Abstract

Protein-engineering methods (phi-values) were used to investigate the folding transition state of a Lysin Motif (LysM) domain from Escherichia coli membrane-bound lytic murein transglycosylase D. This domain consists of just 48 structured residues in a symmetrical beta-alpha-alpha-beta arrangement and is the smallest alpha/beta protein yet investigated using these methods. An extensive mutational analysis revealed a highly robust folding pathway with no detectable transition state plasticity, indicating that LysM is an example of an ‘ideal’ two-state folder. The pattern of phi-values denotes a highly polarised transition state, with significant formation of the helices but no structure within the beta-sheet. Remarkably, this transition state remains polarised after circularisation of the domain, and exhibits an identical phi-value pattern; however, the interactions within the transition state are uniformly weaker in the circular variant. This observation is supported by results from an Eyring analysis of the folding rates of the two proteins, and leads us to propose that the folding pathway of LysM is dominated by enthalpic rather than entropic considerations. We suggest that the lower entropy cost of formation of the circular transition state is balanced, to some extent, by the lower enthalpy of contacts within this structure.