Isotope effects in peptide group hydrogen exchange

Connelly, Gregory P.; Bai, Yawen; Jeng, Mei-Fen; Englander, S. Walter

doi:10.1002/prot.340170111

Cited by 357 publications

(369 citation statements)

References 26 publications

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“…This can be demonstrated using ESI-MS by allowing exchange to occur under conditions where the intrinsic amide exchange rate is significantly lower and hence an initial time point can be measured more readily. At pH 3.5 and 20°C, the average intrinsic exchange rate is reduced to as low as 0.005 s Ϫ1 , with a corresponding half-life of about 2.5 min [39]. Our experiments conducted at pH 3.5 under room temperature show in all cases the 2 min exchange time point for CRABP I is identical regardless the presence of ligands, with 107 Ϯ 3 amides protected, justifying the first restriction above (data not shown).…”

Section: Protein Dynamics and Ligand Binding-hdx Measurements At Neutsupporting

confidence: 76%

Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS

Kaltashov

Eyles

2003

J. Am. Soc. Mass Spectrom.

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Direct mass spectrometric characterization of interactions between proteins and small hydrophobic ligands often poses a serious problem due to the complex instability in the gas phase. We have developed a method that probes the efficacy of ligand-protein interactions indirectly by monitoring changes in protein flexibility. The latter is assessed quantitatively using a combination of charge state distribution analysis and amide hydrogen exchange under both native and mildly denaturing conditions. The method was used to evaluate binding of a model protein cellular retinoic acid binding protein I to its natural ligand all-trans retinoic acid (RA), isomers 13-cis-and 9-cis-RA, and retinol, yielding the following order of ligand affinities: All-trans RA Ͼ 9-cis RA Ͼ 13-cis RA, with no detectable binding of retinol. This order is in agreement with the results of earlier fluorimetric titration studies. Furthermore, binding energy of the protein to each of retinoic acid isomers was determined based on the measured hydrogen exchange kinetics data acquired under native conditions. (J Am Soc Mass Spectrom 2003, 14, 506 -515)

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Section: Protein Dynamics and Ligand Binding-hdx Measurements At Neutsupporting

confidence: 76%

Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS

Kaltashov

Eyles

2003

J. Am. Soc. Mass Spectrom.

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“…These various factors have been accurately calibrated in small structureless amide models (Molday et al 1972;Connelly et al 1993). From these calibrations, the second-order HX rate constant that these factors produce under any given conditions, known as the intrinsic rate constant (k int ), can be computed, and the expected chemical HX rate (k ch ) can be obtained as k int multiplied by the catalyst concentration [k ch =k int (cat)].…”

Section: Hx Chemistrymentioning

confidence: 99%

Protein folding and misfolding: mechanism and principles

Englander

Mayne

Mallela

2007

Quart. Rev. Biophys.

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172

267

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Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors are responsible for 3-state and heterogeneous kinetic folding. The protein folding problemThe search for protein folding pathways and the principles that guide them has proven to be one of the most difficult problems in all of structural biology. Biochemical pathways have almost universally been solved by isolating the pathway intermediates and determining their structures. This approach fails for protein folding pathways. Folding intermediates only live for <1 s and they cannot be isolated and studied by the usual structural methods.The protein folding problem has been attacked by the entire range of fast spectroscopic methods which are able to track folding in real time. Much valuable information has been obtained but mainly of a kinetic nature. Kinetic methods do not describe the structure of the

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“…The intrinsic rate of exchange from the open state, kin,, can be predicted from unstructured peptide data, thus allowing and the determination of the equilibrium constant and the standard free energy change for the opening reaction (Molday et al, 1972;Connelly et al, 1993;Bai et al, 1993): kex = kinr(kop/kcL) = k,,,KOp when kCl kin,,…”

Section: T Kc Kc Imentioning

confidence: 99%

Conformational stability of ribonuclease T1 determined by hydrogen‐deuterium exchange

1997

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The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25,40, 45, and 50 "C at pD 5.6 and at 40 and 45 "C at pD 6.6. The hydrogen exchange rate constants of the hydrogen-bonded residues varied over eight orders of magnitude at 25°C with 13 residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central P-pleated sheet. The residues located in the a-helix and the remaining strands of the P-sheet exhibited exchange behaviors consistent with exchange occumng due to local structural fluctuations. For several residues at 25"C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal/mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively if the cis-trans isomerization of the two cis prolines, Pro-39 and Pro-55, is taken into account. The cis-trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolded state will be 2.6 kcal/mol higher at 25 "C when the two prolines are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schmid FX. 1996. Kinetic analysis of the unfolding and refolding of ribonuclease T1 by a stopped-flow double-mixing technique. Biochemistry 35: 5550-5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free energy unfolded state in which Pro-55 and Pro-39 are still predominantly in the cis conformation. When the conformational stabilities determined by hydrogen exchange are corrected for the proline isomerization equilibrium, the results are in excellent agreement with those from an analysis of urea denaturation curves.Keywords: hydrogen-deuterium exchange; NMR; protein folding; ribonuclease TI The principal factors governing the conformational stability of proteins in solution are still not fully understood. While traditional methods of denaturant titration and calorimetry provide valuable information on global stability, no direct information on the conformational dynamics in solution is gained. The internal mobility of proteins can be measured, however, by following the hydrogendeuterium exchange (HX) kinetics of individual backbone amide hydrogens. Hydrogen exchange experiments take advantage of the fact that buried or hydrogen-bonded protons exchange with solvent several orders of magnitude more slowly than exposed or nonhydrogen-bonded protons. More importantly, large differences are also observed in the exchange kinetics of hydrogen-bonded residues. These differences are a direct reflection of the local stability in the region of the hydrogen bond, thus allowing an assessment of Reprint requests to: Frank M. Raushel, Department of Chemistry, Texas A & M University, College Station, Texas 77843; e-mail: r...

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Isotope effects in peptide group hydrogen exchange

Cited by 357 publications

References 26 publications

Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS

Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS

Protein folding and misfolding: mechanism and principles

Conformational stability of ribonuclease T1 determined by hydrogen‐deuterium exchange

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