Relevant Degree Programs
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
No abstract available.
The Mycobacterium tuberculosis ABC transporter Rv1747 is a putative exporter of cell wall biosynthesis intermediates. Rv1747 has a cytoplasmic regulatory module consisting of two pThr-interacting Forkhead-associated (FHA) domains connected by a conformationally disordered linker with two phospho-acceptor threonines (pThr). In chapter 2, I report the structures of FHA-1 and FHA-2 determined by X-ray crystallography and NMR spectroscopy, respectively. Relative to the canonical 11-strand beta-sandwich FHA domain fold of FHA-1, FHA-2 is circularly permuted and lacking one beta-strand. Nevertheless, the two share a conserved pThr-binding cleft. FHA-2 is less stable and more dynamic than FHA-1, yet binds model pThr peptides with moderately higher affinity (~ 50 uM versus 500 uM equilibrium dissociation constants). Based on NMR relaxation and chemical shift perturbation measurements, when joined within a polypeptide chain, either FHA domain can bind either linker pThr to form intra- and intermolecular complexes. Protein phase separation has been recently shown to be a fundamental mechanism underlying the clustering of some proteins at the eukaryotic cell membrane. In chapter 3, I show that upon multi-site phosphorylation of the linker by several Mtb serine/threonine protein kinases including PknF, the isolated regulatory module readily multimerizes and phase separates into dynamic liquid droplets with diagnostic properties similar to those exhibited by eukaryotic proteins. The process is reversed by the sole Mtb serine/threonine phosphatase PstP. In the absence of phosphorylation, the Rv1747 regulatory module can still undergo phase separation, albeit at higher protein concentrations and into droplets with more fluid properties. This points to a synergy between specific FHA-pThr binding and additional weak association of the ID linker and/or the FHA domains leading to the pre-requisite multivalent interactions for demixing. Droplet formation of the regulatory module was replicated in a pseudo-two-dimensional system on model supported lipid bilayers. Potential clusters of Rv1747 were also detected in Mtb using ultra-high resolution Direct Stochastic Reconstruction Microscopy (dSTORM). This is the first reported example of phase separation by both a bacterial protein and an ABC transporter, and suggests possible mechanisms for the regulation of Rv1747 within Mtb. I hypothesize that the tunable, phosphorylation-dependent multimerization described here regulates Rv1747 transporter activity.
Master's Student Supervision (2010 - 2018)
Amyloid fibril formation, believed to be a generic property of polypeptides, plays major roles in neurodegenerative pathologies such as Alzheimer’s, Parkinson’s and prion diseases, as well as in functional biological processes in many organisms including humans. Revealing specifics of their molecular architecture, conformational stability, mechanisms of formation and physical properties holds clues to devising effective methods to fight their associated pathologies. An increasing requirement has been the development of a detailed understanding of the nanomechanics of amyloid core structures due to their relevance in biomedicine and nanotechnology. Of special significance is the mechanism of fibril fracture and infectivity in disease as well as the mechanical stability for novel biomaterial design. Here, we use a series of steered molecular dynamics simulations on different amyloid fibrils to report a broad spectrum of mechanical properties ranging from a strong and stiff β-helical fibril to weak and soft amyloid such as those formed by the mammalian prion protein. We relate the strength and elastic modulus with hydrogen bond densities and van der Waals energies in the core of the fibrils and show that weakened side-chain interactions lead to fibrils with reduced tensile strengths as a result of modified molecular packing in the fibril core. This modulation might lead to a combination of exceptional mechanical attributes such as those of the human functional amyloids.