Person:

Wagner, Gerhard

A previous study, using capillary electrophoresis (CE) [J. Am. Chem. Soc.2008, 130, 17384–17393], reported that six discrete complexes of ubiquitin (UBI) and sodium dodecyl sulfate (SDS) form at different concentrations of SDS along the pathway to unfolding of UBI in solutions of SDS. One complex (which formed between 0.8 and 1.8 mM SDS) consisted of native UBI associated with approximately 11 molecules of SDS. The current study used CE and 15N/13C–1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy to identify residues in folded UBI that associate specifically with SDS at 0.8–1.8 mM SDS, and to correlate these associations with established biophysical and structural properties of this well-characterized protein. The ability of the surface charge and hydrophobicity of folded UBI to affect the association with SDS (at concentrations below the CMC) was studied, using CE, by converting lys-ε-NH3+ to lys-ε-NHCOCH3 groups. According to CE, the acetylation of lysine residues inhibited the binding of 11 SDS ([SDS] < 2 mM) and decreased the number of complexes of composition UBI-(NHAc)8·SDSn that formed on the pathway of unfolding of UBI-(NHAc)8 in SDS. A comparison of 15N–1H HSQC spectra at 0 mM and 1 mM SDS with calculated electrostatic surface potentials of folded UBI (e.g., solutions to the nonlinear Poisson–Boltzmann (PB) equation) suggested, however, that SDS binds preferentially to native UBI at hydrophobic residues that are formally neutral (i.e., Leu and Ile), but that have positive electrostatic surface potential (as predicted from solutions to nonlinear PB equations); SDS did not uniformly interact with residues that have formal positive charge (e.g., Lys or Arg). Cationic functional groups, therefore, promote the binding of SDS to folded UBI because these groups exert long-range effects on the positive electrostatic surface potential (which extend beyond their own van der Waals radii, as predicted from PB theory), and not because cationic groups are necessarily the site of ionic interactions with sulfate groups. Moreover, SDS associated with residues in native UBI without regard to their location in α-helix or β-sheet structure (although residues in hydrogen-bonded loops did not bind SDS). No correlation was observed between the association of an amino acid with SDS and the solvent accessibility of the residue or its rate of amide H/D exchange. This study establishes a few (of perhaps several) factors that control the simultaneous molecular recognition of multiple anionic amphiphiles by a folded cytosolic protein.

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).