COLD DENATURATION OF BARSTAR - H-1, N-15 AND C-13 NMR ASSIGNMENT AND CHARACTERIZATION OF RESIDUAL STRUCTURE

Detection of residual structure in denatured proteins is of interest b ecause fleetingly structured regions may be initiation points of the f olding pathway. Residual structure in this context is not the definiti on of one stable conformation but a population phenomenon. Acid, therm al and solvent-denatured states have recently been examined by NMR spe ctroscopy, but cold-denatured states have not been characterised to da te. Cold denaturation is a general phenomenon of globular proteins, wh ich provides a convenient route for studying early events in protein f olding: such states can be induced to fold and be monitored on a submi llisecond time scale by temperature-jump methods. Here, we use NMR spe ctroscopy to define residual structure in cold-denatured barstar. The cold-denatured state becomes significantly populated in the presence o f increasing concentrations of urea and lower temperature. In the pres ence of 3 M urea, the double mutant of barstar in which Cys40 and Cys8 2 are both mutated to Ala (C40/82A) is completely and reversibly denat ured at 278 K, a temperature that is accessible to NMR experiments. Th is cold-denatured state of barstar was assigned by heteronuclear NMR e xperiments and structural parameters such as NOE, coupling constants a nd chemical shifts were derived. Based on the excellent dispersion in a HSQC-NOESY-HSQC experiment, d(NN(i,i + 1)) NOEs were observed for al most all residues. This allowed us to normalise NOE intensities as the NOE: diagonal ratio d(NN(i,i + 1)) NOE (sigma(NN)) and the NOE ratio of d(alpha N(i + 1, i + 1)):d(alpha N(i,i + 1)) (sigma(N alpha)/sigma( alpha N)) This approach reveals residual structure populating the alph a-region of the (phi,psi) conformational space in the regions correspo nding to the first and the second helices and near the end of the seco nd beta-strand of native barstar, whereas the C-terminal region that c orresponds to the fourth helix and the third beta-strand is in a rando m coil conformation. The results suggest that the first and the second helices are potential initiation sites for the folding of barstar. Th e details presented here provide the starting point for the study of r apid folding of cold-denatured barstar. (C) 1996 Academic Press Limite d