Advancing the frontiers of biomolecular NMR spectroscopy
Biomembranes are liquid-crystalline systems, and X-ray crystallography provides only a partial view of their functions. Knowledge of both structure and dynamics is needed to understand their mechanisms of action. Our group developed solid-state NMR methods such as order parameter analysis, relaxation methods. There was little understanding of how order parameters are connected to the relaxation times in NMR (or fluorescence) spectroscopy. Subsequently, our solid-state NMR approach has been used to study membrane lipids, and their interactions with cholesterol in raft-like lipid mixtures. Our solid-state NMR approach has recently been extended through separated-local field 13C NMR spectroscopy, which does not require isotopic labeling.
Applications include proteins involved in neuroscience and so-called G-protein–coupled receptors (GPCRs). The GPCRs are the largest protein family in the human genome, and they play a central role in pharmacology. Bacteriorhodopsin works as ion channels and the passive pumps produce life on Earth and are of phenomenal importance in the energy budget of the planet. Misfolding of membrane proteins occurs in neurodegeneration, including Parkinson’s and Alzheimer’s diseases, with profound medical relevance. Membrane-associated peptides and integral membrane proteins are receptors for light, hormones, and neurotransmitters. Proteins are purified from natural sources or expressed using molecular biology techniques.
Our structural work involves organic synthesis for isotopic labeling of the ligands for integral membrane proteins. Angular and distance constraints from NMR spectroscopy are used for molecular modeling of the protein-bound ligands. Currently my students are applying solid-state NMR spectroscopy to study light induced changes of rhodopsin, as well as an amino acid transport protein in plant chloroplasts. We are also using these concepts to illuminate the roles of omega-3 polyunsaturated lipids in membranes. Additional new directions include NMR studies of medically important proteins involved with human health and disease.
Applications of Biomolecular NMR spectroscopy
Knowledge of both structure and dynamics is essential to understand biomolecular functioning. Nuclear magnetic resonance spectroscopy uniquely provides such information. We use multidimensional high-resolution solid-state and solution NMR methods for biomolecular structural determination. We investigate molecular dynamics over wide length and time scales using various NMR relaxation methods. Our solid-state NMR approach has been extended through separated-local field NMR spectroscopy (Leftin et al. 2014).
Applications include proteins involved in neuroscience and so-called G-protein–coupled receptors (GPCRs). The GPCRs are the largest protein family in the human genome, and they play a central role in pharmacology. Membrane-associated peptides and integral membrane proteins are receptors for light, hormones, and neurotransmitters. Furthermore, bacteriorhodopsin works as ion channels and the passive pumps produce life on Earth and are of phenomenal importance in the energy budget of the planet. Misfolding of membrane proteins occurs in neurodegeneration, including Parkinson’s and Alzheimer’s diseases, with profound medical relevance.
For the structural determination, proteins are expressed from bacteria in a 13C and 2H rich medium. Uniformly labeled proteins are used for the structural analysis in lipid bilayers. Angular and distance constraints from NMR spectroscopy are used for molecular modeling of the protein-bound ligands.
NMR Relaxation of Biomolecules:
Nuclear magnetic resonance methods are among the premier experimental technologies for obtaining knowledge of dynamics. We measure the relaxation of deuterium longitudinal (T1Z, T1Q, and T1ρ) and transverse (T2QE, and T2CPMG) relaxation using solid-state NMR methods. We developed a generalized model-free approach for analyzing the relaxation data (Xu et al. 2014). Our experimental measurements of the magnetic field (frequency) dependence of the NMR relaxation rates have been crucial for validating force fields for molecular dynamics (MD) simulations of membrane constituents. In 1982, Brownian dynamics simulations were used to develop a simple model for chain dynamics to help resolve a controversy involving the NMR relaxation of the chains of dipalmitoyl phosphatidylcholine. Later, Feller et al. used this model to generate a starting configuration for an MD simulation and have recently carried out a much longer MD simulation partly to test the validity of their original assumptions.
Furthermore, the solid-state NMR relaxation methods provide the correlation times and the activation energies of the rotation of the retinalidene methyl groups in Rhodopsin. These parameters provide the basis for the MD force fields. One example is that the rotational energy barrier for the 4-methyl-hexatriene is lower than that of the 2-methyl-butadiene. We also developed a theoretical formulation of the nuclear spin relaxation of biomolecules in terms of motional mean-square amplitudes (order parameters), as well as the rates of structural fluctuations. For lipid bilayers, we discovered how the two observables are connected (by a manifestation of Fermi’s Golden Rule).
For lipid bilayers, the new model relates the energy landscape of the molecular fluctuations to the emergence of elastic properties over distances approaching the molecular dimensions. Projects include NMR relaxation studies of collective interactions of membrane lipids, including the role of osmotic stress. An example of how osmotic stress modulate bilayer elastic properties and hence the dynamics is shown in the figure.