Ziad Ganim

Retinal counterion switch in the photoactivation of the G protein-coupled receptor rhodopsin

The biological function of Glu-181 in the photoactivation process of rhodopsin is explored through spectroscopic studies of site-specific mutants. Preresonance Raman vibrational spectra of the unphotolyzed E181Q mutant are nearly identical to spectra of the native pigment, supporting the view that Glu-181 is uncharged (protonated) in the dark state. The pH dependence of the absorption of the metarhodopsin I (Meta I)-like photoproduct of E181Q is investigated, revealing a dramatic shift of its Schiff base pKa compared with the native pigment. This result is most consistent with the assignment of Glu-181 as the primary counterion of the retinylidene protonated Schiff base in the Meta I state, implying that there is a counterion switch from Glu-113 in the dark state to Glu-181 in Meta I. We propose a model where the counterion switch occurs by transferring a proton from Glu-181 to Glu-113 through an H-bond network formed primarily with residues on extracellular loop II (EII). The resulting reorganization of EII is then coupled to movements of helix III through a conserved disulfide bond (Cys110–Cys187); this process may be a general element of G protein-coupled receptor activation.

Transient 2D IR spectroscopy of ubiquitin unfolding dynamics

Transient two-dimensional infrared (2D IR) spectroscopy is used as a probe of protein unfolding dynamics in a direct comparison of fast unfolding experiments with molecular dynamics simulations. In the experiments, the unfolding of ubiquitin is initiated by a laser temperature jump, and protein structural evolution from nanoseconds to milliseconds is probed using amide I 2D IR spectroscopy. The temperature jump prepares a subensemble near the unfolding transition state, leading to quasi-barrierless unfolding (the “burst phase”) before the millisecond activated unfolding kinetics. The burst phase unfolding of ubiquitin is characterized by a loss of the coupling between vibrations of the β-sheet, a process that manifests itself in the 2D IR spectrum as a frequency blue-shift and intensity decrease of the diagonal and cross-peaks of the sheets two IR active modes. As the sheet unfolds, increased fluctuations and solvent exposure of the β-sheet amide groups are also characterized by increases in homogeneous linewidth. Experimental spectra are compared with 2D IR spectra calculated from the time-evolving structures in a molecular dynamics simulation of ubiquitin unfolding. Unfolding is described as a sequential unfolding of strands in ubiquitins β-sheet, using two collective coordinates of the sheet: (i) the native interstrand contacts between adjacent β-strands I and II and (ii) the remaining β-strand contacts within the sheet. The methods used illustrate the general principles by which 2D IR spectroscopy can be used for detailed dynamical comparisons of experiment and simulation.

Solvent and conformation dependence of amide I vibrations in peptides and proteins containing proline

We present a mixed quantum-classical model for studying the amide I vibrational dynamics (predominantly CO stretching) in peptides and proteins containing proline. There are existing models developed for determining frequencies of and couplings between the secondary amide units. However, these are not applicable to proline because this amino acid has a tertiary amide unit. Therefore, a new parametrization is required for infrared-spectroscopic studies of proteins that contain proline, such as collagen, the most abundant protein in humans and animals. Here, we construct the electrostatic and dihedral maps accounting for solvent and conformation effects on frequency and coupling for the proline unit. We examine the quality and the applicability of these maps by carrying out spectral simulations of a number of peptides with proline in D(2)O and compare with experimental observations.

Heterodyne-detected dispersed vibrational echo spectroscopy

We develop heterodyned dispersed vibrational echo spectroscopy (HDVE) and demonstrate the new capabilities in biophysical applications. HDVE is a robust ultrafast technique that provides a characterization of the real and imaginary components of third-order nonlinear signals with high sensitivity and single-laser-shot capability and can be used to extract dispersed pump-probe and dispersed vibrational echo spectra. Four methods for acquiring HDVE phase and amplitude spectra were compared: Fourier transform spectral interferometry, a new phase modulation spectral interferometry technique, and combination schemes. These extraction techniques were demonstrated in the context of protein amide I spectroscopy. Experimental HDVE and heterodyned free induction decay amide I spectra were explicitly compared to conventional dispersed pump-probe, dispersed vibrational echo, and absorption spectra. The new capabilities of HDVE were demonstrated by acquiring single-shot spectra and melting curves of ubiquitin and concentration-dependent spectra of insulin suitable for extracting the binding constant for dimerization. The introduced techniques will prove particularly useful in transient experiments, studying irreversible reactions, and micromolar concentration studies of small proteins.

