Research Topics

• Chemical Exchange Dynamics
• Dynamics of Water
• Proton Transfer Dynamics
• Protein Dynamics and Biological Water
• Complex Liquids

Experimental Methods

• 2D-IR Echo Spectroscopy
• Polarization Selective Pump-Probe
• Optical Kerr Effect Method
• UV-Vis Transient Absorption and Fluorescence

Protein Dynamics and Biological Water--2DIR Vibrational Echo Spectroscopy

Proteins and enzyme are dynamical structures. The biological function of proteins is intimately linked to the ability of proteins to undergo structural changes. A folded protein with a particular structure occupies a minimum on its free energy landscape. However, the minimum is frequently a local minimum. Other minima of similar energy can also exist (see figure 10). When a protein occupies any one of these minima, it has a distinct structure. The different structures are substates of the folded protein. Transitions from one minimum to another correspond to dynamical changes in the protein’s structure that take the protein from one substate to another. Under thermal equilibrium conditions there will be continual conformational switching among substates. In addition to interconversion between substates, proteins undergo continuous structural fluctuations within a particular substate minimum. These fluctuations occur because of transition among shallow minima no the rough energy landscape near a local structural minimum (see figure 10). Such fluctuations within a substate minimum give rise to processes such as small ligand “diffusion” through a protein to an active site.

The ability of proteins to undergo conformational change is central to protein function. When an enzyme binds a substrate, the protein conformation will change. On the path of protein folding, a protein will sample many conformations as it progresses toward the native folded structure. Proteins can undergo large global conformational changes, which occur on long time scales, milliseconds to seconds. However, these large slow conformational changes, such as those that occur following substrate binding to an enzyme, involve a vast number of more local elementary conformational steps.

We are using ultrafast 2D IR vibrational echo spectroscopy as well as other methods to study protein dynamics, structure, and interactions. In addition to the study of biomolecules, we are also investigating biological water. The experimental determination for the time scale of elementary conformational steps (switching from one structural conformation to another) is a long standing problem that we are now been successfully addressed using ultrafast 2D-IR vibrational echo chemical exchange spectroscopy, which was discussed in the Chemical Exchange Dynamics – Solute Solvent Complexes and Other Systems section.

The problem of multiple substates has been studied extensively for the protein myoglobin with the ligand CO bound at the active site (Mb-CO). The infrared spectrum of the heme-ligated CO stretching mode of Mb shows two major absorption bands, denoted A1 (1945 cm-1), and A3 (1932 cm-1) as shown in the upper right portion of figure 11. Mb-CO interconverts between these two conformational substates under thermal equilibrium conditions. The distal histidine, His64, plays a prominent role in determining the conformational substates of Mb. We studied a Mb mutant, L29I (leucine replaced by an isoleucine). The structure and the CO absorption spectrum of L29I-CO are shown in the left hand portion of figure 11. The replacement of leucine with isoleucine makes the A1 and A3 CO bands almost equal in amplitude. Changes in the configuration of the E helix (see figure 11) cause the distal histidine’s imidazole side group to move relative to the CO (see lower right portion of figure 11). The lower frequency of A3 compared to A1 reflects a closer proximity of the protonated epsilon nitrogen of the imidazole side group to the CO in A3. Each A substate exhibits a distinct ligand binding rate. Therefore, the peaks in the FT-IR spectrum of Mb-CO and L29I-CO reflect functionally distinct conformational substates.

Figure 12 displays 2D-IR spectra of CO bound to L29I at several Tws. This data on proteins is equivalent of the chemical exchange data shown in figures 2 and 3 for solute-solvent complex chemical exchange. The red bands are positive going and correspond to the 0-1 vibrational transitions. The blue bands (negative going) are from the 1-2 transition. For Tw = 0.5 ps, only the two diagonal peaks are observed. These correspond to the A1 and A3 bands in the FT-IR spectrum shown in figure 11. As Tw increases, the off-diagonal chemical exchange peaks grow in. By Tw = 48 ps the off-diagonal bands are readily apparent. The band to the upper left in the Tw = 48 ps panel is strong. In contrast to figure 2, because the anharmonicity is not large, the negative going 1-2 diagonal band partially overlaps the positive going off-diagonal chemical exchange peak to the lower right of the two 0-1 diagonal peaks, reducing its amplitude.

The peak volumes were fit, and both the positive (0-1) and negative (1-2) peaks were included in the analysis. The result is a plot like that shown in figure 3. The results of the fitting yield the protein structural substate switching time, ts = 47 ps ± 8 ps. These experiments are the first measurement of the time dependence of a single well defined elementary protein structural change.

