Chemical modification is a simple, useful approach to study membrane protein structure and function. Among amino acids, Cys is average in steric bulk, relatively hydrophobic and amenable to highly specific modification. Cys-scanning mutagenesis takes advantage of these unique features of Cys combined with site-directed mutagenesis1,2
. In order to optimize the approach, it may be necessary to construct a nonreactive or Cys-less mutant without inactivating the protein. On a functional Cys-less background, by systematically mutating each residue to Cys, a library of single Cys-mutant is generated, and the functional role of each position can be assessed by testing activity. A further advantage of the approach is that it enables studies of modification by Cys-specific reagents.
Site-directed sulfhydryl modification of single-Cys mutants in situ
with radioactive N
-ethylmaleimide (NEM) has been particularly useful for studying both static and dynamic features of the lactose permease of Escherichia coli
. In this protocol, LacY is used as a prototype4,5
. Alkylation with NEM is a measure of the reactivity and/or accessibility of a given Cys residue to this small, relatively hydrophobic, membrane-permeant thiol-specific reagent. Reactivity and/or accessibility are dependent primarily on the environment in the vicinity of a given Cys side chain and limited by close tertiary contacts between transmembrane helices and steric constraints of the lipid bilayer. Any change in reactivity of a Cys side chain upon substrate binding is indicative of an alteration in the local environment. Hence, determination of the reactivity of Cys replacement mutants with N
C]NEM) is a convenient way to assess the local environment of specific positions within the tertiary structure of the protein. Furthermore, in situ
site-specific reaction with methanethiosulfonate ethyl-sulfonate (MTSES), a small hydrophilic, membrane-impermeant thiol reagent6,7
, can be utilized to study the accessibility of Cys residues to the aqueous milieu. Cys-scanning mutagenesis and site-directed sulfhydryl modification systematically applied to LacY has provided enormously valuable information with regard to structure, function and dynamics2,3,5,8
Here, we describe a simple, easy-to-handle protocol for measuring the reactivity of single-Cys mutants with various thiol reagents. Application allows ( and ) (i) assessment of Cys reactivity with NEM under various conditions (e.g., absence or presence of ligand and/or an electrochemical proton gradient, temperature) ( and ) and (ii) assessment of Cys reactivity with other nonradioactive thiol reagents by measuring blockade of radioactive NEM labeling ( and ). Once Cys reacts with other thiol reagents, it cannot react with [14
C]NEM. One such application includes the use of impermeant MTSES6,7,9
to study solvent accessibility; (iii) another application is estimation of apparent binding constants for a ligand by measuring ligand protection against alkylation with [14
(), which will not be described here. In principle, NEM labeling and solvent accessibility approaches can be applied to identify residues buried in the core of a soluble protein by carrying out the analyses in the native or denatured condition. Furthermore, it is also useful for identifying positions located in the protein–protein interface of a protein complex by studying the effect of chemical modification on protein–protein interactions, solvent accessibility, as well as the protection against the alkylation of Cys residues.
Diagram for the application of N-ethylmaleimide (NEM) labeling. [14C]NEM, N-[ethyl-1-14C]ethylmaleimide; SDS-PAGE, SDS–polyacrylamide gel electrophoresis.
Figure 2 Diagram for the strategy of testing solvent accessibility of Cys. (a) Chemical reaction of the membrane permeant N-ethylmaleimide (NEM) with Cys. (b) Detection of the reaction of the membrane-impermeant methanethiosulfonate ethylsulfonate (MTSES). When (more ...)
Limitations of this particular protocol include requirement for radioactive NEM, use of an affinity tag fused to the target protein and a target protein containing only a single reactive Cys residue. Several affinity tags can be used to purify the target protein, examples being a His-tag on the target protein and metal affinity chromatography or other commercially availabel tags and protein purification kits. Here, we describe a protocol for purifying LacY containing a biotin acceptor domain at the C terminus by avidin chromatography12,13
It is important that the membrane permeability of the thiol reagent be tested in each system. In E. coli
, NEM and MTSES are demonstrated to be permeant and impermeant, respectively, by analyzing labeling of cytoplasmic proteins9
. It is also highly noteworthy that alternative methods with fluorescent thiol reactive reagents have been utilized14–16