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Single-Molecule Vibrational Spectroscopy Adds Structural Resolution to the Optical Trap (New and Notable Commentary)
Ziad Ganim
Biophys. J., 2013, 104, 4-5 [link: 10.1016/j.bpj.2012.09.044]
The ability to apply forces on single molecules with an optical trap is combined with the endogenous structural resolution of Raman spectroscopy in an article in this issue, and applied to measure the Raman spectrum of ds-DNA during force-extension.
Vibrational excitons in ionophores; Experimental probes for quantum coherence-assisted ion transport and selectivity in ion channels
Ziad Ganim, Andrei Tokmakoff, Alipasha Vaziri
New. J. Phys., 2011, 13, 113030 [link: 10.1088/1367-2630/13/11/113030]
Despite a large body of work, the exact molecular details underlying ion-selectivity and transport in the potassium channel have not been fully laid to rest. One major reason has been the lack of experimental methods that can probe these mechanisms dynamically on their biologically relevant time scales. Recently it was suggested that quantum coherence and its interplay with thermal vibration might be involved in mediating ion-selectivity and transport. In this work we present an experimental strategy for using time resolved infrared spectroscopy to investigate these effects. We show the feasibility by demonstrating the IR absorption and Raman spectroscopic signatures of potassium binding model molecules that mimic the transient interactions of potassium with binding sites of the selectivity filter during ion conduction. In addition to guide our experiments on the real system we have performed molecular dynamic-based simulations of the FTIR and 2DIR spectra of the entire KcsA complex, which is the largest complex for which such modeling has been performed. We found that by combing isotope labeling with 2D IR spectroscopy, the signatures of potassium interaction with individual binding sites would be experimentally observable and identified specific labeling combinations that would maximize our expected experimental signatures.
Transient Two-Dimensional Spectroscopy with Linear Absorption Corrections Applied to Temperature Jump 2D IR
Kevin Jones, Ziad Ganim, Chunte Sam Peng, Andrei Tokmakoff
J. Opt. Soc. Am. B., 2012, 29, 118-129 [link: 10.1364/JOSAB.29.000118]
Multidimensional spectroscopies provide increased spectral information but time resolution is often limited by the picosecond lifetimes of the transitions they probe. At the expense of additional complexity, transient multidimensional techniques extend the accessible timescales for studying nonequilibrium chemical and biophysical phenomena. Transient temperature-jump (T-jump) experiments are particularly versatile, since they can be applied to any temperature-dependent change of state. We have developed a method to correct transient nonlinear techniques for distortions resulting from transient linear absorption of the probing pulses, distortions which can lead to false interpretations of the data. We apply these corrections in the collection of T-jump transient two- dimensional infrared spectra for the peptides diglycine and the β-hairpin peptide trpzip2. For diglycine, the T-jump induces changes in H-bonding, a response which is inherent to all aqueous systems. The trpzip2 results probe the hairpin unfolding kinetics and reveal two time scales: <10ns increased flexibility and 1.1 μs β-hairpin disordering.
Solvent and Conformation Dependence of Amide I Vibrations in Peptides and Proteins Containing Proline
Santanu Roy, Joshua Lessing, Georg Meisl, Ziad Ganim, Andrei Tokmakoff, Jasper Knoester, Thomas L. C. Jansen
J. Chem. Phys., 2011, 135, 234507 [link: 10.1063/1.3665417]
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 D2O and compare with experimental observations.
Melting of a β-Hairpin Peptide Using Isotope-Edited 2D IR Spectroscopy and Simulations
Adam W. Smith, Joshua Lessing, Ziad Ganim, Chunte Sam Peng, Andrei Tokmakoff, Santanu Roy, Thomas L. C. Jansen, Jasper Knoester
J. Phys. Chem. B, 2010, 114, 10913-10924 [link: 10.1021/jp104017h]
Isotope-edited two-dimensional infrared spectroscopy has been used to characterize the conformational heterogeneity of the β-hairpin peptide TrpZip2 (TZ2) across its thermal unfolding transition. Four isotopologues were synthesized to probe hydrogen bonding and solvent exposure of the β-turn (K8), the N-terminus (S1), and the midstrand region (T10 and T3T10). Isotope-shifts, 2D lineshapes, and other spectral changes to the amide I 2D IR spectra of labeled TZ2 isotopologues were observed as a function of temperature. Data were interpreted on the basis of structure-based spectroscopic modeling of conformers obtained from extensive molecular dynamics simulations. The K8 spectra reveal two unique turn geometries, the type I' β-turn observed in the NMR structure, and a less populated disordered or bulged loop. The data indicate that structures at low temperature resemble the folded NMR structure with typical cross-strand hydrogen bonds, although with a subpopulation of misformed turns. As the temperature is raised from 25 to 85 degrees C, the fraction of population with a type I' turn increases, but the termini also fray. Hydrogen bonding contacts in the midstrand region remain at all temperatures although with increasing thermal disorder. Our data show no evidence of an extended chain or random coil state for the TZ2 peptide at any temperature. The methods demonstrated here offer an approach to characterizing conformational variation within the folded or unfolded states of proteins and peptides.