When a protein experiences a perturbation such as substrate binding or a temperature jump that induces folding or unfolding, in accord with linear response, it will respond to the perturbation by fluctuation driven elementary steps (substate changes) that can occur in the absence of the perturbation. However, the sampling of the substates will be biased by the perturbation, and the protein will relax to a new structure. The protein is always executing a multidimensional walk among substates. The walk will be skewed by the perturbation. The skewing can result from the shifting of substate minima and barriers. The relaxation to the new structure can be slow because it requires many elementary steps. A slow response to a perturbation results from structural fluctuation sampling that produces elementary steps, which in turn combine to produce major restructuring. The results shown here provide the time scale for the elementary steps.

In addition to chemical exchange spectroscopy, we are using 2D IR vibrational echo experiments to study protein dynamics through measurements of spectral diffusion. In a 2D IR vibrational echo chemical exchange experiments, the interconversion between two (or more) distinct structures is manifested by the appearance of off-diagonal peaks (see figure 2). When there are a vast number of structures sampled because of transitions on the rough protein energy landscape (see figure 10), the frequency of the vibrational transition under observation will evolve in time. It will take on many values as the protein samples many different structures. The time dependence of the transition frequency is called spectral diffusion. Spectral diffusion is manifested by the time dependence of the 2D IR vibrational echo line shapes.

One example of our studies of protein dynamics using 2D IR vibrational echo spectral diffusion measurement is the influence of substrate binding on the structural dynamics of an enzyme, in this case horseradish peroxidase (HRP). Enzyme-substrate binding is a dynamic process that is intimately coupled to protein structural fluctuations. A complete description of protein-ligand interactions requires information on the modification of protein dynamics when a ligand binds.
HRP is a type III peroxidase family glycoprotein that oxidizes a variety of organic molecules in the presence of hydrogen peroxide as the oxidizing agent. HRP has proven to be amenable to protein engineering and its reactivity towards a wide variety of organic substrates has made it of intense interest in bio-industrial and enantiospecific catalysis applications.

The active site of HRP is comprised of a solvent exposed iron heme prosthetic group (see figure 13) that participates in the enzymatic catalysis cycle. The heme can bind carbon monoxide (CO), which we us as a site specific reporter of protein structure and dynamics. The time dependence of the CO transition frequency measured using 2D IR vibrational echoes is a spectroscopic reporter of protein structural fluctuations. One of the five small molecule substrates that are benzhydroxamic acid analogs that we have studied will be discussed here.

Figure 14 shows the background subtracted linear spectra of HRP-CO without a ligand (top panel) and ligated with BHA. With a ligand, the spectrum changes from two peaks to a single peak centered at 1909 cm-1.97, 98 Spectra with four other substrates show that the ligated HRP spectra are spread around the frequency of the unligated HRP red state (lower frequency peak) and are significantly lower in frequency than the blue unligated state.

Figure 15 shows the 2D vibrational echo spectra of the free form of HRP-CO at several times (Tws,10% contours). The two positive going bands on the diagonal correspond to the two peaks in the linear IR spectrum shown in figure 14 top panel The two off-diagonal negative going bands arise from the two associated 1-2 transitions. As Tw increases, the peaks become more symmetrical. It is the change in shape with time that provides the dynamical information. The low frequency band has a significantly shorter vibrational life time that give rise to its decrease in amplitude relative to the higher frequency band by Tw = 32 ps. The full data sets contain much finer gradations and the signals can be analyzed at the longest Tws.

Figure 16 top shows a measure of the shape of the spectrum as a function of the time, Tw lower frequency peak of free HRP-CO. This is the peak that closely corresponds to the single peak in the IR absorption spectrum when a substrate is bound (see figure 14). The bottom panel shows the shape change with the BHA substrate bound. Thorough theoretical analysis of this data provides quantitative information on the change in dynamics with substrate binding. Lines through the data are obtained from the full 2D-IR calculations that determine the dynamics. The detailed analysis has been published. It is clear from the data that there is a substantial change in HRP’s structural fluctuations when a substrate is bound. The dynamics slow considerably and the time scale of the fluctuations are moved to long times. The detailed analysis of the data and comparison to similar experiments on myoglobin and myoglobin mutants indicate that the change the observed spectral diffusion is caused by changes in the motions of the distal histidine (His 42) and distal arginine (Arg 38) (see figure 13). When the substrate is bound, the distal histidine and arginine are effectively locked up. There motions on the tens of picosecond time scale are greatly reduced.

