Michael Brown

Professor of Chemistry and Biochemistry

The Brown lab conducts research at the cutting edge of molecular spectroscopy and biophysical chemistry as well as nanoscience and naotechnology.   We carry out fundamental research in molecular spectroscopy together with applications to membrane proteins, lipid bilayers, surfactants, and liquid crystals—soft matter in general.  Our work spans fundamental science related to intermolecular interactions through to applied nanoengineering aspects, involving chemically tailored interfaces between lipids, proteins, and water.  Novel experiments are put forth together with theory at the leading edge of macromolecular science, lipid nanotechnology, and (bio)physical chemistry.  Applications encompass surface science, physical chemistry, condensed matter physics, materials science, and lipid nanotechnology.  Our unique focus is on integrating structure, dynamics, and function of proteolipid and surfactant assemblies using NMR spectroscopy and related analytical and biophysical methods.

Our broad goal is to discover how molecular interactions lead to the emergence of material properties and biological function by combining molecular spectroscopy with dynamics simulations and experimental studies.  In the area of molecular spectroscopy, we are applying nuclear magnetic resonance (NMR) technologies to liquid-crystalline materials and biomolecular systems.  A major focus is the use of experimental and theoretical NMR methods to achieve new insights into functional molecular mechanisms.  Indeed NMR is perhaps the most useful spectroscopic method in chemistry and biochemistry—it provides information about structure and dynamics with amazing breadth and versatility!  Applications range from organic synthesis to protein structure elucidation, studies of membranes and (bio)polymers, and magnetic resonance imaging (MRI) of human subjects.  Currently  students are applying solid-state NMR spectroscopy to membrane liquid crystals and proteins using both solution and solid-state NMR methods.  We also use solid-state NMR to investigate small molecule cofactors bound to membrane proteins.  In the area of biophysical chemistry and nanoengineering, our laboratory is engaged in structure-function studies of proteolipid membranes.  Chemical, physical, analytical, and biochemical methods provide a host of opportunities for cross-disciplinary training.  Members of our interdisciplinary team conduct research at US National Laboratories (Oak Ridge, Brookhaven, SLAC), where X-rays and neutrons are used to investigate molecular structure and dynamics.  Femtosecond X-ray nanocrystallography with a free electron laser is used to investigate conformational changes of membrane proteins.  With this approach, we detect the "protein quake" underlying the large-scale conformational changes.  Establishing how proteins like rhodopsin are affected by lipid bilayer deformation is another important emphasis of our highly motivated research team.  Functional work uses rhodopsin as a canonical prototype for G-protein-coupled receptors (GPCRs), the targets of the majority of human pharmaceuticals worldwide.  Notably we have designed lipid formulations using principles of nanoscience and technology that are capable of modulating the activity of membrane proteins like rhodopsin over a wide range.

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Solid-state NMR spectrometer with superconducting magnet and two-dimensional spectrum of a membrane protein. Note that the cross-peaks provide structural information.

Advancing the Frontiers of Biomolecular NMR Spectroscopy.  Biomembranes are liquid-crystalline systems, and X-ray crystallography provides only a partial view of their functions. Our group has devised solid-state NMR methods (order parameter analysis, relaxation methods) in the first detailed studies of membrane structure and dynamics. We have investigated ion-induced changes in the head groups of membrane lipids; their interactions with various sterols; and the structural dynamics of membrane lipids and proteins. Our group achieved a breakthrough by introducing a mean-torque analysis of the order parameters of membrane liquid crystals. Additional structural work involves organic synthesis for isotopic labeling of the ligands for integral membrane proteins. Proteins are purified from natural sources or expressed in bacteria. Our group has studied membrane interactions with antimicrobial peptides and proteins, such as alpha-synuclein (implicated in neurodegenerative disorders), Ras (GTPase oncogene), and ATP-synthase (molecular motor). Currently my students apply solid-state NMR to study light induced changes of rhodopsin (visual receptor), and an amino acid transport protein from 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.

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Laser excitation scheme used for pump-probe studies with femtosecond X-ray nanocrystallography of membrane proteins.

Femtosecond Nanocrystallography of Membrane Proteins.  The advent of X-ray free-electron lasers (XFELs) opens up entirely new horizons in chemistry, biology, and physics. The XFEL pulses have a peak brilliance that is 10⁹ stronger than any synchrotron X-ray radiation and enable time-dependent studies of protein dynamics to be conducted. Our team is currently investigating rhodopsin in nanocrystals using the short intense X-ray pulses of the free-electron laser at the Linac Coherent Light Source (LCLS) at SLAC National Accelerator Laboratory. Pump-probe studies of light activation are carried out in the sub-picosecond to microsecond range, and directly examine the fastest structural events of vision. Soon, we plan to extend the work to the millisecond time range, to probe the changes of rhodopsin that yield interaction with the G-protein, transducin. Current projects involve preparing nano/microcrystals of rhodopsin for pump-probe femtosecond X-ray studies. Additional time-resolved, wide-angle X-ray scattering (WAXS) studies of rhodopsin in detergent solutions investigate light-induced conformational changes ("protein quake"). The ultimate outcome will be a molecular movie of the activation process as a prototype for the Rhodopsin Family of G-protein-coupled receptors (GPCRs).