Insulin dimer dissociation and unfolding revealed by amide I two-dimensional infrared spectroscopy
Ziad Ganim, Kevin C. Jones, Andrei Tokmakoff
Phys. Chem. Chem. Phys., 2010, 12, 3579-3588 [link: 10.1039/b923515a]
The monomer-dimer transition of insulin has been probed with two-dimensional infrared spectroscopy and related infrared spectroscopies to isolate spectral signatures of the conformational changes concomitant with dissociation. These experiments were atomistically interpreted using 2D IR spectra calculated from an ensemble of monomer and dimer structures including the effects of disorder, which provided a complement and a point of comparison to NMR and X-ray crystallography models. The amide I νperp mode, which is delocalized over both monomer units through an intermolecular antiparallel β sheet, was lost upon dimer dissociation and shifts were observed in the β-sheet and α-helix bands. These spectral changes provided a structurally sensitive probe of dimer dissociation, which was used to measure the binding constant, KD, and to parameterize a thermodynamic model for the dimer fraction. The solvent conditions surveyed the effects of ethanol and salt addition on the dimer fraction in acidic, deuterated water as a function of temperature. It was found that addition of ethanol had a significant destabilizing effect on the dimer state, and shifted KD from 70 μM in D2O to 7.0 mM in 20% EtOD at 22 %C. Simulation of the monomer 2D IR spectra indicates that the B-chain C terminus is partially disordered, although not fully solvated by water.
Heterodyne-Detected Dispersed Vibrational Echo Spectroscopy
Kevin C. Jones, Ziad Ganim, Andrei Tokmakoff
J. Phys. Chem. A, 2009, 113 (51), 14060-14066 [link: 10.1021/jp906256s]
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.
Amide I'-II' 2D IR Spectroscopy Provides Enhanced Protein Secondary Structural Sensitivity
Lauren P. DeFlores, Ziad Ganim, Rebecca A. Nicodemus, Andrei Tokmakoff
J. Am. Chem. Soc., 2009, 131 (9), 3385-3391 [link: 10.1021/ja8094922]
We demonstrate how multimode 2D IR spectroscopy of the protein amide I' and II' vibrations can be used to distinguish protein secondary structure. Polarization-dependent amide I'-II' 2D IR experiments on poly-l-lysine in the β-sheet, α-helix, and random coil conformations show that a combination of amide I' and II' diagonal and cross peaks can effectively distinguish between secondary structural content, where amide I' infrared spectroscopy alone cannot. The enhanced sensitivity arises from frequency and amplitude correlations between amide II' and amide I' spectra that reflect the symmetry of secondary structures. 2D IR surfaces are used to parametrize an excitonic model for the amide I'-II' manifold suitable to predict protein amide I'-II' spectra. This model reveals that the dominant vibrational interaction contributing to this sensitivity is a combination of negative amide II'-II' through-bond coupling and amide I'-II' coupling within the peptide unit. The empirically determined amide II'-II' couplings do not significantly vary with secondary structure: -8.5 cm-1 for the β sheet, -8.7 cm-1 for the α helix, and -5 cm-1 for the coil.
Amide I 2D IR Spectroscopy of Proteins
Ziad Ganim, Hoi Sung Chung, Adam W. Smith, Lauren P. DeFlores, Kevin C. Jones, and Andrei Tokmakoff
Acc. Chem. Res., 2008, 41 (3), 432-441 [link: 10.1021/ar700188n]
We review two-dimensional infrared (2D IR) spectroscopy of the amide I protein backbone vibration. Amide I modes are known for secondary structural sensitivity derived from their protein-wide delocalization. However, amide I FTIR spectra often display little variation for different proteins due to the broad and featureless line shape that arises from different structural motifs. 2D IR offers increased structural resolution by spreading the spectra over a second frequency dimension to reveal two-dimensional line shapes and cross-peaks. In addition, it carries picosecond time resolution, making it an excellent choice for understanding protein dynamics.
