Intramolecular motions play an important role in a variety of biological processes including signal transduction, catalysis, and molecular motors. Unfortunately, these motions have proven difficult to study both experimentally and computationally. Standard computational techniques are limited to the nanosecond regime while the above motions occur in micro- to milliseconds. Alternatively, Brownian dynamics (BD) algorithms can reach the necessary timescales, but have traditionally been limited to rigid structures. Therefore, to investigate intramolecular motions from a computational perspective, novel techniques must be developed. In this proposal, we will develop and implement computational strategies to include intramolecular motions in BD simulations. Our approach involves: identifying protein domains, treating them as separate rigid bodies in a BD framework, and tethering them via an empirical energy function. The method, Tethered Brownian dynamics (TBD), will allow long timescale simulations that include intramolecular flexibility of multi-domain proteins. TBD will be initially tested against computational data such as normal mode analysis but then refined by utilizing neutron scattering spectra of Taq DNA polymerase. We will then apply these techniques to study the formation of class I adhesion pili in pyelonephritic E. coli. These structures are necessary and sufficient for adhesion, an important step towards infection. The techniques we will develop are necessary for investigating pili formation because it has been hypothesized that after the monomeric units diffuse through an usher protein in the bacterial outer membrane an internal hinge-bending motion is required to effectively dock the protein in its proper conformation. After validating our TBD algorithm for this system, we will test this docking hypothesis and attempt to identify the molecular mechanism for the structural effects of several mutations. Our simulations will describe an atomic scale model for pilus assembly that may enable the identification of pharmacologic targets for the inhibition of pilus formation and infection.
In this study, computational models that allow the study of flexible proteins will be developed and used to investigate the formation of class I pili, structures that bacteria produce to enable adhesion to human host cells. By elucidating the molecular mechanism of the formation of these structures, we hope to inform strategies to disrupt pili assembly, thus preventing or disabling adhesion and infection.
| Sung, Jeffrey C; Van Wynsberghe, Adam W; Amaro, Rommie E et al. (2010) Role of secondary sialic acid binding sites in influenza N1 neuraminidase. J Am Chem Soc 132:2883-5 |