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Solid-state NMR relaxation reveals phospholipid membrane dynamics over a range of time and length scales.

Relaxation Experiments and Theory Development in NMR Spectroscopy.  Nuclear magnetic resonance (NMR) methods are among the premier experimental tools for obtaining knowledge of dynamics. Our experimental measurements of the magnetic field (frequency) dependence of the NMR relaxation rates are crucial for molecular dynamics (MD) simulations. Before we began our work in this area, interpreting the NMR relaxation times of biomolecules was viewed as too complicated to be solved in a meaningful way. However, we were able to achieve a solution with clear biophysical significance by combining experiment with theory. For lipid bilayers, the new model relates the energy landscape of the molecular fluctuations to the emergence of elastic properties over short distances, approaching the molecular dimensions. Knowing such properties allows a holistic interpretation of membrane structure and dynamics for understanding lipid interactions with cholesterol, peptides, and proteins. Projects include NMR relaxation studies of membrane lipid deformation by osmotic stress. The magnetic field (frequency) dependence of the relaxation is measured to identify the time and length scales of collective interactions. Additional projects involve cholesterol influences in lipid rafts, as well as lipid interactions with membrane peptides and proteins, and NMR relaxation studies of protein dynamics.

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Flexible surface model (FSM) describes how conformational changes of membrane proteins depend on spontaneous curvature of the lipid bilayer.

Biophysics and Biochemistry of Membranes.  Our studies of lipid-protein interactions have significantly advanced the field of biomembranes. Notably, we have extended the standard model (the fluid mosaic model) found in most biochemistry texts. We discovered how membrane lipids govern the conformational energetics of proteins by our innovation of a flexible surface model (FSM). In our picture, frustration (elastic deformation) of the membrane is counterbalanced by the hydrophobic mismatch of the lipids to the protein surface. Our bold new ideas explain how the tightly regulated lipids affect biological functions of proteins in signal transduction, ion conductance, and metabolite transport. Conformational changes involve elastic remodeling of the lipid bilayer that affects lipid-protein and lipid-peptide interactions (described by differential geometry). Allosteric regulation of membrane proteins is linked to influences of bilayer thickness, nonlamellar-forming lipids, detergents, and osmotic stress that are explained by the FSM as a new paradigm. Currently my students are investigating how properties such as curvature stress and membrane surface potential govern light activation of rhodopsin. The role of the membrane lipid composition in controlling activation of the G-protein (transducin) is also under study. Investigations of the roles of omega-3 polyunsaturated lipids in visual function and dysfunction are carried out. Our work illuminates how membrane properties underlie key cellular processes involved with signaling, ion channels, and drug transport.

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Three-dimensional structure of rhodopsin showing how retinal is more elongated in the active structures (purple and tan) than in the dark structure (cyan).

Discovering New Insights into Visual Signaling by Rhodopsin.  Knowing how rhodopsin is activated by light is important for understanding retinal proteins and can contribute added insight into membrane mechanisms.  Prior to our work in this area, rhodopsin was regarded as a simple on-off switch.  Our team determined the solid-state NMR structure of the retinal ligand of rhodopsin, and the changes triggered by light.  Surprisingly, we discovered that activation goes beyond a two-state conformational transition.  To explain our findings, we introduced an ensemble activation model.  We have proposed that the retinal cofactor initiates helix fluctuations on the microsecond-to-millisecond timescale in visual signaling.  Our innovations help to reveal how mutations of rhodopsin are implicated in human diseases.  Currently, we are combining solid-state NMR, electronic (UV-visible), and Fourier transform infrared (FTIR) spectroscopy to investigate how retinal triggers rhodopsin activation in membranes.  By combining spectroscopic methods with functional assays, we aim to achieve a comprehensive understanding of rhodopsin activation as a prototype for various membrane proteins.

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Molecular dynamics (MD) simulation showing an Influx of bulk water into rhodopsin following light-induced isomerization of the retinal cofactor

Research

Unraveling the Molecular Dynamics of G-Protein–Coupled Receptors.  Rhodopsin is the prototype for the Family A G-protein–coupled receptors (GPCRs), which are the targets of the majority of human pharmaceuticals.  When we began our work, the only X-ray structure for a GPCR was rhodopsin in the dark state.  Our group has been combining molecular dynamics (MD) simulations with experimental solid-state NMR data to investigate the triggering of visual signaling by rhodopsin. All-atom MD simulations are conducted using supercomputers and graphics processor units (GPUs) to model rhodopsin activation, and its interactions with signaling proteins like transducin.  We were first to discover a surprising influx of bulk water into the protein core giving activation of rhodopsin.  Currently our research team is testing the relations between hydration, binding affinity, and transducin activation in visual signaling.  Our molecular dynamics simulations and solid-state NMR studies point towards enhanced ligand flexibility, which explains the different retinal orientations seen in X-ray structures of active rhodopsin.

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