contacts, constituting protein loops that significantly constrain regions of the collapsed disordered protein into a native-like low-resolution fluctuating topology of major sections of the backbone. Nature’s establishment of this scaffold of nonlocal contacts is claimed to bypass what would otherwise be a nearly hopeless unaided search for the final three-dimensional structure in proteins longer than ∼100 amino acids. To support this main contention of the Feature Article, the loop hypothesis (LH) description of early folding events is experimentally tested with time-resolved Förster resonance energy transfer techniques for adenylate kinase, and the data are shown to be consistent with theoretical predictions from the sequential collapse model (SCM). The experimentally based LH and the theoretically founded SCM are argued to provide a unified picture of the role of nonlocal contacts as constituting Nature’s Shortcut to protein folding. Importantly, the SCM is shown to reliably predict key nonlocal contacts utilizing only primary sequence information. This view on Nature’s Shortcut is open to the protein community for further detailed assessment, including its practical consequences, by suitable application of advanced experimental and computational techniques.

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“…Nevertheless, we applied mainly the ensemble detection methods for several reasons: deducing the folding pathway requires ensemble characteristics; natural amino acids, or mildly modified residues can be used and the structural perturbation can be minimized; full sets of fluorescence decay curves can be collected in nanosecond time intervals; application of the double kinetics approach is more straightforward compared to the single-molecule FRET-detected (smFRET)-based methods; pairs of probes suitable for monitoring distances in the range corresponding to sub-domain structural dimensions are readily available; and the ability to resolve subpopulations of intramolecular distances, a major strength of the smFRET-based methods, is also readily provided by the ensemble-based trFRET experiments [152]. Analyses of the trFRET measurements do not yield atom-to-atom distances, and depend on the size and dynamics of the probes and on the correct determination of the Förster constant, R o .…”

Section: Mode Of Detectionmentioning

confidence: 99%

“…Five secondary structure elements in the CORE domain were labeled (Table 1), and the folding kinetics were studied by the FRET-detected stopped flow system. All of them were disordered at the initial phase of the folding pathway [110,152]. Analysis of the trFRET double kinetics experiments showed the shift of the mean endto-end distance between the ends of β strands and the rate of reduction of FWHM of the distance distributions.…”

Section: Kinetics Of Folding Of Secondary Structure Elements In the Amentioning

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

Cited by 16 publications

References 117 publications

Folding of a large protein at high structural resolution

Folding of a large protein at high structural resolution

Nature’s Shortcut to Protein Folding

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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