Transient 2D IR Spectroscopy of Ubiquitin Unfolding Dynamics
Hoi Sung Chung*, Ziad Ganim*, Kevin C. Jones, and Andrei Tokmakoff
Proc. Natl. Acad. Sci. USA, 2007, 104 (36), 14237-14242 [link: 10.1073/pnas.0700959104]
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 sheet's 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 ubiquitin's 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.
Spectral Signatures of Heterogeneous Protein Ensembles Revealed by MD Simulations of 2DIR Spectra
Ziad Ganim and Andrei Tokmakoff
Biophys. J., 2006, 91 (7), 2636-2646 [link: 10.1529/biophysj.106.088070]
A model for the calculation of amide I FTIR and 2DIR spectra taking into account fluctuations in hydrogen bonding and structure from molecular dynamics (MD) simulations is tested on three systems. It is found that although the homogeneous lineshape approximation yields satisfactory FTIR spectra, 2DIR spectra are sensitive to the inhomogeneity naturally present in any solvated protein and the common approximations of a static structure and averaged-effect solvent are invalid. By building on the local amide Hamiltonian and incorporating site energy variation with electrostatic-based models and disorder from MD trajectories, good agreement is obtained between calculated and measured 2DIR spectra. The largest contribution to the observed inhomogeneity is found to be the fluctuating site energies, which in turn are most sensitive to the water solvent. With the ability to accurately predict 2DIR spectra from atomistic simulations, new opportunities to test force fields and mechanistic predictions from MD are revealed.
The Anharmonic Vibrational Potential and Relaxation Pathways of the Amide I and II Modes of N-Methylacetamide
Lauren P. Deflores, Ziad Ganim, Sarah F. Ackley, Hoi Sung Chung, and Andrei Tokmakoff
J. Phys. Chem. B, 2006, 110 (38), 18973-18980 [link: 10.1021/jp0603334]
We investigate the influence of isotopic substitution and solvation of N-methylacetamide (NMA) on anharmonic vibrational coupling and vibrational relaxation of the amide I and amide II modes. Differences in the anharmonic potential of isotopic derivatives of NMA in D2O and DMSO-d6 are quantified by extraction of the anharmonic parameters and the transition dipole moment angles from cross-peaks in the two-dimensional infrared (2D-IR) spectra. To interpret the effects of isotopic substitution and solvent interaction on the anharmonic potential, density functional theory and potential energy distribution calculations are performed. It is shown that the origin of anharmonic variation arises from differing local mode contributions to the normal modes of the NMA isotopologues, particularly in amide II. The time domain manifestation of the coupling is the coherent exchange of excitation between amide modes seen as the quantum beats in femtosecond pump-probes. The biphasic behavior of population relaxation of the pump-probe and 2D-IR experiments can be understood by the rapid exchange of strongly coupled modes within the peptide backbone, followed by picosecond dissipation into weakly coupled modes of the bath.
Residual Native Structure in a Thermally Denatured β-Hairpin
Adam W. Smith, Hoi Sung Chung, Ziad Ganim, and Andrei Tokmakoff
J. Phys. Chem. B, 2005, 109 (36), 17025-17027 [link: 10.1021/jp053949m]
We investigate the thermal denaturation of trpzip2 between 15 and 82 oC using two-dimensional infrared (2D IR) vibrational spectroscopy, dispersed vibrational echo (DVE) spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. The FTIR and DVE spectra of trpzip2 show in the amide I region of the spectrum two resonances, which arise primarily from the interstrand coupling between local amide I oscillators along the peptide backbone. The coupling is seen directly in the 2D IR spectra as the formation of cross-peak ridges. Although small shifts of these frequencies occur on heating the sample, the existence of cross-peak ridges at all temperatures indicates that stable hydrogen bond interactions persist between the two β-strands. These observations indicate a significant amount of native structure in the thermally denatured state of trpzip2.
Conformational Changes During the Nanosecond-To-Millisecond Unfolding of Ubiquitin
Hoi Sung Chung, Munira Khalil, Adam W. Smith, Ziad Ganim, and Andrei Tokmakoff
Proc. Natl. Acad. Sci. USA, 2005, 102 (3), 612-617 [link: 10.1073/pnas.0408646102]
Steady-state and transient conformational changes upon the thermal unfolding of ubiquitin were investigated with nonlinear IR spectroscopy of the amide I vibrations. Equilibrium temperature-dependent 2D IR spectroscopy reveals the unfolding of the β-sheet of ubiquitin through the loss of cross peaks formed between transitions arising from delocalized vibrations of the β-sheet. Transient unfolding after a nanosecond temperature jump is monitored with dispersed vibrational echo spectroscopy, a projection of the 2D IR spectrum. Whereas the equilibrium study follows a simple two-state unfolding, the transient experiments observe complex relaxation behavior that differs for various spectral components and spans 6 decades in time. The transient behavior can be separated into fast and slow time scales. From 100 ns to 0.5 ms, the spectral features associated with β-sheet unfolding relax in a sequential, nonexponential manner, with time constants of 3 μs and 80 μs. By modeling the amide I vibrations of ubiquitin, this observation is explained as unfolding of the less stable strands III-V of the β-sheet before unfolding of the hairpin that forms part of the hydrophobic core. This downhill unfolding is followed by exponential barrier-crossing kinetics on a 3-ms time scale.
