UC San Diego

Often, proteins are studied using bulk techniques, in which the average properties of large ensembles of molecules are studied. Although this has led to substantial new knowledge, the use of single-molecule biophysics and computational techniques to understand individual molecular actions and dynamics and the role of each residue allows for a more complete picture of activity in some cases. We present two applications of such techniques, first to determine the relationship between structure and function of the DNA translocation motor gp17 in Bacteriophage T4, and second to study looping of DNA mediated by the tumor suppressor protein p53. Specifically, we studied the role of an interface between the N- and C-terminal subdomains in generation of the high packaging forces and translocation velocities using a dual-beam optical trap. Mutation of charged amino acids located within this interface region confirmed that electrostatic forces play a role in force and velocity generation, with mutants showing a reduction in forward velocity, average velocity, and percentage of time spent packaging at different applied forces. To explain these experimental results, we generated a two-state computational model to calculate the free energy of the translocation step in gp17. We found excellent correlation between experimental data and calculated free energy change of translocation. Decomposition of the free energy change allowed for the identification of key residues involved in gp17-mediated packaging, and the role of each was explained. These results reveal that the power stroke of the motor requires substantial contributions from charged residues, hydrophobic residues, and polar residues instead of charged residues alone. Finally, we propose that several of these key residues may be hot spot residues, contributing a significant portion of the free energy used to package DNA. p53-mediated loop formation in DNA was studied using direct measurement with a dual optical tweezer setup. Looping of DNA by p53 has previously been demonstrated qualitatively using cryo- electron microscopy as well as transcription assays. we demonstrated formation of loops in purified human Col18A promoter containing five p53 binding sites. This looping may be directly related to p53 activity at transcriptional start sites