Rapid cooperative two-state folding of a miniature α-β protein and design of a thermostable variant

There is a great deal of interest in developing small stably folded miniature proteins. A limited number of these molecules have been described, however they typically have not been characterized in depth. In particular, almost no detailed studies of the thermodynamics and folding kinetics of these proteins have been reported. Here we describe detailed studies of the thermodynamics and kinetics of folding of a 39 residue mixed α-β protein (NTL9_1-39) derived from the N-terminal domain of the ribosomal protein L9. The protein folds cooperatively and rapidly in a two-state fashion to a native state typical of those found for normal globular proteins. At pH 5.4 in 20mM sodium acetate, 100mM NaCl the temperature of maximum stability is 6°C, the t_m is 65.3°C, ΔH°(t_m) is between 24.6kcalmol^-1 and 26.3kcalmol^-1, and ΔC_p° is 0.38kcalmol^-1deg^-1. The thermodynamic parameters are in the range expected on the basis of per residue values determined from databases of globular proteins. H/²H exchange measurements reveal a set of amides that exchange via global unfolding, exactly as expected for a normal cooperatively folded globular protein. Kinetic measurements show that folding is two-state folding. The folding rate is 640s^-1 and the value of ΔG° calculated from the folding and unfolding rates is in excellent agreement with the equilibrium value. A designed thermostable variant, generated by mutating K12 to M, was characterized and found to have a t_m of 82°C. Equilibrium and kinetic measurements demonstrate that its folding is cooperative and two-state.