Effects of denaturants on amide proton exchange rates: a test for structure in protein fragments and folding intermediates

Loftus, D. J.; Gbenle, George O.; Kim, Peter; Baldwin, Robert L.

doi:10.1021/bi00354a036

Cited by 58 publications

(40 citation statements)

References 41 publications

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“…1C) is consistent with the small (less than an order of magnitude) effect of 8 M urea on k HX for the BPTI amides that exchange by subglobal fluctuations (29). Because urea has a similarly small effect on k int (61,62), this observation indicates that the C⇄O equilibrium is much less sensitive to urea than the global N⇄U equilibrium, consistent with a modest increase of solvent exposure in the O state.…”

Section: Discussionsupporting

confidence: 62%

How amide hydrogens exchange in native proteins

Persson

Halle

2015

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

127

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Amide hydrogen exchange (HX) is widely used in protein biophysics even though our ignorance about the HX mechanism makes data interpretation imprecise. Notably, the open exchange-competent conformational state has not been identified. Based on analysis of an ultralong molecular dynamics trajectory of the protein BPTI, we propose that the open (O) states for amides that exchange by subglobal fluctuations are locally distorted conformations with two water molecules directly coordinated to the N-H group. The HX protection factors computed from the relative O-state populations agree well with experiment. The O states of different amides show little or no temporal correlation, even if adjacent residues unfold cooperatively. The mean residence time of the O state is ∼100 ps for all examined amides, so the large variation in measured HX rate must be attributed to the opening frequency. A few amides gain solvent access via tunnels or pores penetrated by water chains including native internal water molecules, but most amides access solvent by more local structural distortions. In either case, we argue that an overcoordinated N-H group is necessary for efficient proton transfer by Grotthuss-type structural diffusion.protein dynamics | hydration | proton transfer | MD simulation | BPTI B efore the tightly packed and densely H-bonded structure of globular proteins had been established, Hvidt and Linderstrøm-Lang (1) showed that all backbone amide hydrogens of insulin exchange with water hydrogens, implying that all parts of the polypeptide backbone are, at least transiently, exposed to solvent. In the following 60 y, hydrogen exchange (HX), usually monitored by NMR spectroscopy (2) or mass spectrometry (3), has been widely used to study protein folding and stability (4-10), structure (11, 12), flexibility and dynamics (13-15), and solvent accessibility and binding (16,17), often with single-residue resolution. However, because the exchange mechanism is unclear, HX data from proteins can, at best, be interpreted qualitatively (18)(19)(20)(21)(22)(23)(24)(25).Under most conditions, amide HX is catalyzed by hydroxide ions (26, 27) at a rate that is influenced by inductive and steric effects from adjacent side chains (28). For unstructured peptides, HX is a slow process simply because the hydroxide concentration is low. For example, at 25°C and pH 4, HX occurs on a time scale of minutes. Under similar conditions, amides buried in globular proteins exchange on a wide range of time scales, extending up to centuries. HX can only occur if the amide is exposed to solvent, so conformational fluctuations must be an integral part of the HX mechanism (18).Under sufficiently destabilizing conditions HX occurs from the denatured-state ensemble, but under native conditions few amides exchange by such global unfolding (9, 29-31). For example, in bovine pancreatic trypsin inhibitor (BPTI), 8 amides in the core β-sheet exchange by global unfolding under native conditions (7, 32), whereas the remaining 45 amides require less extensive conforma...

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Section: Discussionsupporting

confidence: 62%

How amide hydrogens exchange in native proteins

Persson

Halle

2015

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

127

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“…An average k int value for ubiquitin was determined from the pH (i.e., where k int ϭ 10 pH-5 min Ϫ1 or 1.7 s Ϫ1 at pH 7.0) [11]. Values for k int , have been shown to very slightly change (e.g., Ͻ10-fold) with denaturant concentration [14]. However, for the purposes of this work, no denaturant dependence was assigned to k int values.…”

Section: Ubiquitinmentioning

confidence: 99%

Accuracy of SUPREX (stability of unpurified proteins from rates of H/D exchange) and MALDI mass spectrometry-derived protein unfolding free energies determined under non-EX2 exchange conditions

