Poster Sessions

Biophys-6

Timothy Stasevich

T. J. Stasevich, F. Mueller, D. T. Brown, J. G. McNally

Quantifying the Cooperativity of Protein Binding Domains In Vivo Using FRAP: Sequential Binding of H1 to Chromatin

Protein binding is a complex process that can involve many intermediate steps and often relies on strong cooperation between different parts of the protein. Traditionally, in vitro biochemistry was the only way to quantify cooperative effects and distinguish protein-substrate binding pathways. Here we show how this analysis can be done in live cells using quantitative FRAP. We developed a novel FRAP protocol that produces high-resolution spatio-temporal images of recoveries to better visualize protein dynamics. In these images, fast and slow binding components appear as distinct features separable by eye. Now, instead of fitting FRAP recoveries to one-dimensional curves, we fit the entire spatio-temporal image. This method produces accurate bound fraction estimates that can then be used to deduce the most likely pathway for a multi-domain protein to bind its substrate. We used our technique to dissect the binding of the linker histone H1 to chromatin. H1 plays a crucial role in the folding of chromatin into higher order structures and is thought to be a general repressor of transcription. How H1 accomplishes this remains unclear. Earlier studies found evidence for three important chromatin binding domains on the H1 molecule: the S1 and S2 subdomains in the central globular domain, as well as the unstructured C terminal tail. We decomposed the binding process by analyzing proteins defective in all combinations of these three domains. Taken together, we obtained a matrix of binding rates and bound fractions that is remarkably consistent, with different rates and fractions attributable to different binding domains. Our analysis reveals complex sequential H1-chromatin binding that is most often initiated by the C terminal tail, followed by S1 or S2 with near equal affinity. Interestingly, we find little cooperation between the S1 and S2 subdomains by themselves, but they both cooperate significantly with the C terminal tail. This suggests the tail “bridges the gap” between the S1 and S2 binding sites so they can be simultaneously bound. Once this happens, a transition to an even tighter state then occurs in roughly 20% of the H1 population. These results paint a complex, highly dynamic picture of H1-chromatin binding, with the majority of H1 only partially bound in metastable states. Such states may enhance binding flexibility, allowing for partial dissociation of H1 from DNA during processes such as transcription and replication.