Resonance Raman Analysis of the Mechanism of Energy Storage and Chromophore Distortion in the Primary Visual Photoproduct
Elsa C. Y. Yan, Ziad Ganim, Manija A. Kazmi, Belinda S. W. Chang, Thomas P. Sakmar, Richard A. Mathies
Biochemistry, 2004, 43 (34), 10867-10876 [link: 10.1021/bi0400148]
The vibrational structure of the chromophore in the primary photoproduct of vision, bathorhodopsin, is examined to determine the cause of the anomalously decoupled and intense C11=C12 hydrogen-out-of-plane (HOOP) wagging modes and their relation to energy storage in the primary photoproduct. Low-temperature (77 K) resonance Raman spectra of Glu181 and Ser186 mutants of bovine rhodopsin reveal only mild mutagenic perturbations of the photoproduct spectrum suggesting that dipolar, electrostatic, or steric interactions with these residues do not cause the HOOP mode frequencies and intensities. Density functional theory calculations are performed to investigate the effect of geometric distortion on the HOOP coupling. The decoupled HOOP modes can be simulated by imposing ~40o twists in the same direction about the C11=C12 and C12-C13 bonds. Sequence comparison and examination of the binding site suggests that these distortions are caused by three constraints consisting of an electrostatic anchor between the protonated Schiff base and the Glu113 counterion, as well as steric interactions of the 9- and 13-methyl groups with surrounding residues. This distortion stores light energy that is used to drive the subsequent protein conformational changes that activate rhodopsin.
Retinal Counterion Switch in the Photoactivation of the G Protein-Coupled Receptor Rhodopsin
Elsa C. Y. Yan, Manija A. Kazmi, Ziad Ganim, Jian-Min Hoi, Douhai Pan, Belinda S. W. Chang, Thomas P. Sakmar, and Richard A. Mathies
Proc. Natl. Acad. Sci. USA, 2003, 100 (16), 9262-9267 [link: 10.1073/pnas.1531970100]
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.
Time-Resolved Resonance Raman Analysis of Chromophore Structural Changes in the Formation and Decay of Rhodopsin's BSI Intermediate
Duohai Pan, Ziad Ganim, Judy E. Kim, Michiel A. Berhoeven, Johan Lugtenburg, Richard A. Mathies
J. Am. Chem. Soc., 2002, 124 (17), 4857-4864 [link: 10.1021/ja0126663]
Time-resolved resonance Raman microchip flow experiments are performed to obtain the vibrational spectrum of the chromophore in rhodopsin's BSI intermediate and to probe structural changes in the bathorhodopsin-to-BSI and BSI-to-lumirhodopsin transitions. Kinetic Raman spectra from 250 ns to 3 μs identify the key vibrational features of BSI. BSI exhibits relatively intense HOOP modes at 886 and 945 cm-1 that are assigned to C14H and C11H=C12H Au wags, respectively. This result suggests that in the bathorhodopsin-to-BSI transition the highly strained all-trans chromophore has relaxed in the C10-C11=C12-C13 region, but is still distorted near C14. The low frequency of the 11,12 Au HOOP mode in BSI compared with that of lumirhodopsin and metarhodopsin I indicates weaker coupling between the 11H and 12H wags due to residual distortion of the BSI chromophore near C11=C12. The C=NH+ stretching mode in BSI at 1653 cm-1 exhibits a normal deuteriation induced downshift of 23 cm-1, implying that there is no significant structural rearrangement of the Schiff base counterion region in the transition of bathorhodopsin to BSI. However, a dramatic Schiff base environment change occurs in the BSI-to-lumirhodopsin transition, because the 1638 cm-1 C=NH+ stretching mode in lumirhodopsin is unusually low and shifts only 7 cm-1 in D2O, suggesting that it has essentially no H-bonding acceptor. With these data we can for the first time compare and discuss the room temperature resonance Raman vibrational structure of all the key intermediates in visual excitation.
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