Flexibility and bioactivity of insulin: an NMR investigation of the solution structure and folding of an unusually flexible human insulin mutant withincreased biological activity

The structure and folding of a novel human insulin mutant. [Thr(B27) --> Pr o, Pro(B28) --> Thr]insulin (PT insulin), in aqueous solution and in mixtur es of water and 2,2,2-trifluoroethanol (TFE) have been studied by NMR spect roscopy. It was found that PT insulin has a highly flexible structure in pu re water and is present in at least two different conformations, although w ith an overall tertiary structure similar to that of native insulin. Furthe rmore, the native helical structures are poorly defined. Surprisingly, the mutant has a biological activity about 50% higher than native insulin. In c ontrast, in TFE/water solution the mutant reveals a propensity of forming a well-defined structure at the secondary structure level, similar to monome ric native insulin. Thus, as shown by a detailed determination of the struc ture from 208 distance restraints and 52 torsion angle restraints by distan ce geometry, simulated annealing, and restrained energy minimization, the n ative insulin helices (A2-A7, A13-A19, and B10-B19) as well as the beta -tu rn (B20-B23) are formed in 35% TFE. However. the amount of tertiary structu re is decreased significantly in TFE/water solution. The obtained results s uggest that only an overall tertiary fold, as observed for PT insulin in pu re water, is necessary for expressing the biological activity of insulin, a s long as the molecule is flexible and retains the propensity, to form the secondary structure required for its receptor binding. In contrast, a compa ct secondary structure, as found for native insulin in solution, is unneces sary for the biological activity. A model for the receptor binding of insul in is suggested that relates the increased bioactivity to the enhanced flex ibility of the mutant.