STRUCTURE AND STABILITY OF MONOMERIC LAMBDA-REPRESSOR - NMR EVIDENCE FOR 2-STATE FOLDING

The absence of equilibrium intermediates in protein folding reactions (i.e., two-state folding) simplifies thermodynamic and kinetic analyse s but is difficult to prove rigorously. We demonstrate a sensitive met hod for detecting partially folded species based on using proton chemi cal shifts as local probes of structure. The coincidence of denaturati on curves for probes throughout the molecule is a particularly stringe nt test for two-state folding. In this study we investigate a new form of the N-terminal domain of bacteriophage lambda repressor consisting of residues 6-85 (lambda(6-85)) using nuclear magnetic resonance (NMR ) and circular dichroism (CD). This truncated version lacks the residu es required for dimerization and is monomeric under the conditions use d for NMR. Heteronuclear NMR was used to assign the H-1, N-15, and bac kbone C-13 resonances. The secondary and tertiary structure of lambda( 6-85) is very similar to that reported for the crystal structure of th e DNA-bound 1-92 fragment [Beamer, L. J., and Pabo, C. O. (1992) J. Mo l. Biol. 227, 177-196], as judged by analysis of chemical shifts, amid e hydrogen exchange, amide-alpha coupling constants, and nuclear Overh auser enhancements. Thermal and urea denaturation studies were conduct ed using the chemical shifts of the four aromatic side chains as local probes and the CD signal at 222 nm as a global probe. Plots of the fr action denatured versus denaturant concentration obtained from these s tudies are identical for all probes under all conditions studied. This observation provides strong evidence for two-state folding, indicatin g that there are no populated intermediates in the folding of lambda(6 -85).