GuHCl and NaCl-dependent Hydrogen Exchange in MerP Reveals a Well-defined Core with an Unusual Exchange Pattern

Brorsson, Ann-Christin; Lundqvist, Martin; Sethson, Ingmar; Jonsson, Bengt‐Harald

doi:10.1016/j.jmb.2006.01.090

Cited by 5 publications

(6 citation statements)

References 58 publications

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“…It is a contradictory behavior to the normal denaturant‐dependent unfolding process. But such an upward curvature with increase of denaturant concentration was observed in the 72‐amino acid protein MerP by Brosson et al 51. They suggested that such stabilization of the residues can originate from the selective destabilization of the unfolded state by guanidinium ions and/or due to the selective stabilization of these exchanging protons in the native state by the chloride ions.…”

Section: Discussionmentioning

confidence: 93%

Residue‐wise conformational stability of DLC8 dimer from native‐state hydrogen exchange

2009

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Dynein light chain (DLC8) is the smallest subunit of the dynein motor complex, which is known to act as a cargo adaptor in intracellular trafficking. The protein exists as a pure dimer at physiological pH and a completely folded monomer below pH 4. Here, we have determined the energy landscape of the dimeric protein using a combination of optical techniques and native-state hydrogen exchange of amide groups, the former giving the global features and the latter yielding the residue level details. The data indicated the presence of intermediates along the equilibrium unfolding transition. The hydrogen exchange data suggested that the molecule has differential stability in its various segments. We deduce from the free energy data that the antiparallel beta-sheets (beta4 and beta5) that form the hydrophobic core of the protein and the alpha2 helix, all of which are highly protected with regard to hydrogen exchange, contribute significantly to the initial step of the protein folding mechanism. Denaturant-dependent hydrogen exchange indicated further that some amides exchange via local fluctuations, whereas there are others which exchange via global unfolding events. Implications of these to cargo adaptability of the dimer are discussed.

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Section: Discussionmentioning

confidence: 93%

Residue‐wise conformational stability of DLC8 dimer from native‐state hydrogen exchange

2009

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“…We hope that a more complete theory of hydrogen exchange in folded polypeptides might explain some of the surprising and sometimes confusing results of H/D exchange experiments that have accumulated for over 50 years in biochemistry and in organic and polymer chemistry 24–27,40–42,89,90…”

Section: Resultsmentioning

confidence: 99%

“…This study has, nevertheless, shown that protein charge ladders offer a unique tool to use in understanding the structural and electrostatic factors that govern the rate of hydrogen exchange in folded proteins (and that have, so far, been intractably difficult to explore experimentally, and hence largely ignored). We hope that a more complete theory of hydrogen exchange in folded polypeptides might explain some of the surprising and sometimes confusing results of H/D exchange experiments that have accumulated for over 50 years in biochemistry and in organic and polymer chemistry. − ,− ,, …”

Section: Discussionmentioning

confidence: 99%

Neutralizing Positive Charges at the Surface of a Protein Lowers Its Rate of Amide Hydrogen Exchange without Altering Its Structure or Increasing Its Thermostability

et al. 2010

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This paper combines two techniques—mass spectrometry and protein charge ladders—to examine the relationship between the surface charge and hydrophobicity of a representative globular protein (bovine carbonic anhydrase II; BCA II) and its rate of amide hydrogen-deuterium (H/D) exchange. Mass spectrometric analysis indicated that the sequential acetylation of surface lysine-ε-NH3+ groups—a type of modification that increases the net negative charge and hydrophobicity of the surface of BCA II without affecting its 2° or 3° structure—resulted in a linear decrease in the aggregate rate of amide H/D exchange at pD 7.4, 15 °C. According to analysis with MS, the acetylation of each additional lysine generated between 1.4 and 0.9 additional hydrogens that are protected from H/D exchange during the 2 h exchange experiment at 15 °C, pD 7.4. NMR spectroscopy demonstrated that none of the hydrogen atoms which became protected upon acetylation were located on the side chain of the acetylated lysine residues (i.e., lys-ε-NHCOCH3) but were instead located on amide NHCO moieties in the backbone. The decrease in rate of exchange associated with acetylation paralleled a decrease in thermostability: the most slowly exchanging rungs of the charge ladder were the least thermostable (as measured by differential scanning calorimetry). This observation—that faster rates of exchange are associated with slower rates of denaturation—is contrary to the usual assumptions in protein chemistry. The fact that the rates of H/D exchange were similar for perbutyrated BCA II (e.g., [lys-ε-NHCO(CH2)2CH3]18) and peracetylated BCA II (e.g., [lys-ε-NHCOCH3]18) suggests that the electrostatic charge is more important than the hydrophobicity of surface groups in determining the rate of H/D exchange. These electrostatic effects on the kinetics of H/D exchange could complicate (or aid) the interpretation of experiments in which H/D exchange methods are used to probe the structural effects of non-isoelectric perturbations to proteins (i.e., phosphorylation, acetylation, or the binding of the protein to an oligonucleotide or to another charged ligand or protein).