We are using protein spectral diffusion measurements in a variety of contexts. We are studying the influence of protein unfolding on protein structural dynamics by examining spectral diffusion using denaturants to produce different partially unfolded protein states. We are also using spectral diffusion measurements to understand structural changes in protein, such as the disruption of a disulfide bond. In addition, we are studying biological water, that is, water in well defined biological environments such as the surfaces of model membranes. The boundary between a living cell and its surroundings is the plasma membrane with a thickness ranging from 7-10 nm. This nanoscale structure is primarily composed of phospholipids and embedded proteins. The membrane controls the flow of materials into and out of a cell, and it senses and controls the response of cells to hormones and other external signals. Biological membranes have a basic bilayer structure where the nonpolar chains of the phospholipids form the interior of a molecular bilayer. The organization of phospholipid bilayers and water is important because lipid-water interactions play a key role in native membrane functioning, stability of bilayers, water permeation, and fusion-related repulsive forces between bilayers. As discussed in the Dynamics of Water and Nanoscopic Water section, we are using 2D IR vibrational echoes and polarization selective IR pump-probe experiments to directly examine the dynamics of structure in nanoscopic environments. In addition to water at the surfaces of membranes, we are also interested in water-protein interactions.

Protein Dynamics and Biological Water 2D IR Vibrational Echo Spectroscopy

306. “Myoglobin-CO Substate Structures and Dynamics: Multidimensional Vibrational Echoes and Molecular Dynamics Simulations,” Kusai A. Merchant, W. G. Noid, Ryo Akiyama, Ilya Finkelstein, Alexei Goun, Brian L. McClain, Roger F. Loring, and M. D. Fayer, J. Am. Chem. Soc. 125, 13804-13818 (2003).

320. “Dynamics of Hemoglobin in Human Erythrocytes and in Solution: Influence of Viscosity Studied by Ultrafast Vibrational Echo Experiments,” Brian L. McClain, Ilya J. Finkelstein, and M. D. Fayer, J. Am. Chem. Soc. 126, 15702-15710 (2004).

341. “Dynamics of Proteins Encapsulated in Silica Sol-gel Glasses Studied with IR Vibrational Echo Spectroscopy,” Aaron M. Massari, Ilya J. Finkelstein, and M. D. Fayer, J. Am. Chem. Soc. 128, 3990-3997 (2006).

347. “Viscosity Dependent Protein Dynamics,” Ilya J. Finkelstein, Aaron M. Massari, and M. D. Fayer, Biophys. J. 92, 3652-3662 (2007).

355. “Substrate Binding and Protein Conformational Dynamics Measured via 2D-IR Vibrational Echo Spectroscopy,” Ilya J. Finkelstein, Haruto Ishikawa, Seongheun Kim, Aaron M. Massari, and M. D. Fayer Proc. Nat. Acad. Sci. 104, 2637-2642, (2007).

366. “Neuroglobin Dynamics Observed with Ultrafast 2D-IR Vibrational Echo Spectroscopy,” Haruto Ishikawa, Ilya J. Finkelstein, Seongheun Kim, Kyungwon Kwak, Jean K. Chung, Keisuke Wakasugi, Aaron M. Massari, and M. D. Fayer Proc. Nat. Acad. Sci. U.S.A. 104, 16116-16121 (2007).

367. “Disulfide Bonds’ Influence on Protein Structural Dynamics Probed with 2D-IR Vibrational Echo Spectroscopy,” Haruto Ishikawa, Seongheun Kim, Kyungwon Kwak, Keisuke Wakasugi, and M. D. Fayer Proc. Nat. Acad. Sci. U.S.A. 104, 19309-19314 (2007).

372. “Direct Observation of Fast Protein Conformational Switching,” Haruto Ishikawa, Kyungwon Kwak, Jean K. Chung, Seongheun Kim and M. D. Fayer Proc. Nat. Acad. Sci. U.S.A. 105, 8619-8624 (2008).

373. “Native and Unfolded Cytochrome C – Comparison of Dynamics using 2D-IR Vibrational Echo Spectroscopy,” Seongheun Kim, Jean K. Chung, Kyungwon Kwak, Sarah E. J. Bowman, Kara L. Bren, Biman Bagchi, and M. D. Fayer J. Phys. Chem. B ASAP (2008).
 

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