WFU Joint Colloquium -- Center for Structural Biology and Department of Physics

TITLE: Using repeat proteins to dissect the energy landscape and probe the basis for cooperativity in protein folding

SPEAKER: Professor Doug Barrick ,

TIME: Tuesday May 19, 2009 at 4:00 PM

PLACE: Room 101 in Olin Physical Laboratory

High cooperativity is a hallmark of protein folding, although its origin and its consequences in terms of protein folding kinetics remains poorly understood. My laboratory has been studying linear repeat proteins, which possess modular architecture, to obtain a quantitative picture of how folding energies are distributed (the energy landscape), to better understand the origins of cooperativity, and to explore the relationships between the landscape and cooperativity. We have made progress in these areas by studying both naturally occurring repeat proteins and non-natural "consensus" repeat proteins.

We have found that naturally occurring ankyrin and leucine-rich repeat proteins show a high level of cooperativity in folding, which is surprising given their size and architecture. The distribution of stability in naturally occurring repeat proteins is complex, and creates channels in the energy landscape. For YopM, a large LRR protein of ~400 residues, we find local stability to be so variable that large segments of the polypeptide become unfolded in the absence of internal capping sequences. Moreover, stability can be increased by internal deletion of a well-folded pair of repeats. The kinetics of folding of these naturally occurring repeat proteins appears to be somewhat more complex than for equilibrium folding, but shows preferred pathways that follow the low energy folding channels to the native state described above. This pathway preference demonstrates local stability to be an important factor in determining folding, in addition to topology.

By studying consensus repeat proteins composed of units of identical sequence, we can accurately separate the energetics of long-range coupling from local interactions using an Ising formalism, and are using these constructs to determine the origins of local versus cooperative interactions. Consistent with previous results by Pluckthun and coworkers, we find highly stabilizing nearest-neighbor ankyrin interactions (~11 kcal/mol) which drive the unfavorable folding of individual repeats. Based on guanidine and thermal denaturation, we find the interfacial interaction to be less sensitive to denaturant than the individual repeat units, but to contribute nearly all the heat capacity decrease upon folding. Using this system, we are dissecting the structural basis of cooperative long-range versus short-range interactions via site-specific substitutions, and are examining how such interactions influence the kinetics of folding.