The goal of Core 4 of the HARC Center is to generate recombinant antibody fragments (Fabs), single chain Fvs (scFvs) and nanobodies that bind to different conformational states of HIV accessory and regulatory proteins to enable and accelerate activities in all Projects and Cores. We will generate multiple Fabs to stabilize protein complexes, to serve as fiducial markers in single particle cryo-EM studies and to act as binding partners to accelerate crystallization studies. Information gained from Fab studies including binding epitopes, structural data, and the pharmacologic effect of scFvs or nanobodies will be inputs for molecular modeling to provide insights into the mechanism of HIV-host interactions. The Craik lab has a successful record of generating recombinant Fabs to a variety of challenging targets. For example, together with Yifan Cheng, we have demonstrated the utility of Fabs as fiducial markers for single particle cryo-EM studies of proteins and with John Gross we have probed the activity of the CRL5-Vif-CBF? E3 ligase in vitro and in cells using Fabs and scFvs, which are described as examples. Given availability of reagent quality and quantity of proteins from Projects 1-7 that include Vif, Vpu, Nef, Tat, Rev and APOBEC3s, and other targets from additional collaborators. Identification of Fabs will enable similar studies to be performed in conjunction with the individual Projects and Cores 3, 5 and 6 to determine structures and functions of the protein complexes.