Vibrational excitons in ionophores: experimental probes for quantum coherence-assisted ion transport and selectivity in ion channels

Despite there being a large body of work, the exact molecular details underlying ion selectivity and transport in the potassium channel have not been fully uncovered. One major reason has been the lack of experimental methods that can probe these mechanisms dynamically on their biologically relevant timescales. Recently, it was suggested that quantum coherence and its interplay with thermal vibration might be involved in mediating ion selectivity and transport. In this paper, we present an experimental strategy for using time-resolved infrared spectroscopy to investigate these effects. We show the feasibility by demonstrating the infrared (IR) absorption and Raman spectroscopic signatures of the potassium-binding model molecules that mimic the transient interactions of potassium with binding sites of the selectivity filter during ion conduction. In addition to guiding our experiments on the real system, we have performed molecular dynamic-based simulations of the FTIR and two-dimensional IR (2DIR) spectra of the entire KcsA complex, which is the largest complex for which such modeling has been performed. We found that by combining isotope labeling with 2DIR spectroscopy, the signatures of potassium interaction with individual binding sites would be experimentally observable, and we identified specific labeling combinations that would maximize our expected experimental signatures.

Mechanically switching single-molecule fluorescence of GFP by unfolding and refolding

Significance Green fluorescent protein (GFP) is widely used as a tag to watch otherwise invisible proteins and as a sensor of its local chemical environment. Since GFP can form many partially folded states, it is critical to know how these structural changes affect its signature fluorescence. We use optical tweezers to force single molecules of GFP into folding and unfolding intermediate states and simultaneously probe single-molecule fluorescence from each state. It was found that GFP fluorescence requires complete structural integrity; none of the unfolding or refolding intermediates were observed to fluoresce, but fluorescence could be recovered by complete refolding. This feature was exploited to reversibly, mechanically switch GFP between on and off fluorescence states. Green fluorescent protein (GFP) variants are widely used as genetically encoded fluorescent fusion tags, and there is an increasing interest in engineering their structure to develop in vivo optical sensors, such as for optogenetics and force transduction. Ensemble experiments have shown that the fluorescence of GFP is quenched upon denaturation. Here we study the dependence of fluorescence on protein structure by driving single molecules of GFP into different conformational states with optical tweezers and simultaneously probing the chromophore with fluorescence. Our results show that fluorescence is lost during the earliest events in unfolding, 3.5 ms before secondary structure is disrupted. No fluorescence is observed from the unfolding intermediates or the ensemble of compact and extended states populated during refolding. We further demonstrate that GFP can be mechanically switched between emissive and dark states. These data definitively establish that complete structural integrity is necessary to observe single-molecule fluorescence of GFP.

An Optical Tweezers Platform for Single Molecule Force Spectroscopy in Organic Solvents

Observation at the single molecule level has been a revolutionary tool for molecular biophysics and materials science, but single molecule studies of solution-phase chemistry are less widespread. In this work we develop an experimental platform for solution-phase single molecule force spectroscopy in organic solvents. This optical-tweezer-based platform was designed for broad chemical applicability and utilizes optically trapped core-shell microspheres, synthetic polymer tethers, and click chemistry linkages formed in situ. We have observed stable optical trapping of the core-shell microspheres in ten different solvents, and single molecule link formation in four different solvents. These experiments demonstrate how to use optical tweezers for single molecule force application in the study of solution-phase chemistry.

Octave-spanning mid-infrared pulses by plasma generation in air pumped with an Yb:KGW source

Femtosecond mid-infrared (IR) supercontinuum generation in gas media provides a broadband source suited for time-domain spectroscopies and microscopies. This technology has largely utilized <100  fs Ti:sapphire pump lasers. In this Letter, we describe the first plasma generation mid-IR source based on a 1030 nm, 171 fs Yb:KGW laser system; when its first three harmonics are focused in air, a conical mode supercontinuum is generated that spans <1000 to 2700  cm-1 with a 190 pJ pulse energy and 0.5% RMS stability.

Thermal denaturing of proteins: Equilibrium and transient studies using nonlinear infrared probes

Thermal unfolding of β-sheets in ribonuclease A and ubiquitin is revealed by disappearance of cross peaks in 2D IR spectra. Transient unfolding probed with vibrational echoes following a temperature jump reveals nanosecond to millisecond dynamics.

Force-detected nanoscale absorption spectroscopy in water at room temperature using an optical trap

Measuring absorption spectra of single molecules presents a fundamental challenge for standard transmission-based instruments because of the inherently low signal relative to the large background of the excitation source. Here we demonstrate a new approach for performing absorption spectroscopy in solution using a force measurement to read out optical excitation at the nanoscale. The photoinduced force between model chromophores and an optically trapped gold nanoshell has been measured in water at room temperature. This photoinduced force is characterized as a function of wavelength to yield the force spectrum, which is shown to be correlated to the absorption spectrum for four model systems. The instrument constructed for these measurements combines an optical tweezer with frequency domain absorption spectroscopy over the 400-800 nm range. These measurements provide proof-of-principle experiments for force-detected nanoscale spectroscopies that operate under ambient chemical conditions.