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dc.contributor.authorWhitesides, George M.
dc.contributor.authorShaw, Bryan F.
dc.contributor.authorArthanari, Haribabu
dc.contributor.authorLee, Andrew
dc.contributor.authorDurazo, Armando
dc.contributor.authorFrueh, Dominique P.
dc.contributor.authorPollastri, Michael P.
dc.contributor.authorBilgicer, Basar
dc.contributor.authorGygi, Steven P.
dc.contributor.authorWagner, Gerhard
dc.contributor.authorNarovlyansky, Max
dc.date.accessioned2012-11-06T16:24:10Z
dc.date.issued2010
dc.identifier.citationShaw, Bryan F., Haribabu Arthanari, Max Narovlyansky, Armando Durazo, Domique P. Frueh, Michael P. Pollastri, Andre Lee, et al . 2010. Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability. Journal of the American Chemical Society 132(49): 17411-17425.en_US
dc.identifier.issn0002-7863en_US
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:9871957
dc.description.abstractThis paper combines two techniques—mass spectrometry and protein charge ladders—to examine the relationship between the surface charge and hydrophobicity of a 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-\(\epsilon\)-NH\(_{3}\)\(^{+}\) 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 increase in the total number of backbone amide hydrogen that are protected from exchange with solvent (2 h, pD 7.4, 15 ºC). Each successive acetylation produced BCA II proteins with one additional hydrogen protected after two hours in deuterated buffer (pD 7.4, 15 ºC). NMR spectroscopy demonstrated that these protected hydrogen atoms were not located on the side chain of the acetylated lysine residues (i.e., lys-\(\epsilon\)-NHCOCH\(_{3}\)). The decrease in rate of exchange associated with acetylation paralleled a decrease in thermostability: the most slowly exchanging rungs were the least thermostable (as measured by differential scanning calorimetry). The fact that the rates of H/D exchange were similar for perbutyrated BCA II (e.g., [lys-\(\epsilon\)-NHCO(CH\(_{2}\))\(_{2}\)CH\(_{3}\)]\(_{18}\)) and peracetylated BCA II (e.g., [lys-\(\epsilon\)-NHCOCH\(_{3}\)]\(_{18}\)) suggests that the charge is more important than the hydrophobicity of surface groups in determining the rate of H/D exchange. These kinetic electrostatic effects could complicate 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 another charged ligand or protein).en_US
dc.description.sponsorshipChemistry and Chemical Biologyen_US
dc.language.isoen_USen_US
dc.publisherAmerican Chemical Societyen_US
dc.relation.isversionofdoi:10.1021/ja9067035en_US
dash.licenseOAP
dc.subjectamide H/D exchangeen_US
dc.subjectlysine acetylationen_US
dc.subjectmass spectrometryen_US
dc.subjectprotein foldingen_US
dc.subjectcarbonic anhydrase IIen_US
dc.subjectprotein charge ladderen_US
dc.subjecthydrogen/deuteriumen_US
dc.subjectelectrostatic potentialen_US
dc.titleNeutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostabilityen_US
dc.typeJournal Articleen_US
dc.description.versionAccepted Manuscripten_US
dc.relation.journalJournal of the American Chemical Societyen_US
dash.depositing.authorWhitesides, George M.
dc.date.available2012-11-06T16:24:10Z
dc.identifier.doi10.1021/ja9067035*
dash.authorsorderedfalse
dash.contributor.affiliatedWagner, Gerhard
dash.contributor.affiliatedWhitesides, George
dash.contributor.affiliatedGygi, Steven
dash.contributor.affiliatedArthanari, Haribabu


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