Kinetic Studies of the Folding of Heterodimeric Monellin: Evidence for Switching between Alternative Parallel Pathways

Aghera, Nilesh; Udgaonkar, Jayant B.

doi:10.1016/j.jmb.2012.04.019

Cited by 18 publications

(47 citation statements)

References 82 publications

(111 reference statements)

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“…where [8] (see SI for the fits for several mutants of I27 using the double exponential model) and for monellin [9], that there is upward curvature in the (Table S1) suggests that although the double exponential model above fits the data, inferring the nature of the TSE requires entirely new set of experiments along the lines reported by Clarke and coworkers [8].…”

Section: Smd In Denaturant-induced Unfoldingmentioning

confidence: 84%

Force-dependent switch in protein unfolding pathways and transition-state movements

Zhuravlev

Hinczewski

Chakrabarti

et al. 2016

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

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Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments. Unfolding rate, k u (f ), as a function of force f, obtained in single-molecule pulling experiments on src SH3 domain, exhibits upward curvature on a log k u (f ) plot. Similar observations were reported for other proteins for the unfolding rate k u ([C]). These findings imply unfolding in these single-domain proteins involves a switch in the pathway as f or [C] is increased from a low to a high value. We provide a unified theory demonstrating that if log k u as a function of a perturbation (f or [C]) exhibits upward curvature then the underlying energy landscape must be strongly multidimensional. Using molecular simulations we provide a structural basis for the switch in the pathways and dramatic shifts in the transition-state ensemble (TSE) in src SH3 domain as f is increased. We show that a single-point mutation shifts the upward curvature in log k u (f ) to a lower force, thus establishing the malleability of the underlying folding landscape. Our theory, applicable to any perturbation that affects the free energy of the protein linearly, readily explains movement in the TSE in a β-sandwich (I27) protein and single-chain monellin as the denaturant concentration is varied. We predict that in the force range accessible in laser optical tweezer experiments there should be a switch in the unfolding pathways in I27 or its mutants.protein folding | parallel pathways | single-molecule force spectroscopy T hat single-domain proteins should fold by parallel or multiple pathways emerges naturally from theoretical considerations (1-3) and computational studies (4-7). The generality of the conclusions reached in the theoretical studies is sufficiently compelling, which makes it surprising that they are not routinely demonstrated in typical ensemble folding experiments. The reasons for the difficulties in directly observing parallel folding or unfolding pathways in monomeric proteins can be appreciated based on the following arguments. Consider a protein that reaches the folded state by two different pathways. The ratio of flux through these pathways is proportional to exp, where ΔG ‡ L and ΔG ‡ H are, respectively, the free energy barriers separating the folded and unfolded states along the two pathways, k B is the Boltzmann constant, and T is the temperature. Ifis large compared with k B T then the experimental detection of flux through the high free energy barrier pathway (H) is unlikely. External perturbations (such as mechanical force f or denaturants ½C ) could reduce ΔΔG ‡ . However, the values of f (or ½C ) should fall in an experimentally accessible range for detecting a potential switch between pathways. Despite these inherent limitations, Clarke and coworkers showed in a most insightful study that unfolding of Ig domain (I27) induced by denaturants occurs by parallel routes (8). Subsequently, additional experiments on single-chain monellin (9), ...

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Section: Smd In Denaturant-induced Unfoldingmentioning

confidence: 84%

Force-dependent switch in protein unfolding pathways and transition-state movements

Zhuravlev

Hinczewski

Chakrabarti

et al. 2016

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

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“…The sweet plant protein monellin and its single‐chain derivatives have recently become attractive model systems to study dynamic processes such as protein folding/unfolding and aggregation . Natural monellin is a heterodimeric protein that was originally isolated from the berry of the African plant Dioscoreophyllum cuminisii .…”

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confidence: 99%

“…At low pH, they refold easily even after boiling. The reliability and reversibility of this process was the reason for monellins to be successfully used in many folding studies . The results of these studies have also been instrumental to depict the correlation between folding intermediates and the beginning of aggregation processes.…”

