Study of protein topography with flash photolytically generated nonspecific surface-labeling reagents: surface labeling of ribonuclease A

Abstract

A method for nonspecifically labeling essentially all exposed residues of a protein is described. A reactive aryl nitrene is generated from N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-Taurine), within 500 mus by flash photolysis in the presence of protein. The reactive nitrene is inserted in about 2 ms into those carbon-hydrogen bonds of the protein that are exposed to the solvent. The method is applied here to ribonuclease A to demonstrate the different degree of labeling of the native and denatured protein. On the basis of amino acid analysis, it appears that residues of the native protein that are buried in the interior of the molecule (as judged from the x-ray structure) do not react with the nitrene. However, when these residues (even nonreactive ones such as valine and proline) are exposed by denaturation of the protein, they do react with the nitrene. It is shown that native ribonuclease A retains 90% of its enzymatic activity when flashed in the absence of NAP-Taurine. This small loss in activity arises from the disruption of a limited portion of the native enzyme structure, as judged by circular dichroism, ultraviolet, and Raman spectra. The site of this limited disruption may be a portion of the enzyme surface near the Cys-26-Cys-84 disulfide bond. The utility of this surface labeling technique for studying the pathways of protein folding or unfolding is discussed.

Steps in the pathway of the thermal unfolding of ribonuclease A. A nonspecific photochemical surface-labeling study

“…If a residue is labeled it must be accessible to the label (and solvent). However, if it is not labeled, no definite conclusions can be drawn because labeling is not 100% efficient, the extent of modification is not related in a simple way to either the type or degree of exposure of a residue, and only modifications which are not reversed by acid hydrolysis are detected (Matheson et al, 1977b).…”

“…His and Cys are not included because the reliability of their determinations was affected adversely by an artifact (unidentified) which overlapped the His peak and by losses of Cys from both hydrolysis (Rupley & Scheraga, 1963) and photolysis (Matheson et al, 1977b). A few labeling experiments were carried out at higher temperatures (up to 90 "C) and higher ionic strengths (up to 0.2 M KC1).…”

“…The nitrogen-saturated solutions of RNase A (1 pM) and NAP-taurine (250-300 pM) were adjusted to the desired ionic strength (in the range of 0.001 to 0.1 M ) with KCI and a p H of 4.9-5.3 (measured at 25 "C) with dilute HCl. Samples (5.8 mL) were thermally equilibrated (at temperatures in the range of 12 to 78 "C) for 10 min and flash-photolyzed in the apparatus described previously (Matheson et al, 1977b). Three or four such samples, flashed at a given temperature, were pooled in order to obtain sufficient material for amino acid analysis.…”

Binding of photoreactive lysozyme peptides to murine histocompatibility class II molecules.

“…Although differently substituted arylnitrenes, or their rearranged arylazirines, have different chemical reactivities and reaction kinetics (13), these differences are unlikely to account for the differences seen in photoaffinity labeling ( Table 1). The chemical reactivities of these intermediates are, in any case, exceptionally high (14), and the kinetics, even of slow photoreactions, occur in time intervals negligibly small in comparison with the class II proteinligand binding event. The other possibility, according to which the native HEL peptides and their photoreactive derivatives do not bind at the same site on I-Ak, is more difficult to evaluate.…”

“…In contrast, the nitrenes generated by photolysis from compounds 1-6 ( Fig. 1) will react within a few milliseconds with potentially any amino acid residue of IAk except glycine (14). The linkage furthermore involves a short and defined distance given by the crosslinker.…”