We report experiments to investigate the role of the physiologically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T-cell receptor f chain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the f chain. Preliminary 1 H-NMR studies of recombinant f chain suggested that it is essentially unstructured and therefore that peptide mimics would serve as useful models for investigating individual ITAM tyrosines. Phosphorylation kinetics were determined for each tyrosine by assaying the transfer of 32 P by recombinant Lck on to each of the peptides. The rates of phosphorylation were found to depend on the location of the tyrosine, leading to the proposal that Lck phosphorylates the six f chain ITAM tyrosines in the order 1N (first) > 3N > 3C > 2N > 1C > 2C (last) as a result of differences in the amino-acid sequence surrounding each tyrosine. This proposal was then tested on cytosolic, recombinant T-cell receptor f chain. After in vitro phosphorylation by Lck, the partially phosphorylated f chain was digested with trypsin. Separation and identification of the f chain fragments using LC-MS showed, as predicted by the peptide phosphorylation studies, that tyrosine 1N is indeed the first to be phosphorylated by Lck. We conclude that differences in the amino-acid context of the six f chain ITAM tyrosines affect the efficiency of their phosphorylation by the kinase Lck, which probably contributes to the distinct patterns of phosphorylation observed in vivo.

Protein L is a multidomain cell wall bound protein found in ϳ10% of the Peptostreptococcal isolates (1) and contains a series of repeated domains, some of which are able to bind to immunoglobulins without initiating an immune response. Expression of this protein has been correlated to the virulence of these opportunistic pathogenic bacteria (1) that are found in the gastrointestinal and urogenital tracts (2). Its presence has been found to cause cellular responses such as histamine release from basophils and mast cells (3) presumably by crosslinking IgE molecules bound to surface Ig receptors.1 H NMR spectroscopy of a single Ig-binding domain of protein L (isolated from strain 312, PpL 312 ) 1 revealed that PpL 312 is a rigid structure consisting of a ␤-sheet, formed by two pairs of antiparallel ␤-strands, lying on top of a single ␣-helical section (4), with a flexible N terminus (5). Further NMR studies (6) led to the proposal that the binding site of PpL 312 for -chain involves residues from the second ␤-strand and from the loop between the third ␤-strand and the ␣-helix and located the binding site for PpL 312 on the -chain to the second ␤-strand and the two ␤-strands located on the outer surface of the framework region of the V L domain (7).Parallel studies on the binding interaction of a single Igbinding domain of protein L from strain 3316 (PpL 3316 ) found that it forms a high affinity complex with -chains with a K d of 112 nM (8, 9). The x-ray crystallographic structure of this domain has been determined in complex with the human antibody fragment, Fab 2A2, and revealed that two Fab 2A2 fragments can in fact bind to sites on opposite faces of PpL 3316 (10). Site 1 of PpL 3316 is that characterized by Beckingham et al. (9) and equivalent to the site studied on PpL 312 using NMR by Wikström et al. (5). Site 2 of PpL 3316 , involving part of the helix and strands 3 and 4, was identified for the first time by the crystallographic study (10). This site has not yet been characterized although preliminary studies suggested that the affinity of site 2 is lower than that of site 1 (10).By use of a program of site directed mutagenesis we have been able to derive the relative binding affinities of sites 1 and 2 and propose a mechanism by which PpL 3316 binds light chains that is consistent with published data. The hydroxyl group of Tyr 53 is important for the formation of a high affinity complex at site 1. Previous enzyme-linked immunosorbent assay experiments (11) have shown that nitration of Tyr 53 or its mutation to Phe dramatically increases the K d of the -chainPpL 3316 interaction. Another important residue at site 1 is Leu 57 . No binding of the 1 -chain used in these studies can be detected at site 1 of the mutant Y53F/L57H by fluorimetry or isothermal titration calorimetry, and this mutant is thus ideal for measuring the affinity at site 2.Important residues for binding at site 2 are Asp 55 and Ala 66 . The mutation D55A eliminates a salt bridge and dramatically weakens or eliminates binding (12), and co...

The activity of proteases chymotrypsin and subtilisin dissolved in a range of protic hydroxylalkylammonium ionic liquids was tested against the model substrate APEE (N-acetyl-L-phenylalanine ethyl ester); activity was only observed for subtilisin in diethanolammonium chloride (DEA Cl), while chymotrypsin was not active in any PIL tested.