Mechanisms and uses of hydrogen exchange

Englander, S. Walter; Sosnick, Tobin R.; Englander, Joan J.; Mayne, Leland

doi:10.1016/s0959-440x(96)80090-x

Cited by 358 publications

(363 citation statements)

References 91 publications

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“…If amide proton exchange occurs within the lifetime of the soluble asp-C, additional signal attenuation is expected for crosspeaks involving labile protons. Hydrogen exchange experiments (Hvidt & Nielsen, 1966) have a long tradition for probing protein conformational equilibria (for recent reviews, see Englander et al, 1996;Chamberlain & Marqusee, 1997;Clarke et al, 1997). The backbone conformation as well as the average degree of deuteration of the backbone amide groups of SP-C in the insoluble aggregate were analysed by CD and FTIR spectroscopy.…”

Section: Resultsmentioning

confidence: 99%

Pulmonary surfactant‐associated polypeptide C in a mixed organic solvent transforms from a monomeric α‐helical state into insoluble β‐sheet aggregates

Szyperski¹,

Vandenbussche²,

Curstedt

et al. 1998

Protein Science

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In the 35-residue pulmonary surfactant-associated lipopolypeptide C (SP-C), the stability of the valyl-rich tu-helix comprising residues 9-34 has been monitored by circular dichroism, nuclear magnetic resonance, and Fourier transform infrared spectroscopy in both a mixed organic solvent and in phospholipid micelles. The a-helical form of SP-C observed in freshly prepared solutions in a mixed solvent of CHC13/CH30H/O.I M HCI 32:64:undergoes within a few days an irreversible transformation to an insoluble aggregate that contains P-sheet secondary structure. Hydrogen exchange experiments revealed that this conformational transition proceeds through a transition state with an Eyring free activation enthalpy of about 100 kJ mol", in which the polypeptide segment 9-27 largely retains a helical conformation. In dodecylphosphocholine micelles, the helical form of SP-C was maintained after seven weeks at 50°C. The a-helical form of SP-C thus seems to be the thermodynamically most stable state in this micellar environment, whereas its presence in freshly prepared samples in the aforementioned mixed solvent is due to a high kinetic bamer for unfolding. These observations support a previously proposed pathway for in vivo synthesis of SP-C through proteolytic processing from a 21-kDa precursor protein.

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Section: Resultsmentioning

confidence: 99%

Pulmonary surfactant‐associated polypeptide C in a mixed organic solvent transforms from a monomeric α‐helical state into insoluble β‐sheet aggregates

Szyperski¹,

Vandenbussche²,

Curstedt

et al. 1998

Protein Science

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“…where k U and k R represent the unfolding and refolding rate constants, respectively (38,39). The equilibrium constant (K U ) between N and U is related to the microscopic rate constants by K U ϭ k U /k R .…”

Section: Discussionmentioning

confidence: 99%

Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange

Lee

Tamura

Hoshino

et al. 2007

Journal of Biological Chemistry

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NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residuebased conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP ؉ reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP ؉ )-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD r values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD r values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP ؉ -binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP ؉ -binding domain at pD r 8.0, the daytime pH in chloroplasts, than at pD r 6.0 is likely to be involved in the increased binding of NADP ؉ for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.Protein conformations as shown by x-ray crystallography or nuclear magnetic resonance spectroscopy are virtually dynamic entities over various time ranges in solution (1, 2). Clarifying such conformational motions at the level of the atom or residue is essential for understanding the structural stabilities of proteins and the relationships between protein structures and functions (3-5). The hydrogen/deuterium (H/D) 4 exchange of amide protons in backbones has become an important way to address the motions of a protein from small or relatively large scale motions involved in biological functions such as binding of a substrate or releasing a product to much more large scale motions like a global unfolding process (4, 6). Among several approaches, the H/D exchange combined with heteronuclear NMR spectroscopy is the most convenient and powerful way because it can provide residue-specific information for most residues (7). For many globular proteins, this approach has identified protected cores, which are often composed of secondary structures buried inside the molecules and the cooperative interactions with partner molecules (4, 8), leading to allostery (9). Detailed analyses of exchanges in the native state of cytochrome c in the presence of various concentrations of denaturants suggested a pathway to unfolding, in which groups of secondary structural elements (i.e. foldons) are unfolded sequentially through distin...

