Identification of protein binding partners is one of the key challenges of proteomics. We recently introduced a screen for detecting protein-protein interactions based on reassembly of dissected fragments of green fluorescent protein fused to interacting peptides. Here, we present a set of comaintained Escherichia coli plasmids for the facile subcloning of fusions to the green fluorescent protein fragments. Using a library of antiparallel leucine zippers, we have shown that the screen can detect very weak interactions (K(D) approximately 1 mM). In vitro kinetics show that the reassembly reaction is essentially irreversible, suggesting that the screen may be useful for detecting transient interactions. Finally, we used the screen to discriminate cognate from noncognate protein-ligand interactions for tetratricopeptide repeat domains. These experiments demonstrate the general utility of the screen for larger proteins and elucidate mechanistic details to guide the further use of this screen in proteomic analysis. Additionally, this work gives insight into the positional inequivalence of stabilizing interactions in antiparallel coiled coils.

Protein design aims to understand the fundamentals of protein structure by creating novel proteins with pre-specified folds. An equally important goal is to understand protein function by creating novel proteins with pre-specified activities. Here we describe the design and characterization of a tetratricopeptide (TPR) protein, which binds to the C-terminal peptide of the eukaryotic chaperone Hsp90. The design emphasizes the importance of both direct, short-range protein-peptide interactions and of long-range electrostatic optimization. We demonstrate that the designed protein binds specifically to the desired peptide and discriminates between it and the similar C-terminal peptide of Hsp70.

Assembly of the transcription repression complex at the Escherichia coli biotin biosynthetic operon occurs via coupled protein-protein and protein-DNA interactions in which the holoBirA dimer binds to the forty base pair biotin operator sequence. The thermodynamic driving forces for the assembly process have been dissected using sedimentation equilibrium measurements and DNaseI footprint titrations. Measurements of the temperature dependence of dimerization indicate that this process is strongly enthalpically opposed and is driven by a very favorable entropy. By contrast, the DNA binding step is enthalpically driven and opposed by a modest entropy. Neither step is accompanied by a heat capacity change. The convoluted protein-protein and protein-DNA binding reaction is dominated by the thermodynamic signature of the dimerization step. This observed dominance of the dimerization step illustrates the importance of dissecting complex DNA binding reactions into their constituent steps in elucidation of the thermodynamic driving forces for these processes. Measurements of the salt dependence of dimerization and DNA binding indicate modest contributions of electrostatic interactions to each contributing step as well as the total assembly of the repression complex. In light of the known structural features of this system, this modest dependence of the DNA binding equilibrium on salt concentration was unanticipated.