This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Sequence and structure analysis of proteins reveals that they are frequently comprised of modular units. Such organization provides opportunity to create novel functions by recombining domains; indeed, this phenomenon occurs in both nature and the biotech laboratory. However, the function of a given domain in a new context cannot be reliably predicted from studies of the domain in isolation or in the context of other intact proteins, using the LacI/GalR family of transcription regulators. We are studying and beginning to understand how the function of a single DNA-binding domain is altered when the regulatory domain to which it is joined normally is replaced with homologous domains. The two functional domains of these homodimeric proteins do not directly contact each other. Instead, interactions are mediated through an ~18 amino acid linker, which contacts (1) the DNA-binding domain, (2) DNA ligand, (3) the linker of the partner monomer, and (4) one surface of the regulatory domain. Changes in these interfaces may be one mechanism by which domain function is altered in the context of a new protein. We have designed and constructed novel transcription repressors comprising the DNA-binding domain/linker of LacI and the regulatory domains of other E. coli homologues. This design modifies the interface between the linker and the regulatory domain. We are first determining which features of protein function are altered by this process: Preliminary results show that DNA-affinity, DNA-specificity, and allostery can be differentially affected in the presence of different regulatory domains. We will next determine which specific residues of the linker and of the regulatory domains impart these unique aspects of function. We will reconcile these results with several predictions about residues that impart unique function to individual members.
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