Rapid Mapping of Protein Structure, Interactions, and Ligand Binding by Misincorporation Proton-Alkyl Exchange

Abstract

Understanding protein conformation, interactions, and ligand binding is essential to all biological inquiry. We report a novel biochemical technique, called misincorporation proton-alkyl exchange (MPAX), that can be used to footprint protein structure at single amino acid resolution. MPAX exploits translational misincorporation of cysteine residues to generate probes for physical analysis. We apply MPAX to the triosephosphate isomerase (␤/␣) 8 barrel, accurately determining its substrate-binding site, a protein-protein interaction surface, the solvent-accessible protein surface, and the stability of the barrel. Because MPAX requires only microgram quantities of material and is not limited by protein size, it is ideally suited for proteins not amenable to conventional structural methods, such as membrane proteins, partially folded or insoluble proteins, and large protein complexes.

Expanding the Repertoire of an ERK2 Recruitment Site: Cysteine Footprinting Identifies the D-Recruitment Site as a Mediator of Ets-1 Binding

“…Typically, little variation in k alk is expected, suggesting that the magnitude of k 1 /k −1 usually determines cysteine reactivity. This is supported by the correlation between k a and solvent accessibility that holds over a reactivity range of 5 orders of magnitude (31). We reasoned that cysteine residues placed within the DRS of ERK2 would serve to report the engagement of ligands at this locus through a decrease in their observed rate of alkylation.…”

“…In contrast, the cysteine footprinting approach described here provides narrow coverage, but at the resolution of a single amino acid. An analysis termed misincorporation protein-alkyl exchange (MPAX), which utilizes the misincorporation of cysteine residues into proteins through the use of mutant t-RNAs, is an interesting methodology that potentially expands the footprinting approach by encompassing the advantages of both approaches (31). More studies are required to compare the ability of each approach to accurately define a protein-ligand interaction.…”

“…After the addition of loading dye (5 μL), the radioactivity of each sample was counted using a Packard 1500 scintillation counter to facilitate the addition of equal quantities of protein to a 15% or 20% SDS-PAGE gel. The proteins were then fractionated and the gels dried using a Gel Dryer (Biorad) before being exposed on image plates (Amersham Pharmacia) for 0.5-12 h. The gels were quantified using a PhosphorImager (Molecular Dynamics) and analyzed using ImageQuant software (30,31). Data obtained from the gel analysis were fitted to a first-order decay curve and generally found to reproducible to within 20%.…”

The histidine kinase inhibitor Sda binds near the site of autophosphorylation and may sterically hinder autophosphorylation and phosphotransfer to Spo0F

“…The alkylation protection assays were performed essentially as described (Silverman and Harbury, 2002). See Supporting information for experimental details.…”

Structural and kinetic mapping of side-chain exposure onto the protein energy landscape

“…This 155-amino acid protein is a well characterized model system known to populate a kinetic folding intermediate that resembles a high-energy conformation detected by NSHX (15,41,42). To expedite the SX characterization, we employ a modified version of Silverman's gel-based methodology to simultaneously monitor alkylation at multiple sites (43). The folding kinetics of the previously characterized variant D10A (44) relative to the intrinsic alkylation rate with the modifying agent iodoacetamide (IAM) allow us to apply a mixed EX1/EX2 approach to identify species on both sides of the rate-limiting barrier in a single experiment, characterize unique partially unfolded forms present on the folded side, and interrogate the rate-limiting folding barrier at the residue level.…”