Investigating PIPs in Lipid Bilayers with Solid-State NMR
Phosphatidylinositol phosphates (PIPs) are a class of mammalian lipid that are based off of phosphorylation of the headgroup of phosphoinoisitol (PI).
- Mis-regulation of PIP pathways leads to disease (even cancer)
- Bind with specificity to 100s of proteins, including PH domains
- Goal: Establish a ssNMR methodology for observing PIPs and their interactions with Kindlin-2 (K2) PIP3-binding domains to gain insight into structural and dynamical changes which occur in the lipid and protein
The atomic resolution insight provided by solid-state NMR is vital for expanding our understanding of PIP-protein binding. In particular, we utilize 1H and 31P for studying our lipid systems. Both are naturally 100% abundant, therefore not needing any further labeling, and have high gyromagnetic ratios, a measure of magnetic susceptibility.
We seek to study membrane proteins in their native lipids, and determine the effects the lipids bilayer itself has on the protein and understand why they occur via the direct investigation of membranes by solid-state NMR. To do this, we continue to develop techniques for directly probing the structure and dynamics of lipid bilayers by solid-state NMR as well as the effects that different lipid species and small molecules can have on them. At Rutgers we have improved the 31P spectroscopy used to directly observe lipid head-groups. We use very-fast MAS and proton detection to observe lipid-lipid and lipid-protein interactions. We are developing pulse sequences and data analysis workflows to measure precise lipid-protein distances using 1H, 13C, 15N and 31P.
Molecular Dynamics to Study Peripheral Membrane Protein Binding
In conjunction with our ssNMR studies of biomimetic membranes, we use multiresolution molecular dynamics (MD) simulations to understand the binding mechanisms of peripheral membrane proteins (PMPs) to lipid bilayers. This involves coarse grained through quantum-scale simulation of PMPs and their effector lipids. More specifically, we are interested in the Integrin signaling pathway which has been implicated in diverse disease processes including aggregation of inflammatory cell recruitment and tumor metastasis. Kindlin-2 is a protein involved in initial integrin signaling events and has been found to enhance talin-mediated activation of integrin. However, the basis for recruitment of Kindlin-2 by phosphatidylinositol phosphates (PIPs) and its impact on Integrin activation is still poorly understood. We employ computational methods to study the biochemical factors which effect PIP dynamics at the lipid bilayer and their role in PMP binding.
Figure: Course grained simulations of K2 bound to PIP containing membranes. Using the INSANE framework for course graining, we were able to run longer simulation times with CPUs nodes. This allows us to sample inter-domain orientations relative to the bilayer.
NMR Method Development
We seek to apply the power of 1H-detected ssNMR to growing numbers of areas in ssNMR. Due to the fact 1H’s gyromagnetic ratio is 4 times that of 13C, experiments detecting 1H are more sensitive and therefore faster. While 1H detection is increasing well developed in terms of chemical shift assignments, there is still a wide array of areas where it has not yet been deployed. In particular, we are interested on using ultrafast MAS with 1H detection to gain structural insight without deuterated samples. We are developing new pulse sequences to determine intermolecular distances both within a protein and intermolecularly, as well as study dynamics at many timescales. We have a MAS probe which can spin at 40 kHz and another which spins at 100 kHz is on the way.
In addition to 1H detected methods, we will use 31P detected methods. 31P is spin 1/2, 100% abundant, and relatively high gamma (17.235 MHz/T). It has good lineshape and sensetivity, a wide chemical shift range, and a large dipolar coupling range (~ 8 A). In addition, we expect to be able to filter out the lipid phosphate signal to obtain spectra with good signal-to-noise of specific phosphates we are interested in.
NMR of Extremophile Bacteria
Thermopiezophiles are microorganisms that grow optimally at temperature between 50 and 100°C and at high pressures (> 200 bar), and are typically isolated from deep-sea hydrothermal vents. The details of their biochemistry may hold significance for new biotechnological applications and for our understanding of the origins of life, but relatively little is currently known about their biochemistry due to the challenges associated with observing them under conditions relevant to their growth and reproduction. We seek to apply NMR as a tool to reveal new biochemical details about the structure and dynamics of the membranes, cell wall, and metabolism of thermopiezophiles, and to develop new methods and instrumentation in order to observe these details in situ under high pressure and high temperature conditions. This project is a collaboration with the group of Prof. Costa Vetriani from the Rutgers School of Environmental and Biological Sciences (group website link: https://marine.rutgers.edu/deep-seamicrobiology/).
ssNMR of Ionic Liquids
Ionic liquids (ILs) are molten salts that are liquid near room temperature. They have distinct structural motifs. Each of these motifs has its relaxation and is associated with different length scales and can be described by the peaks associated with the structure-function. We conduct high-energy x-ray scattering experiments to obtain the structure-function and Quasi Elastic Neutron Scattering (QENS) and Neutron Spin Echo (NSE) experiments to study the microscopic dynamics of ILs in the space and time domain. On the other hand, we study the structure of the glassy state of ILs using solid-state Nuclear Magnetic Resonance (ssNMR) spectroscopy. Our experiments focus on understanding the connection between structure, local friction, and the overall viscosity of ILs. This project is a collaboration with Prof. (emeritus) Edward W. Castner, from Rutgers University, Prof. Claudio Margulis’ group from the University of Iowa, Prof. Sharon Lall-Ramnarine group from Queensborough Community College, Prof. Hideaki Shirota from Chiba University Japan and Dr. James Wishart group from Brookhaven National Laboratory.
13C 1D spectra of an ionic liquid (Pyrr14+ NTf2-) sample show splitting from 1H in the 19F decoupled spectrum (blue) and from the 19F in the 1H decoupled spectrum (red).