Dai

Fitzgerald

2006

J. Am. Soc. Mass Spectrom.

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Described here is the impact of so-called non-EX2 exchange behavior on the accuracy of protein unfolding free energies (i.e., ⌬G u values) and m values (i.e.,-␦⌬G u /␦[denaturant] values) determined by an H/D exchange and mass spectrometry-based technique termed stability of unpurified proteins from rates of H/D exchange (SUPREX). Both experimental and theoretical results on a model protein, ubiquitin, reveal that reasonably accurate thermodynamic parameters for its folding reaction can be determined by SUPREX even when H/D exchange data is collected in a non-EX2 regime. Not surprisingly, the theoretical results reported here on a series of hypothetical protein systems with a wide range of biophysical properties show that the accuracy of SUPREX-derived ⌬G u and m values is compromised for many proteins when analyses are performed at high pH (e.g., pH 9) and for selected proteins with specific biophysical parameters (e.g., slow folding rates) when analyses are performed at lower pH. Of more significance is that the experimental and theoretical results reveal a means by which problems with non-EX2 exchange behavior can be detected in the SUPREX experiment without prior knowledge of the protein's biophysical properties.

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“…The random coil exchange rate constants, k x u ( i ) for each residue were calculated as described by Bai et al (1993) and Connelly et al (1993) taking into account temperature, primarystructure effects, the pH of the exchange pulse, and the isotope effect of D-H versus H-D exchange. Although the effect of the presence of 0.15 M GuDCl in the exchange mixture on the amide exchange rates is difficult to quantify, previous studies have indicated that at most an increase of 5-10% in the random coil exchange rate constants may be observed (Loftus et al, 1986). Consequently, no attempt was made to correct for the presence of 0.…”

Section: Analysis Of the Quenched-flow D-h Exchange Kineticsmentioning

confidence: 99%

Fast folding of a prototypic polypeptide: The immunoglobulin binding domain of streptococcal protein G

1994

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The folding of the small (56 residues) highly stable B1 immunoglobulin binding domain (GBl) of streptococcal protein G has been investigated by quenched-flow deuterium-hydrogen exchange. This system represents a paradigm for the study of protein folding because it exhibits no complicating features superimposed upon the intrinsic properties of the polypeptide chain. Collapse to a semicompact state exhibiting partial order, reflected in protection factors for ND-NH exchange up to 10-fold higher than that expected for a random coil, occurs within the dead time ( 5 1 ms) of the quenched flow apparatus. This is followed by the formation of the fully native state, as monitored by the fractional proton occupancy of 26 backbone amide groups spread throughout the protein, in a single rapid concerted step with a half-life of 5.2 ms at 5 "C. Keywords: folding intermediates; protein folding; quenched-flow deuterium-hydrogen exchangeAlthough folding intermediates have attracted considerable attention over the last few years (Kuwajima, 1989;Kim & Baldwin, 1990;Matthews, 1993;Fersht & Dill, 1994;Ptitsyn, 1994), new experimental and theoretical studies have cast doubt on their importance as obligate states on the folding pathway of proteins. In particular, theoretical studies and computer simulations of the folding of simplified models for proteins has led to the proposal of a 3-stage random search mechanism of folding, involving the initial rapid collapse from a random coil state to a random semicompact globule, followed by a rate-determining search for one of the many possible transition states from which the chain rapidly folds to the native state (Camacho & Thirumalai, 1993; Chan & Dill, 1994;Sali et al., 1994). The results of this theoretical study further stimulate the debate (Baldwin, 1994;Creighton, 1994), initiated by a provocative paper by Sosnick et al. (1994), challenging the widely held assumption that transient, partly folded intermediates are responsible for the rapid folding of proteins. In 2 related studies (Elove et al., 1994; Sosnick et al., 1994), it was shown that for cytochrome cat least, folding can occur extremely rapidly if trapped, incorrectly ligated heme intermediates are avoided. Indeed, the authors demonstrated that the experimental conditions could be adjusted such that up to 50-70% of the protein can attain the correct heme ligation directly and thus fold rapidly to the native state.These results reopen the debate of whether slow processes observed in folding reactions are of any significance kinetically for native protein folding or are simply the result of species trapped by optional barriers created by conditions specific to the folding solution. Such misorganization of the polypeptide chain may be connected to particular chemical requirements for the folding of the protein under investigation. These include side-chain protonation states, sulfhydryl chemistry, cis-trans proline isomerizations, and the presence of prosthetic groups or metal binding sites. To date, all experimental stu...

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Effects of denaturants on amide proton exchange rates: a test for structure in protein fragments and folding intermediates

Cited by 58 publications

References 41 publications

How amide hydrogens exchange in native proteins

How amide hydrogens exchange in native proteins

Accuracy of SUPREX (stability of unpurified proteins from rates of H/D exchange) and MALDI mass spectrometry-derived protein unfolding free energies determined under non-EX2 exchange conditions

Fast folding of a prototypic polypeptide: The immunoglobulin binding domain of streptococcal protein G

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