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Hwang, Tsang‐Lin; Zijl, Peter van; Mori, Susumu

doi:10.1023/a:1008276004875

Cited by 329 publications

(256 citation statements)

References 13 publications

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“…HX rates were determined with 1D spectra by using two sequences. At pH Ͼ9.5, at which exchange occurs faster than 0.5 s Ϫ1 , rates were measured by using the CLEANEX pulse sequence (19). This sequence acts by dephasing the protein NMR resonances while maintaining coherence of solvent protons.…”

Section: Methodsmentioning

confidence: 99%

Barrier-limited, microsecond folding of a stable protein measured with hydrogen exchange: Implications for downhill folding

Meisner

Sosnick

2004

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

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Folding experiments are conducted to test whether a covalently cross-linked coiled-coil folds so quickly that the process is no longer limited by a free-energy barrier. This protein is very stable and topologically simple, needing merely to ''zipper up,'' while having an extrapolated folding rate of kf ‫؍‬ 2 ؋ 10 5 s ؊1 . These properties make it likely to attain the elusive ''downhill folding'' limit, at which a series of intermediates can be characterized. To measure the ultra-fast kinetics in the absence of denaturant, we apply NMR and hydrogen-exchange methods. protein folding ͉ EX1 ͉ stretched exponential ͉ coiled-coil T he two-state approximation of protein folding, in which the unfolded and native states are separated by a single freeenergy barrier and no other species accumulate to a significant degree, appears to be adequate for most small proteins (1). Nevertheless, it is unsatisfying because it generally precludes identifying the individual steps involved in the folding process. To overcome this problem, experiments (2-6) have pursued the elusive theoretical prediction of ''downhill folding'' (7-9) (Fig. 1). If attainable, a downhill energy surface may allow the experimental identification of a series of intermediate states along the folding pathways that likely exists but largely has been observed only in computer simulations.Downhill-folding behavior is predicted to occur when folding rates approach the value of the attempt frequency of the barrier crossing process,Here, the barrier height does not contribute to the rate, and ⌬G ‡ Ϸ0. This limit may be likened to extreme Hammond behavior (10) in which the folding transition state moves so far to the starting condition in response to heightened stability that the transition state coincides with the unfolded state.A barrier-free reaction must be extremely rapid, but how fast is fast enough? Estimates from measured rates of helix, loop, and hairpin formation, reaction-rate and polymer-collapse theories, and folding simulations suggest that the minimum possible folding time constant is on the order of 1 s (6). Because of the increased ruggedness of the energy landscape, downhill folders of high stability would have proportionally slower folding rates, as would those with longer sequences and greater ␤-sheet content. After correcting for length and stability, 12 proteins with predicted barrier-free time constants of Ͻ100 s have been identified as potential downhill folders (6).The folding speed and stability of the covalently cross-linked variant of the dimeric yeast transcription factor GCN4 coiledcoil places it with the top members of this group (11,12). For the version GCN4p2C, with the Gly-Gly-Cys tether located at the C terminus, the extrapolated folding time constant is f Ϸ 10 s and Ͻ1 s when normalized for the high stability of the molecule (6). This fast folding rate may be due to a simple topology that requires that the helices only ''zipper-up.'' These qualities identify it among the most likely candidates for downhill folding. Here, we invest...

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Section: Methodsmentioning

confidence: 99%

Barrier-limited, microsecond folding of a stable protein measured with hydrogen exchange: Implications for downhill folding

Meisner

Sosnick

2004

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

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“…Inertia moments were calculated with the program pdb_inertia [39]. CLEANEX experiments [40] were performed in order to monitor the accessibility of amide moieties to the solvent in the two separate proteins and in equimolar mixtures of them.…”

Section: Nmr Experimentsmentioning

confidence: 99%

“…CLEANEX experiments [40] were performed to monitor the change in hydration properties of the two proteins upon complex formation. Indeed, it is expected that amide moieties in regions involved in specific intermolecular contacts would become protected from exchange with H 2 O upon interaction with the partner.…”

Section: Nmr Experimentsmentioning

confidence: 99%

A further investigation of the cytochrome b 5–cytochrome c complex

et al. 2003

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The interaction of reduced rabbit cytochrome b 5 with reduced yeast iso-1 cytochrome c has been studied through the analysis of 1 H-15 N HSQC spectra, of 15 N longitudinal (R 1 ) and transverse (R 2 ) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b 5 has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b 5 .

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“…CLEANEX-PM experiment, as described by Hwang et al (28). Additional experimental procedures are described in detail in SI Text.…”

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confidence: 99%

Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

Murphy

Korzhnev

Ceccarelli

et al. 2007

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

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The Par-1/MARK protein kinases play a pivotal role in establishing cellular polarity. This family of kinases contains a unique domain architecture, in which a ubiquitin-associated (UBA) domain is located C-terminal to the kinase domain. We have used a combination of x-ray crystallography and NMR dynamics experiments to understand the interaction of the human (h) MARK3 UBA domain with the adjacent kinase domain as compared with ubiquitin. The x-ray crystal structure of the linked hMARK3 kinase and UBA domains establishes that the UBA domain forms a stable intramolecular interaction with the N-terminal lobe of the kinase domain. However, solution-state NMR studies of the isolated UBA domain indicate that it is highly dynamic, undergoing conformational transitions that can be explained by a folding-unfolding equilibrium. NMR titration experiments indicated that the hMARK3 UBA domain has a detectable but extremely weak affinity for monoubiquitin, which suggests that conformational instability of the isolated hMARK3 UBA domain attenuates binding to ubiquitin despite the presence of residues typically involved in ubiquitin recognition. Our data identify a molecular mechanism through which the hMARK3 UBA domain has evolved to bind the kinase domain, in a fashion that stabilizes an open conformation of the Nand C-terminal lobes, at the expense of its capacity to engage ubiquitin. These results may be relevant more generally to the 30% of UBA domains that lack significant ubiquitin-binding activity, and they suggest a unique mechanism by which interaction domains may evolve new binding properties.catalytic domain ͉ dynamics ͉ Par-1 ͉ relaxation ͉ ubiquitin-associated

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Cited by 329 publications

References 13 publications

Barrier-limited, microsecond folding of a stable protein measured with hydrogen exchange: Implications for downhill folding

Barrier-limited, microsecond folding of a stable protein measured with hydrogen exchange: Implications for downhill folding

A further investigation of the cytochrome b 5–cytochrome c complex

Conformational instability of the MARK3 UBA domain compromises ubiquitin recognition and promotes interaction with the adjacent kinase domain

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