NLM DIR Seminar Schedule

Using ProteinMPNN to Dissect RfaH CTD Refolding

Although most proteins adopt a single energetically favorable fold, some proteins have been evolutionarily selected to reversibly interconvert between distinct folds that regulate biological processes or perform different functions. One such fold-switching protein is Escherichia coli RfaH, a member of the only known family of universally conserved transcription factors. RfaH is composed of an N-terminal NGN domain and a C-terminal KOW domain expected to fold into a beta-roll structure. Strikingly, RfaH’s KOW domain adopts an alpha-helical fold bound to the NGN domain in its apo form, but upon binding its target DNA sequence and RNA polymerase, RfaH KOW dissociates from the NGN domain and reversibly switches to the expected beta-roll topology. There are numerous challenges in studying this transition, both experimentally and computationally, including the large complex required to drive the switch, the timescale of the folding event, and the low solubility of the transition state. Using ProteinMPNN, I have designed a binding-competent NGN domain to measure the transition from the unfolded intermediate to bound state by nuclear magnetic resonance. Binding results show that helix 5 forms as expected, but further work is required to reveal the formation of the entire complex. However, the approach developed will permit more streamlined design of future systems that are more friendly for biophysical investigation.

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