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.
abstract: 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 secondary or tertiary 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).
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, USA. bryan_shaw@baylor.edu
authors.forename: Bryan F
authors.initials: BF
authors.lastname: Shaw
authors.forename: Haribabu
authors.initials: H
authors.lastname: Arthanari
authors.forename: Max
authors.initials: M
authors.lastname: Narovlyansky
authors.forename: Armando
authors.initials: A
authors.lastname: Durazo
authors.forename: Dominique P
authors.initials: DP
authors.lastname: Frueh
authors.forename: Michael P
authors.initials: MP
authors.lastname: Pollastri
authors.forename: Andrew
authors.initials: A
authors.lastname: Lee
authors.forename: Basar
authors.initials: B
authors.lastname: Bilgicer
authors.forename: Steven P
authors.initials: SP
authors.lastname: Gygi
authors.forename: Gerhard
authors.initials: G
authors.lastname: Wagner
authors.forename: George M
authors.initials: GM
authors.lastname: Whitesides
mesh_terms: D000107:Acetylation; D000577:Amides / Q000737:chemistry*; D000818:Animals; D024402:Carbonic Anhydrase II / Q000737:chemistry*; D002417:Cattle; D006859:Hydrogen / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D055550:Protein Stability; D055672:Static Electricity; D013696:Temperature
chemical_list: D000577:Amides; D006859:Hydrogen; D024402:Carbonic Anhydrase II; D008239:Lysine