A General Strategy for Site-Specific Double Labeling of Globular Proteins for Kinetic FRET Studies

Abstract

Site-directed mutagenesis provides a straightforward means of creating specific targets for chemical modifications of proteins. This capability enhanced the applications of spectroscopic methods adapted for addressing specific structural questions such as the characterization of partially folded and transient intermediate structures of globular proteins. Some applications such as the steady state or time-resolved fluorescence resonance energy transfer (FRET) detection of the kinetics of protein folding require relatively large quantities (approximately 10-100 mg) of site-specific doubly labeled protein samples. Engineered cysteine residues are common targets for labeling of proteins. The challenge here is to develop methods for selective modification of one of two reactive sulfhydryl groups in a protein molecule. A general systematic procedure for selective labeling of each of two cysteine residues in a protein molecule was developed, using Escherichia coli adenylate kinase (AKe) as a model protein. Potential sites for insertion of cysteine residues were selected by examination of the crystal structure of the protein. A series of single-cysteine mutants was prepared, and the rates of the reaction of each engineered cysteine residue with a reference reagent [5,5'-dithiobis(2-nitrobenzoic acid) (DTNB)] were determined. Two-cysteine mutants were prepared by selection of pairs of sites for which the ratio of this reaction rate constant was high (>80). The conditions for the selective labeling reaction were optimized. In a first cycle of labeling, the more reactive cysteine residue was labeled with a fluorescent probe (donor). The second probe was attached to the less reactive site under unfolding conditions in the second cycle of labeling. The doubly and singly labeled mutants retained full enzymatic activity and the capacity for a reversible folding-unfolding transition. High yields (70-90%) of the preparation of the pure, site-specific doubly labeled AK mutant were obtained. The procedure described herein is a general outline of procedures, which can meet the double challenge of both site specificity and large-scale preparation of doubly labeled proteins.

Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein

“…Extensive efforts to remove the intrinsic cysteines, which are highly buried and inaccessible to solvent, were unsuccessful. The significant difference in solvent accessibility, however, allowed selective labeling with IAE-DANS of the exposed artificially introduced cysteine, which has a reactivity Ϸ80-fold higher than the three highly buried native cysteines (43). Standard labeling protocols were followed and are detailed in SI Text.…”

Fluorescent labeling and modification of proteins

“…Other approaches to deal with this problem are based upon measuring the reactivity of each surface-exposed cysteine and then optimizing conditions to selectively label the desired residue. Obviously, this relies upon having differently reactive cysteines which is not always possible [50]. If structural information is available, then it may be possible to selectively label a cysteine following selection of different conformations [4].…”

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

“…Labeling of Csp was accomplished by introducing two complementary fluorophores at two engineered cysteine (Cys) residues at the N and C terminus (wild-type Csp is devoid of Cys) using the sequential labeling method pioneered by Haas and co-workers. 163 Although the size of the polypeptide is (in principle) not a limiting factor, this method usually results in sample heterogeneity, unless the D/A-and the dye-permutated A/D-labeled analogues can be chromatographically separated. Such mixtures can lead to unwanted sample heterogeneity, as the conjugated dyes can exert a positional-dependent perturbation of the energy landscape of the modified protein or exhibit photophysical properties dependent on the local environment.…”