Our research focuses on characterizing the molecular interactions that drive protein folding and association, determine specificity in ligand-receptor interactions, and mediate lipid domain formation in biological membranes. We develop and apply new optical tools based on ultrafast two-dimensional infrared (2D IR) spectroscopy and tip-enhanced spectral microscopy to directly visualize protein-lipid and lipid-lipid interactions in model membranes. Our current research builds upon our expertise in ultrafast optics, non-linear spectroscopy and microscopy, molecular modeling of vibrational spectra, and protein biophysics.
Peptide folding and membrane translocation
Three-dimensional protein structures are the end result of a complex, multi-step folding process. Spontaneous folding depends on a subtle enthalpic and entropic balance between contact formation, desolvation, and electrostatics. Lipid membranes add an extra layer of complexity as the environment changes significantly between the hydrophilic, solvent-exposed environment of the disordered peptide and the hydrophobic, membrane-embedded folded state. The figure shows possible pathways for the coupled folding-translocation of a small peptide into a lipid membrane. Our group uses a combination of transient two-dimensional infrared spectroscopy, peptide synthesis and isotope labeling of the peptide backbone, and molecular dynamics simulations to map protein-membrane interactions and track the folding pathways from the initial disordered state to the final folded structure. Future triggered experiments will reveal the underlying mechanisms of folding and insertion of pH-modulated peptides.
Biophysics of lipid membranes
Mixed lipid membranes are highly compartmentalized. Phase-separation, mediated by the cholesterol-assisted packing of the rigid acyl chains, results in the formation of nanometer scale domains enriched in rigid lipids. Membrane proteins, in turn, have the ability to self-organize into specific domains, producing short- and long-range order within the bilayer. The thermodynamics, composition, and lifetimes of these domains remain to be fully characterized, and while expected to be of key importance for a multitude of biological processes, the specific implications of membrane organization remain to be fully explored. Our experiments are aimed at identifying the molecular nature of specific lipid-lipid and protein-lipid interactions that result in phase-separation in model membranes. We are also interested in characterizing the mechanisms and biophysical properties of colocalized versus raft-associated proteins. We use a combination of isotope-edited 2D IR spectroscopy, tip-enhanced microscopy, and spectral simulations to map the molecular environment of specific lipids in heterogeneous systems.
Ultrafast 2D IR spectroscopy
Two-dimensional infrared spectroscopy is an ultrafast optical technique that reveals molecular structure and fast dynamics by using a sequence of femtosecond (10-15 s) laser pulses to measure frequency correlations between (and within) vibrational modes in a sample. In brief, a pair of pulses excites specific vibrational modes in the sample, then, following a short waiting time, a third laser pulse measures the response of the sample. Analogous to 2D NMR, a 2D IR spectrum is, in essence, a two-dimensional excitation-detection frequency correlation map. Cross peaks are observed when two vibrational modes are able to exchange vibrational energy, for instance, two normal modes involving the same atoms, or molecules able to chemically interconvert within the timescales measurement, such as two rapidly-interconverting molecular conformations. The degree of diagonal elongation (frequency correlation) is a direct measure of the frequency fluctuations of a vibrational mode, which in turn reports on the environment around a molecule. In general, the diagonal and off-diagonal lineshapes and peak intensities provide a detailed, bond-specific, view of molecular structure and dynamics.
Protein structure is reflected on the backbone C=O stretching vibrations, known as amide I modes. The periodic arrangement of residues along a protein backbone, gives rise to characteristic vibrational modes which appear in specific regions of the IR spectrum, but the broad, often featureless, peaks that arise from structural disorder and solvent exposure complicate the interpretation of traditional FTIR spectra. Two-dimensional infrared spectroscopy spreads the spectral information onto two frequency axes, thus providing an additional degree of structural characterization of the protein ensemble. In addition, the excitation-waiting-detection sequence of interactions serves to map the fast hydrogen-bonding dynamics of the backbone.
Computational modeling of IR spectra
Vibrational frequencies are particularly sensitive to the environment of a molecule, but lineshapes are often broad and difficult to interpret. Computational models are useful in helping us interpret the measured spectra (frequencies, linewidths, timescales). We develop models that produce accurate results at little computational expense. Particularly, semiclassical models take the advantage of the sensitivity of vibrational frequencies to the electrostatic environment. Our group is currently extending these models to understand how the spectra of lipids report on the overall structure and hydrogen-bonding environment, specifically the degree of hydration inside lipid bilayers.
To learn more about protein 2D IR spectroscopy please refer to:
C. Baiz, M. Reppert and A. Tokmakoff, An Introduction to Protein 2D IR Spectroscopy, in Ultrafast Infrared Vibrational Spectroscopy, ed. by Michael D. Fayer, pp. 361-404 [PDF]