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This protocol describes a detailed method to study the static and dynamic features of membrane proteins, as well as solvent accessibility, by utilizing the lactose permease of Escherichia coli (LacY) as a model. The method relies on the use of functional single-Cys mutants, an affinity tag and a PhosphoImager. The membrane-permeant, radioactive thiol reagent N-[ethyl-1-14C]ethylmaleimide ([14C]NEM) is used to detect site-directed alkylation of engineered single-Cys mutants in situ. The solvent accessibility of the Cys residues is also determined by blockage of [14C]NEM labeling with membrane-impermeant thiol reagents such as methanethiosulfonate ethylsulfonate (MTSES). The labeled proteins are purified by mini-scale affinity chromatography and analyzed by gel electrophoresis. Gels are dried and exposed to a PhosphoImager screen for 1–5 d, and incorporation of radioactivity is visualized. Initial results can be obtained in 24 h.
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 (LacY)3. 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-[ethyl-1-14C]ethylmaleimide ([14C]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 (Fig. 1 and Fig. 2) (i) assessment of Cys reactivity with NEM under various conditions (e.g., absence or presence of ligand and/or an electrochemical proton gradient, temperature) (Fig. 1a and Fig. 2a) and (ii) assessment of Cys reactivity with other nonradioactive thiol reagents by measuring blockade of radioactive NEM labeling (Fig. 1b and Fig. 2b). Once Cys reacts with other thiol reagents, it cannot react with [14C]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 [14C]NEM10,11 (Fig. 1c), 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.
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.
Column wash buffer 50 mM NaPi (pH 7.4)/0.1 M NaCl/0.02 % DDM.
Elution buffer 5 mM Biotin in the column wash buffer given above; adjust to pH 7.5.
Troubleshooting advice can be found in Table 1.
The reactivity of Cys residues with NEM as well as the effect of ligand can be easily visualized by comparing the density of radioactive bands (Fig. 4a, upper panel). The total protein loaded, as visualized from western blot (Fig. 4a, lower panel) and data collected from a positive and a negative control for protein labeling are needed for a correct interpretation of the results. The following are examples for the interpretation of results as reported from studies on solvent accessibility of single-Cys residues determined by blockade of NEM labeling with MTSES following the protocol described above (Fig. 4a). The position studied is mapped in an x-ray crystal structure of LacY (Fig. 4b,c).
The authors acknowledge support from National Institutes of Health (NIH) grants DK51131 and DK06946, GM074929 and National Science Foundation (NSF) grant 0450970 (to H.R.K.).
COMPETING INTERESTS STATEMENT The authors declare no competing financial interests.
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