Many cellular functions rely on interactions among proteins and between proteins and nucleic acids. The overall goal of this project, jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Biological Physics Program in the Division of Physics in the Mathematical and Physical Sciences Directorate, is to decipher the first principles of self-assembly processes of biomolecules with the ultimate aim to understand the principles of cellular network organization from a physical viewpoint. Energy landscape theory and the funnel concept have advanced the understanding of the robust self-assembly of a single protein chain into its uniquely native structure. For small proteins, the reduced models that are minimally energetically frustrated will be generalized to study the effects of local geometrical details, non-native interactions, and desolvation on folding mechanisms. Larger proteins, which have much more complex energy landscapes, will be studied to characterize their folding mechanisms, kinetics, pathways, and intermediates. Folding alone, however, is not sufficient for a full picture of function. Function requires change of structure and specific recognition to form complexes and to enable the wireless communication in the cell. This research will address these two aspects by: (1) Exploring the conformational transitions associated with function using generalized reduced models that incorporate the multiple stable conformations of the protein. Unlike macroscopic machines, proteins are biological machines that can locally break and then reassemble during function. Models for global structural transformations, such as allostery and molecular communication, will be developed. They involve large-scale motions and possible partial unfolding. (2) Exploring the physical and chemical principles of protein binding that govern the protein-protein interactions in the context of the cell. Such principles have to be formulated before one turns to study specificity, affinity, and cross-reactivity. Understanding these global/functional motions of proteins will not only provide an understanding of the biological phenomena but, by learning the function and designing principles of these nanoscale molecular machines, it will lead to a major impact in nanoscience. Using designing principles that mimic these biomolecular machines will allow us to develop an entirely new family of materials, which function at the nanoscale, with applications such as sensing and catalysis.
The NSF support has been vital for training students and postdoctoral fellows in the highly interdisciplinary field of molecular biophysics. Besides theoretical training, a strong understanding of the experiments is essential. Five former postdoctoral fellows and two graduate students are now professors at major universities. The PI has always had a good representation of women and members from underrepresented groups in his research.
Using the power of theoretical and computational models we can now understand how proteins use not only their final folded structures but other partially disordered states to achieve their functions. These ensemble of possible conformations (called the protein landscape) is not only important to help the protein to fold on its native structure but also to achieve its function. Below we show a few examples of what has been achieved using this theoretical and computational framework. In terms of broader impact, our trained scientists, which were initially only trained in physics, were exposed on the life sciences and able to make major contributions to molecular biophysics using ideas and tools coming from physics. Also we made our software publicly available and now is widely used by the experimental and theoretical protein community . Our SMOG folding/function software has proven to be a powerful tool to study protein folding and connections to function of single proteins as well as large biomachines such as the ribosome. 1. Protein Complex Formation and Function The Rop-homodimer (Repressor of Primer), Rop regulates replication in {it E. coli} through RNA binding. While WT-Rop (un)folds very slowly, mutations in the protein core can increase the folding/unfolding rates by up to four orders of magnitude. Surprisingly, these changes in kinetic behavior are not accompanied by strong changes in thermal and chemical stability. As most mutants are still able to bind RNA, it was assumed that Rop kept the same binding interface and global fold ({it syn} conformation - see fig. 1). Using a combined theoretical and experimental approach, we have shown that the hydrophobic core mutations undo the evolutionary bias towards function and enable competition between the syn and anti conformations in which Rop's constituent monomers may arrange (fig. 1). The dual existence of the two conformations and the ability of proteins to switch between different structures is totally novel discovery and opens many different ways of understanding protein signaling and function. 2. Biomolecular Motors In the presence of ATP, kinesin proceeds along the protofilament of microtubules by alternated binding of two motor domains on the tubulin binding sites. Because the processivity of kinesin is much higher than other motor proteins, it has been speculated that there exists allosteric regulation between the two monomers. Recent experiments suggest that ATP binding to the leading head (L) domain in kinesin is regulated by the rearward strain built on the neck-linker. We have shown that this function ability comes from the competition between the tendency of the protein motor to fold to its native structure and the desire of the motor heads to bind to the microtubule. Both cannot be completely satisfied simultaneously. Because the protein motor cannot satisfy both conditions, the equilibrium structures of kinesin on the microtubule show disordered and ordered neck-linker configurations for the L and trailing head (T), respectively. The comparison of the structures between the two heads shows that several native contacts present at the nucleotide binding site in L are less intact than those in the binding site of T. The network of native contacts obtained from this comparison provides the internal tension propagation pathway, which leads to the disruption of the nucleotide binding site in the L. This strain (or local cracking) avoids the early binding of ATP, which would lead to destruction of processivity, i.e., the protein motor would detach from the microtubule instead of repeating the functional cycle.