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“…The proton exchange rate of water is also sensitive to the salt concentration in the media (Hertz et al 1983), suggesting an equivalent dependence for the amide proton exchange rate. An increase of the solution ionic strength can alter the exchange rate for the amide protons belonging to charged residues (Kim and Baldwin 1982), and sodium chloride can also modulate the exchange rate due to nonspecific weak binding to the protein (Laurents et al 2005; Brorsson et al 2006). Nevertheless, the use of other inorganic salts in the proton amide exchange rate has not been investigated in detail, and, in this study, we have explored the influence of low to moderate concentrations of several anions on the exchange rate of the IGg‐binding domain of protein L (ProtL; 64 residues) (Kim et al 2000) and the glucose–galactose‐binding protein (GGBP; 309 residues) (Shilton et al 1996).…”

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confidence: 99%

Anion modulation of the ¹H/²H exchange rates in backbone amide protons monitored by NMR spectroscopy

2007

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The ability of three anionic cosolutes (sulfate, thiocyanate, and chloride) in modulating the 1 H/ 2 H exchange rates for backbone amide protons has been investigated using nuclear magnetic resonance (NMR) for two different proteins: the IGg-binding domain of protein L (ProtL) and the glucosegalactose-binding protein (GGBP). Our results show that moderate anion concentrations (0.2 M-1 M) regulate the exchange rate following the Hofmeister series: Addition of thiocyanate increases the exchange rates for both proteins, while sulfate and chloride (to a less extent) slow down the exchange reaction. In the presence of the salt, no alteration of the protein structure and minimal variations in the number of measurable peaks are observed. Experiments with model compounds revealed that the unfolded state is modulated in an equivalent way by these cosolutes. For ProtL, the estimated values for the local free energy change upon salt addition (m 3,DG ) are consistent with the previously reported free energy contribution from the cosolute's preferential interaction/exclusion term indicating that nonspecific weak interactions between the anion and the amide groups constitute the dominant mechanism for the exchange-rate modulation. The same trend is also found for GGBP in the presence of thiocyanate, underlining the generality of the exchange-rate modulation mechanism, complementary to more investigated effects like the electrostatic interactions or specific anion binding to protein sites.Keywords: amide proton exchange; Hofmeister series; sulfate; thiocyanate; cosolute; anion Supplemental material: see www.proteinscience.org Solvent composition can significantly alter the constitutive properties of the biomolecules and, in particular, their biological activity (Record Jr. et al. 1998;Timasheff 1998;Fayos et al. 2005;Ellis and Minton 2006;Holthauzen and Bolen 2007). Early works of Hofmeister demonstrated that the addition of moderate amounts of inorganic salts to the media (up to 1 M) efficiently modulates protein stability and solubility, dividing these ions between ''kosmotropic'' (well-solvated cosolutes with a large negative DG hydr value) and ''chaotropic'' (poorly solvated cosolutes) (Hofmeister 1888). Although there is no simple model to explain such effects, different mechanisms seem to be directly related, including a reduction in the translational Reprint requests to: Oscar Millet, Structural Biology Unit, CIC bioGUNE, Bizkaia Technology Park, Building 800, 48160 Derio, Spain; e-mail: omillet@cicbiogune.es; fax: 34-944-061-301.Abbreviations: DG hydr , free energy of hydration; NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantum correlation; ProtL, IGg-binding domain of peptostreptococcal protein; GGBP, glucose-galactose-binding protein; k ex , experimental exchange rate; m 3,ex , slope for the variation of the exchange rate with cosolute concentration; k cl , closing rate; k op , opening rate; k rc , intrinsic exchange rate in the unfolded state; DG ex , local free energy change; m 3,DG , slop...

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GuHCl and NaCl-dependent Hydrogen Exchange in MerP Reveals a Well-defined Core with an Unusual Exchange Pattern

Cited by 5 publications

References 58 publications

Residue‐wise conformational stability of DLC8 dimer from native‐state hydrogen exchange

Residue‐wise conformational stability of DLC8 dimer from native‐state hydrogen exchange

Neutralizing Positive Charges at the Surface of a Protein Lowers Its Rate of Amide Hydrogen Exchange without Altering Its Structure or Increasing Its Thermostability

Anion modulation of the ¹H/²H exchange rates in backbone amide protons monitored by NMR spectroscopy

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GuHCl and NaCl-dependent Hydrogen Exchange in MerP Reveals a Well-defined Core with an Unusual Exchange Pattern

Cited by 5 publications

References 58 publications

Residue‐wise conformational stability of DLC8 dimer from native‐state hydrogen exchange

Residue‐wise conformational stability of DLC8 dimer from native‐state hydrogen exchange

Neutralizing Positive Charges at the Surface of a Protein Lowers Its Rate of Amide Hydrogen Exchange without Altering Its Structure or Increasing Its Thermostability

Anion modulation of the 1H/2H exchange rates in backbone amide protons monitored by NMR spectroscopy

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Anion modulation of the ¹H/²H exchange rates in backbone amide protons monitored by NMR spectroscopy