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confidence: 99%

Influence of pH on the structure and stability of the sweet protein MNEI

et al. 2016

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Edited by Stuart FergusonMNEI is a single-chain derivative of the sweet protein monellin that, in recent years, has become an accepted model for studying protein dynamic properties such as folding and aggregation. Although MNEI is very resistant at acidic pH, exposure to neutral or alkaline pH strongly affects its stability. We have performed a thorough NMR study of the dynamic properties of MNEI at different pHs. The results demonstrate that, at physiological temperature, exposure to higher pH increases MNEI flexibility. The changes, originating from a well-defined region in the protein, are transmitted to the whole structure and are likely to be the key for triggering unfolding processes.

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“…(i) Previously, we showed that there are two unfolding pathways even if F is applied between the N and C termini (8). The same inferences were drawn by noting upward curvature in the denaturant-dependent unfolding rates of I27 (6) and monellin (7). (ii) The major reason why Alberti questions the relevance of several experiments, which have used multiple pulling directions to map the folding landscape of proteins, is that in the proteins he lists (1), the N and C termini are solventexposed.…”

mentioning

confidence: 59%

Reply to Alberti: Are in vitro folding experiments relevant in vivo?

Zhuravlev¹,

Hinczewski²,

Chakrabarti³

et al. 2016

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

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In his letter, Alberti (1) does not challenge any of the central results in our paper (2), including the main proof that upward curvature in the logarithm of the unfolding rate of a protein as a function of an applied mechanical force implies that underlying energy landscape is multidimensional. However, he wonders if the switch in pathway in Src tyrosine kinase SH3 (Src homology 3) domain discovered in single-molecule pulling experiments (3, 4) applies to "living cells/ physiological settings," an issue that is not germane to our work (2), and, by inference, to innumerable in vitro ensemble (5-7) and single-molecule studies on proteins. Although Alberti's view point has merits, it can be asserted that such biophysics studies and countless others have literally revolutionized modern biology.A few other points are worth making. (i) Previously, we showed that there are two unfolding pathways even if F is applied between the N and C termini (8). The same inferences were drawn by noting upward curvature in the denaturant-dependent unfolding rates of I27 (6) and monellin (7). (ii) The major reason why Alberti questions the relevance of several experiments, which have used multiple pulling directions to map the folding landscape of proteins, is that in the proteins he lists (1), the N and C termini are solventexposed. Although this fact generally appears to be correct, there are a number of proteins in which this fact is not the case (9). (iii) The proposed link (1) between chaperones and pulling experiments is even more tenuous. The mechanism of how eukaryotic Hsp 70 interacts with substrate proteins (SPs) has not been quantitatively elucidated. In bacterial chaperonin (GroEL/GroES), we showed that SPs could experience an unfolding force of ≈ 10 pN (10) [confirmed recently (11)] due to ATP-driven GroEL domain movements. It is unlikely that this force is directed along the N and C termini of the misfolded SP. (iv) The transit through the tunnel of the ribosome is facilitated by the nascent protein experiencing a mechanical force. However, because the translational rate is slow, force direction on the protein could vary, especially as it reaches the vestibule of the ribosome. Subsequent folding is a complicated process involving trigger factor and folding in confined spaces, with force being irrelevant.It is a matter of opinion whether precise in vitro studies are valuable in understanding physiological processes. Only by using theory, experiments, and simulations in well-defined systems can quantitative understanding of the functions of proteins under cellular conditions be achieved.

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Kinetic Studies of the Folding of Heterodimeric Monellin: Evidence for Switching between Alternative Parallel Pathways

Cited by 18 publications

References 82 publications

Force-dependent switch in protein unfolding pathways and transition-state movements

Force-dependent switch in protein unfolding pathways and transition-state movements

Influence of pH on the structure and stability of the sweet protein MNEI

Reply to Alberti: Are in vitro folding experiments relevant in vivo?

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