Overview
Using recently developed nuclear magnetic resonance methods, the McDermott lab studies the structure, function, solvation, and conformational dynamics of proteins in native-like environments. Efforts of the research group demonstrated that NMR spectra of “solid state” proteins, such as intrinsic membrane proteins in native lipid bilayers, or protein assemblies like viral coats, can be spectrally assigned and serve as the basis for structural and dynamic investigations. NMR studies clarified the coordination of enzymatic function to protein conformational exchange, including active site loop motions and substrate repositioning.
Research Projects
Membrane Proteins
Membrane proteins are important players in many physiological processes such as transmembrane signaling, energy transduction, and ion transport and comprise ~30% of the genes encoded in eukaryotic genomes. Solid-state NMR offers the unique opportunity to study membrane proteins at atomic resolution while they are in a native-like lipid bilayer. We study the structure and dynamics of several such systems. These include KcsA, a bacterial potassium channel, E.Coli Subunit c, which is part of the ATP synthase machinery, and the photosynthetic light harvesting protein LH1.
Cytochrome P450
Cytochromes P450 are heme monooxygenases that play critical roles in the biosynthesis of lipids, steroids, antibiotics, and drug metabolism. Understanding the active site structure and dynamics is important for drug discovery and providing insight on biological processes. Cryogenic crystal structures of numerous P450 isoforms show the substrate positioned too far away from the heme to be the catalytically relevant binding mode. Molecular dynamics simulations have shown that at room temperature the ligand is position correctly for chemistry. We use solid state NMR to collect structural information of N-palmitoylglycine (NPG) bound to cytochrome P450 BMP in order to confirm the low temperature binding mode and prove the room temperature conformation.
Antifreeze Proteins and Protein Solvent Interactions
Antifreeze proteins (AFPs) can bind specifically to ice crystals and thereby lower the freezing point of a given solution below its melting point (thermal hysteresis). Although the soluble form of these proteins is very well characterized, little is known about their structure and dynamics when they are bound to ice. Therefore, we are applying solid-state NMR to a type III AFP to investigate the structural details of its ice-bound form. The ice affinity of AFPs distinguishes them from other soluble proteins, which are, even in the presence of ice, surrounded by a hydration shell that is crucial for their dynamics and function. We are, therefore, also investigating the interaction of soluble proteins such as ubiquitin with their hydration shell using frozen solution solid-state NMR.
Filamentous Phage Pf1
With the ultimate goal of developing an atomic-resolution structure for an entire intact virion, we are working toward assigning the DNA residues of the Pf1 bacteriophage (the coat protein already having been previously assigned almost to completion) and establishing distance restraints by solid-state NMR. We are primarily interested in the protein-DNA interactions which hold the massive 36 MDa virion together; understanding these interactions in detail would likely yield a model of virus structure and assembly that can be applicable to a broad range of filamentous bacteriophages and perhaps other classes of viruses. To this end, and to overcome the challenges of working with such a large system, we have employed novel techniques such as dynamic nuclear polarization (DNP)-enhanced solid-state NMR and innovative chemical labeling schemes. Besides purely structural studies, we are also investigating the role of hydration in Pf1 structure and dynamics. Water is thought to be an important structural element, mediating the interactions between the DNA and capsid (coat protein) and allowing the DNA some degree of mobility. We are interested in whether sub-populations of water (i.e. bulk vs. interfacial water) exist within the virion, and, if so, what their dynamics tell us about viral function
Protein Dynamics
For most enzymes and drug targets, ligand binding is associated with the motion of a flexible loop or domain and the restructuring of hydrogen bonds and other contacts. The characteristic timescales of an active-site flexible loop in TIM is under investigation. Similarly, metal-substrate geometry as well as conformational exchange rates are studied for metalloenzymes, such as the important drug target cytochrome P450. An advantage of solid-state NMR is that anisotropic interactions such as dipolar, quadrupolar, and chemical shift anisotropy (CSA) are retained, providing abundant dynamic information for proteins. With steady advances being made in signal-to-noise and resolution in the solid state, it is very promising to extract site-specific dynamics information through specific excitation schemes (pulse sequences). Currently, we study slow conformational change (millisecond-to-second timescale) of proteins by refocusing dipolar interactions through a very high-efficiency 1D dynamics detection scheme (CODEX), described by Schmidt-Rohr. Intermediate timescale (millisecond-to-microsecond) dynamics are studied through T1ρ measurements monitoring the motion of CSA tensors. We also study the fast conformational dynamics (faster than microsecond) of proteins through deuterium lineshape analysis. The quantitative application of these techniques to proteins and improvement of the pulse sequences are two main directions of our research.
Functional Prions
In a recent collaboration with the groups of Kandel and Hendrickson, the laboratory is studying the Aplysia Cytoplasmic Polyadenylation Element Binding protein (ApCPEB) which is involved in synaptic plasticity in the context of the molecular basis for memory. Structural and dynamic characterization of ApCPEB address the surprising idea that conversion to an amyloid form with enhanced function could provide the basis for the persistence of memory. Solid-state NMR and other biophysical methods provide support for this general model, and raise interesting questions about the contrast between this amyloid and those that are disease related.