Seongheun Kim

Direct observation of fast protein conformational switching.

Folded proteins can exist in multiple conformational substates. Each substate reflects a local minimum on the free-energy landscape with a distinct structure. By using ultrafast 2D-IR vibrational echo chemical-exchange spectroscopy, conformational switching between two well defined substates of a myoglobin mutant is observed on the ≈50-ps time scale. The conformational dynamics are directly measured through the growth of cross peaks in the 2D-IR spectra of CO bound to the heme active site. The conformational switching involves motion of the distal histidine/E helix that changes the location of the imidazole side group of the histidine. The exchange between substates changes the frequency of the CO, which is detected by the time dependence of the 2D-IR vibrational echo spectrum. These results demonstrate that interconversion between protein conformational substates can occur on very fast time scales. The implications for larger structural changes that occur on much longer time scales are discussed.

Substrate binding and protein conformational dynamics measured by 2D-IR vibrational echo spectroscopy

Enzyme structural dynamics play a pivotal role in substrate binding and biological function, but the influence of substrate binding on enzyme dynamics has not been examined on fast time scales. In this work, picosecond dynamics of horseradish peroxidase (HRP) isoenzyme C in the free form and when ligated to a variety of small organic molecule substrates is studied by using 2D-IR vibrational echo spectroscopy. Carbon monoxide bound at the heme active site of HRP serves as a spectroscopic marker that is sensitive to the structural dynamics of the protein. In the free form, HRP assumes two distinct spectroscopic conformations that undergo fluctuations on a tens-of-picoseconds time scale. After substrate binding, HRP is locked into a single conformation that exhibits reduced amplitudes and slower time-scale structural dynamics. The decrease in carbon monoxide frequency fluctuations is attributed to reduced dynamic freedom of the distal histidine and the distal arginine, which are key residues in modulating substrate binding affinity. It is suggested that dynamic quenching caused by substrate binding can cause the protein to be locked into a conformation suitable for downstream steps in the enzymatic cycle of HRP.

Neuroglobin dynamics observed with ultrafast 2D-IR vibrational echo spectroscopy

Neuroglobin (Ngb), a protein in the globin family, is found in vertebrate brains. It binds oxygen reversibly. Compared with myoglobin (Mb), the amino acid sequence has limited similarity, but key residues around the heme and the classical globin fold are conserved in Ngb. The CO adduct of Ngb displays two CO absorption bands in the IR spectrum, referred to as N3 (distal histidine in the pocket) and N0 (distal histidine swung out of the pocket), which have absorption spectra that are almost identical with the Mb mutants L29F and H64V, respectively. The Mb mutants mimic the heme pocket structures of the corresponding Ngb conformers. The equilibrium protein dynamics for the CO adduct of Ngb are investigated by using ultrafast 2D-IR vibrational echo spectroscopy by observing the CO vibrations spectral diffusion (2D-IR spectra time dependence) and comparing the results with those for the Mb mutants. Although the heme pocket structure and the CO FTIR peak positions of Ngb are similar to those of the mutant Mb proteins, the 2D-IR results demonstrate that the fast structural fluctuations of Ngb are significantly slower than those of the mutant Mbs. The results may also provide some insights into the nature of the energy landscape in the vicinity of the folded protein free energy minimum.

Disulfide bond influence on protein structural dynamics probed with 2D-IR vibrational echo spectroscopy

Intramolecular disulfide bonds are understood to play a role in regulating protein stability and activity. Because disulfide bonds covalently link different components of a protein, they influence protein structure. However, the effects of disulfide bonds on fast (subpicosecond to ≈100 ps) protein equilibrium structural fluctuations have not been characterized experimentally. Here, ultrafast 2D-IR vibrational echo spectroscopy is used to examine the constraints an intramolecular disulfide bond places on the structural fluctuations of the protein neuroglobin (Ngb). Ngb is a globin family protein found in vertebrate brains that binds oxygen reversibly. Like myoglobin (Mb), Ngb has the classical globin fold and key residues around the heme are conserved. Furthermore, the heme-ligated CO vibrational spectra of Mb (Mb-CO) and Ngb (Ngb-CO) are virtually identical. However, in contrast to Mb, human Ngb has an intramolecular disulfide bond that affects its oxygen affinity and protein stability. By using 2D-IR vibrational echo spectroscopy, we investigated the equilibrium protein dynamics of Ngb-CO by observing the CO spectral diffusion (time dependence of the 2D-IR line shapes) with and without the disulfide bond. Despite the similarity of the linear FTIR spectra of Ngb-CO with and without the disulfide bond, 2D-IR measurements reveal that the equilibrium sampling of different protein configurations is accelerated by disruption of the disulfide bond. The observations indicate that the intramolecular disulfide bond in Ngb acts as an inhibitor of fast protein dynamics even though eliminating it does not produce significant conformational change in the proteins structure.

