Technology Development Programs (TDP)
TDP1- MicroElectron Diffraction, directed by Guillermo Calero.
X-ray crystallography over the last 50 years has been the most successful method to obtain atomic structures of biological molecules. Crystallization of large multi-protein complexes is challenging, and even if successful, the resulting crystals are usually small, delicate and present multiple challenges. New developments such as serial femtosecond crystallography using a free electron laser (FEL) and electron crystallography or micro-electron diffraction (microED) are alternative approaches to obtain structures, using nano-meter or low micro-meter sized crystals (nanocrystals). Solving structures of bio-macromolecules using FELs requires billions of nanocrystals, amounts of material that frequently is not available. Solving structures with microED, on the other hand, may, in principle, be possible with only a few nanocrystals, thus overcoming problems related to sample quantity. The Calero laboratory at the University of Pittsburgh has pioneered nanocrystal discovery and optimization using transmission electron microscopy (TEM). In our MicroED technology program, new methodologies will be developed to determine structures of protein complexes of HIV-1 proteins and their human binding partners by exploring nano crystallization space and optimizing stabilizing conditions for the application of microED approaches for structure determination.
TDP2- Magic-Angle Spinning NMR Spectroscopy, co-directed by Angela Gronenborn and Tatyana Polenova
It has become clear that dynamics within protein molecules comprising HIV-1 assemblies play critical roles in regulating viral infectivity, including uncoating and maturation. Current progress in the field is hampered by the paucity of structural-biological methods that yield atomic-level information into structure and dynamics simultaneously, particularly when large-amplitude conformational rearrangements take place. Magic angle spinning (MAS) NMR is uniquely positioned to yield such information and, when integrated with molecular dynamics (MD) simulations, provides dynamic information inaccessible by any other means. While MAS NMR is a very powerful technique, it currently suffers from three major drawbacks: i) inherent low sensitivity, resulting in long measurement times; ii) high spectral congestion for large assemblies due to numerous overlapping signals; and iii) extensive and time-consuming data analysis for large systems (resonance assignments and structure
calculation), hampering the widespread use of the method.
Our technology program in MAS NMR integrates high magnetic fields (17.6–28.2 T) with ultrafast MAS frequencies, proton detection, streamlined data acquisition, processing, and analysis. It also employs dynamic nuclear polarization (DNP)-based experiments for analysis of low-concentration species as well as for investigations of the conformational space accessible to the dynamically disordered states. One goal is to develop new experiments suitable for studies of large assemblies of HIV-1 proteins and their complexes with host proteins and small-molecule interactors, in a fraction of time and with a small fraction of material that is required for conventional experiments.