Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability

DSpace/Manakin Repository

Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability

Show simple item record

dc.contributor.author Whitesides, George M.
dc.contributor.author Shaw, Bryan F.
dc.contributor.author Arthanari, Haribabu
dc.contributor.author Lee, Andrew
dc.contributor.author Durazo, Armando
dc.contributor.author Frueh, Dominique P.
dc.contributor.author Pollastri, Michael P.
dc.contributor.author Bilgicer, Basar
dc.contributor.author Gygi, Steven P.
dc.contributor.author Wagner, Gerhard
dc.contributor.author Narovlyansky, Max
dc.date.accessioned 2012-11-06T16:24:10Z
dc.date.issued 2010
dc.identifier.citation Shaw, 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.issn 0002-7863 en_US
dc.identifier.uri http://nrs.harvard.edu/urn-3:HUL.InstRepos:9871957
dc.description.abstract This 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.sponsorship Chemistry and Chemical Biology en_US
dc.language.iso en_US en_US
dc.publisher American Chemical Society en_US
dc.relation.isversionof doi:10.1021/ja9067035 en_US
dash.license OAP
dc.subject amide H/D exchange en_US
dc.subject lysine acetylation en_US
dc.subject mass spectrometry en_US
dc.subject protein folding en_US
dc.subject carbonic anhydrase II en_US
dc.subject protein charge ladder en_US
dc.subject hydrogen/deuterium en_US
dc.subject electrostatic potential en_US
dc.title Neutralizing positive charges at the surface of a protein lowers its rate of amide hydrogen exchange without altering its structure or increasing its thermostability en_US
dc.type Journal Article en_US
dc.description.version Accepted Manuscript en_US
dc.relation.journal Journal of the American Chemical Society en_US
dash.depositing.author Whitesides, George M.
dc.date.available 2012-11-06T16:24:10Z

Files in this item

Files Size Format View
Whitesides_Neutralizing.pdf 3.240Mb PDF View/Open
Whitesides_Neutralizing.doc 4.327Mb Microsoft Word View/Open

This item appears in the following Collection(s)

  • FAS Scholarly Articles [7219]
    Peer reviewed scholarly articles from the Faculty of Arts and Sciences of Harvard University

Show simple item record

 
 

Search DASH


Advanced Search
 
 

Submitters