Native and Unfolded Cytochrome c-Comparison of Dynamics using 2D-IR Vibrational Echo Spectroscopy

Unfolded vs native CO-coordinated horse heart cytochrome c (h-cyt c) and a heme axial methionine mutant cyt c552 from Hydrogenobacter thermophilus ( Ht-M61A) are studied by IR absorption spectroscopy and ultrafast 2D-IR vibrational echo spectroscopy of the CO stretching mode. The unfolding is induced by guanidinium hydrochloride (GuHCl). The CO IR absorption spectra for both h-cyt c and Ht-M61A shift to the red as the GuHCl concentration is increased through the concentration region over which unfolding occurs. The spectra for the unfolded state are substantially broader than the spectra for the native proteins. A plot of the CO peak position vs GuHCl concentration produces a sigmoidal curve that overlays the concentration-dependent circular dichroism (CD) data of the CO-coordinated forms of both Ht-M61A and h-cyt c within experimental error. The coincidence of the CO peak shift curve with the CD curves demonstrates that the CO vibrational frequency is sensitive to the structural changes induced by the denaturant. 2D-IR vibrational echo experiments are performed on native Ht-M61A and on the protein in low- and high-concentration GuHCl solutions. The 2D-IR vibrational echo is sensitive to the global protein structural dynamics on time scales from subpicosecond to greater than 100 ps through the change in the shape of the 2D spectrum with time (spectral diffusion). At the high GuHCl concentration (5.1 M), at which Ht-M61A is essentially fully denatured as judged by CD, a very large reduction in dynamics is observed compared to the native protein within the approximately 100 ps time window of the experiment. The results suggest the denatured protein may be in a glassy-like state involving hydrophobic collapse around the heme.

Direct observation of ligand rebinding pathways in hemoglobin using femtosecond mid-IR spectroscopy.

The dynamics of NO rebinding in hemoglobin (Hb) was directly observed using femtosecond mid-IR spectroscopy after photodeligation of NO from HbNO in D(2)O at 283 K. Time-resolved spectra of bound NO appeared to have a single feature peaked at 1616 cm(-1) but were much better described by two Gaussians with equal intensities but different rebinding kinetics, where the feature at 1617 cm(-1) rebinds faster than the one at 1614 cm(-1). It is possible that the two bands each correspond to one of two subunit constituents of the tetrameric Hb. Transient absorption spectra of photodeligated NO revealed three evolving bands near 1858 cm(-1) and their red-shifted replicas. The red-shifted replicas arise from photodeligated NO in the vibrationally excited v = 1 state. More than 10% of the NO was dissociated into the vibrationally excited v = 1 state when photolyzed by a 580 nm pulse. The three absorption bands for the deligated NO could be attributed to three NO sites in or near the heme pocket. The kinetics of the three transient bands for the deligated NO, as well as the recovery of the bound NO population, was most consistent with a kinetics scheme that incorporates time-dependent rebinding from one site that rapidly equilibrates with the other two sites. The time dependence results from a time-dependent rebinding barrier due to conformational relaxation of protein after deligation. By assigning each absorption band to a site in the heme pocket of Hb, a pathway for rebinding of NO to Hb was proposed.

Protein conformation-controlled rebinding barrier of NO and its binding trajectories in myoglobin and hemoglobin at room temperature.

The effect of the solvent viscosity on the dynamics of NO rebinding to myoglobin (Mb) and hemoglobin (Hb) was examined by femtosecond (fs) time-resolved vibrational spectroscopy after photodeligation of NO from MbNO and HbNO in various viscous solutions at 283 K using a 580 nm excitation pulse. The rebinding kinetics of NO to both Mb and Hb were nonexponential, but their dependence on the solvent viscosity was different. The rate of NO rebinding to Mb increased with increasing solution viscosity, which was achieved by increasing the glycerol content in glycerol/water mixture. In contrast, the rate of NO rebinding to Hb was independent of the solution viscosity but faster than the fastest rate of NO rebinding observed in Mb. The dynamics of conformational relaxation of the protein after deligation were also measured by probing the evolution of the amide band. The effect of the solvent viscosity on the kinetics of conformational relaxation in both proteins was also quite different. The conformational relaxation of Mb became slower with increasing solution viscosity. On the other hand, the conformational relaxation of Hb was independent of the solution viscosity but slower than the slowest kinetics of Mb. The inverse correlation in the kinetics of conformational relaxation and NO rebinding suggests that the barrier of NO rebinding increases as the conformation of the protein relaxes toward the deligated structure after NO dissociation. The rebinding kinetics of NO to both proteins was well described by a kinetic model incorporating a time-dependent barrier for rebinding and exponential translocations between three states for dissociated NO.