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“…In fact, EX1 reactions are rarely seen in stable proteins, occurring mostly under the conditions used in some protein refolding experiments [43][44][45][46]. Under the EX2 regime, the exchange rates are described by Eqn (1):…”

Section: H-2 H Amide Exchangementioning

confidence: 99%

Protein stabilization by compatible solutes

Lamosa

Turner

Ventura

et al. 2003

European Journal of Biochemistry

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Heteronuclear NMR relaxation measurements and hydrogen exchange data have been used to characterize protein dynamics in the presence or absence of stabilizing solutes from hyperthermophiles. Rubredoxin from Desulfovibrio gigas was selected as a model protein and the effect of diglycerol phosphate on its dynamic behaviour was studied. The presence of 100 mM diglycerol phosphate induces a fourfold increase in the half-life for thermal denaturation of D. gigas rubredoxin [Lamosa, P., Burke, A., Peist, R., Huber, R., Liu, M.Y., Silva, G., Rodrigues-Pousada, C., LeGall, J., Maycock, C. & Santos, H. (2000) Appl. Environ. Microbiol. 66, 1974Microbiol. 66, -1979. A model-free analysis of the protein backbone relaxation parameters shows an average increase of generalized order parameters of 0.015 reflecting a small overall reduction in mobility of fast-scale motions. Hydrogen exchange data acquired over a temperature span of 20°C yielded thermodynamic parameters for the structural opening reactions that allow for the exchange. This shows that the closed form of the protein is stabilized by an additional 1.6 kJAEmol )1 in the presence of the solute. The results seem to indicate that the stabilizing effect is due mainly to a reduction in mobility of the slower, larger-scale motions within the protein structure with an associated increase in the enthalpy of interactions.Keywords: chemical exchange; compatible solutes; protein dynamics; rubredoxin; thermostability.Protein stability, activity and dynamics are interrelated issues with great importance not only in physiological processes but also in protein engineering. The evolution of protein structures towards extreme thermostability was vital for hyperthermophiles, microorganisms thriving near the boiling point of water. In general, the proteins of these organisms are intrinsically resistant to heat denaturation. However, hyperthermophiles also possess intracellular proteins that are not particularly stable, implying the existence of alternative strategies for their stabilization in vivo [1,2]. Hyperthermophiles accumulate high levels of charged organic osmolytes in response to supra-optimal growth temperatures, and this observation led to the hypothesis that these compounds play a role in thermoprotection of macromolecules in vivo [3,4]. This view is supported by in vitro studies showing that these osmolytes protect proteins against heat [1,[5][6][7][8]. Nevertheless, the molecular basis for this well established stabilization phenomenon remains elusive.Several possible mechanisms for protein stabilization by osmolytes have been proposed [9][10][11]. Arakawa and Timasheff [12,13] proposed a preferential hydration model to explain protein stabilization by compatible solutes: solute molecules are excluded from the protein surface, thereby making denaturation entropically less favourable. In conformity, exclusion factors have been measured for a variety of organic solutes and salts [14][15][16], however, the correlation between exclusion factors and the degree of protection ...

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Mechanisms and uses of hydrogen exchange

Cited by 358 publications

References 91 publications

Pulmonary surfactant‐associated polypeptide C in a mixed organic solvent transforms from a monomeric α‐helical state into insoluble β‐sheet aggregates

Pulmonary surfactant‐associated polypeptide C in a mixed organic solvent transforms from a monomeric α‐helical state into insoluble β‐sheet aggregates

Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange

Protein stabilization by compatible solutes

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