The overall goals of this study are to probe the dynamics of molecular rearrangements in both the protein and the DNA during complex formation and to provide a physical basis for elucidating the molecular origins of sequence- and structure-specificity. This project will focus on two classes of proteins: (i) three closely related eubacterial DNA-bending proteins involved in DNA packaging and gene regulation that dramatically bend the DNA, by nearly 180 degrees: E. coli Integration Host Factor (IHF), histone-like protein from Anabeana (AHU), and Hbb from the Lyme-disease causing spirochete Borrelia burgdorferi; and (ii) a DNA-repair protein, MutS, that recognizes and binds to DNA sites with a mismatch, and bends the DNA by about 60 degrees, thus initiating the DNA repair machinery. These proteins recognize their binding sites primarily by an indirect readout mechanism, in which the sequence-dependent structure and flexibility/bendablity of the DNA play a key role. A ~10 nanosecond laser temperature-jump (T-jump) will be used to perturb the protein-DNA complex, and the bending/unbending dynamics of the bound DNA substrate will be monitored with time-resolved FRET on end-labeled DNA substrates. In addition, protein conformational changes in response to the T-jump will be monitored with Trp fluorescence changes of intrinsic or introduced Trp residues. Single-molecule FRET measurements will also be carried out on immobilized DNA substrates with bound protein to probe the distribution of bent conformations in the complex, and to provide dynamics information at the single-molecule level. The specific aims of this study are to (i) probe the role of DNA flexibility/bendability in the recognition mechanism by measuring the kinetics of DNA bending/unbending for a range of substrates with inserted distortions such as mismatches or single-T insertions that bind with widely varying affinities to the proteins in the IHF/HU family; (ii) probe directly the conformational changes in the protein to address the question: do protein conformational changes occur concurrently with the DNA bending/unbending step or in a distinct kinetic step; (iii) probe the nature of the transition state along the reaction coordinate for complex formation by investigating the effect of mutations that perturb specific protein-DNA interactions on the DNA bending/unbending rates; (iv) probe the DNA bending kinetics in mismatched substrates bound to MutS to investigate how DNA bending dynamics influence mismatch recognition by MutS and subsequent ATPase-driven steps in the DNA repair mechanism.
A novel aspect of this project is the application of laser T-jump techniques to probe the dynamics of protein-DNA interactions with submicrosecond time-resolution. The broader impact of this work is in the potential for extending these kinetics measurements to a wider class of protein-DNA systems, including other regulatory and DNA-repair proteins, for a deeper understanding of the underlying mechanisms. The primary educational goal is to establish an undergraduate Biophysics major at UIC, which will provide a multidisciplinary education with a strong analytical component. This project is being jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences and the Biological Physics Program in the Physics Division.
Many important biological functions such as gene regulation and DNA repair are initiated by the binding of special proteins that recognize specific DNA sequences. These "site-specific" proteins typically bind their target sites with thousand- or even million-fold higher affinities relative to random DNA sequences. Many of these proteins distort the DNA structure when they bind, by bending, kinking or twisting the DNA at that site, and rearrange their conformations for an optimal fit. Thus, sequence-dependent DNA deformability plays an important role in the recognition mechanism. Nevertheless, the underlying mechanism by which DNA-bending proteins recognize their target sites among a sea of nonspecific sites remains a fundamental problem in biology. Proteins need to rapidly scan the DNA for their target sites, and hence recognition of binding site must be fast compared to one-dimensional diffusion of the protein on DNA. How rapid is this recognition step is virtually unknown for almost any protein. The goals of this project were to measure the kinetics of conformational rearrangements in protein and DNA during binding site recognition, to improve our understanding of the role of sequence-dependent DNA structure in binding site recognition, and to elucidate the underlying physical principles that govern the recognition process. The experimental difficulty has been in capturing the kinetics of conformational rearrangements in protein and DNA during binding site recognition, because of the micro- to millisecond time-scales on which these conformational rearrangements are known to occur. In this project, laser temperature-jump techniques were used to probe the DNA bending dynamics with microsecond time-resolution. This study focused on two classes of DNA-bending proteins: (i) eubacterial DNA-bending protein Integration Host Factor (IHF) involved in DNA packaging and gene regulation that dramatically bends the DNA, by nearly 180°, at its binding site, and (ii) a DNA-repair protein, MutS, that recognizes and binds to DNA sites that contain a mismatch, bends the DNA at that site by about 60°, and initiates the DNA repair machinery. Measurements were designed to address the question: does the protein bend the DNA (protein-induced bending) or, alternatively, are "prebent" DNA conformations thermally accessible, which the protein captures to form the specific complex (conformational capture). To distinguish between these mechanisms requires characterization of reaction intermediates, and, in particular, snapshots of the "transition state" or bottlenecks along the recognition pathway. We have obtained such a snapshot, from measurements on the IHF-DNA complex, which indicate that the bottleneck in the recognition step for IHF is spontaneous kinking of cognate DNA to adopt a partially prebent conformation and point to conformational capture as the underlying mechanism of initial recognition, with additional protein-induced bending occurring after the transition state. Measurements on MutS bound to DNA containing an extra nucleotide in one strand revealed for the first time the conformational rearrangements in mismatch recognition by a DNA repair protein, which holds promise for a more thorough investigation of the rates of mismatch recognition, for different types of mismatches, and their correlation with mismatch repair efficiency. The broader impact of this work is in setting the stage for extending these kinetics measurements to a wider class of protein-DNA systems, including other regulatory and DNA-repair proteins, for a deeper understanding of the underlying mechanisms. This project has also contributed to the professional training of undergraduates, graduates, and postdoctoral students by their involvement in research, using state-of-the-art biophysical approaches designed to unveil elusive protein-DNA dynamics. Aspects of the research have been integrated in classroom teaching at the interface of biology and physics. A new introductory biophysics course, aimed primarily for biological science majors, which introduces quantitative approaches to the study of biological systems, has been developed to augment a previously developed upper-level molecular biophysics course.
