Cheolhee Yang

Protein Structural Dynamics of Photoactive Yellow Protein in Solution Revealed by Pump–Probe X-ray Solution Scattering

Photoreceptor proteins play crucial roles in receiving light stimuli that give rise to the responses required for biological function. However, structural characterization of conformational transition of the photoreceptors has been elusive in their native aqueous environment, even for a prototype photoreceptor, photoactive yellow protein (PYP). We employ pump-probe X-ray solution scattering to probe the structural changes that occur during the photocycle of PYP in a wide time range from 3.16 μs to 300 ms. By the analysis of both kinetics and structures of the intermediates, the structural progression of the protein in the solution phase is vividly visualized. We identify four structurally distinct intermediates and their associated five time constants and reconstructed the molecular shapes of the four intermediates from time-independent, species-associated difference scattering curves. The reconstructed structures of the intermediates show the large conformational changes such as the protrusion of N-terminus, which is restricted in the crystalline phase due to the crystal contact and thus could not be clearly observed by X-ray crystallography. The protrusion of the N-terminus and the protein volume gradually increase with the progress of the photocycle and becomes maximal in the final intermediate, which is proposed to be the signaling state. The data not only reveal that a common kinetic mechanism is applicable to both the crystalline and the solution phases, but also provide direct evidence for how the sample environment influences structural dynamics and the reaction rates of the PYP photocycle.

Conformational Substates of Myoglobin Intermediate Resolved by Picosecond X-ray Solution Scattering

Conformational substates of proteins are generally considered to play important roles in regulating protein functions, but an understanding of how they influence the structural dynamics and functions of the proteins has been elusive. Here, we investigate the structural dynamics of sperm whale myoglobin associated with the conformational substates using picosecond X-ray solution scattering. By applying kinetic analysis considering all of the plausible candidate models, we establish a kinetic model for the entire cycle of the protein transition in a wide time range from 100 ps to 10 ms. Four structurally distinct intermediates are formed during the cycle, and most importantly, the transition from the first intermediate to the second one (B → C) occurs biphasically. We attribute the biphasic kinetics to the involvement of two conformational substates of the first intermediate, which are generated by the interplay between the distal histidine and the photodissociated CO.

Coherent Oscillations in Chlorosome Elucidated by Two-Dimensional Electronic Spectroscopy.

Chlorosomes are the most efficient photosynthetic light-harvesting complexes found in nature and consist of many bacteriochlorophyll (BChl) molecules self-assembled into supramolecular aggregates. Here we elucidate the presence and the origin of coherent oscillations in chlorosome at cryogenic temperature using 2D electronic spectroscopy. We observe coherent oscillations of multiple frequencies superimposed on the ultrafast amplitude decay of 2D spectra. Comparison of oscillatory features in the rephasing and nonrephasing 2D spectra suggests that an oscillation of 620 cm(-1) frequency arises from electronic coherence. However, this coherent oscillation can be enhanced by vibronic coupling with intermolecular vibrations of BChl aggregate, and thus it might originate from vibronic coherence rather than pure electronic coherence. Although the 620 cm(-1) oscillation dephases rapidly, the electronic (or vibronic) coherence may still take part in the initial step of energy transfer in chlorosome, which is comparably fast.

Protein Folding Dynamics of Cytochrome c Seen by Transient Grating and Transient Absorption Spectroscopies

We investigate optically triggered protein folding dynamics of cytochrome c (Cytc) using transient grating (TG) and transient absorption (TA) spectroscopies. Despite many studies on protein folding dynamics of Cytc, a well-known model protein, direct spectroscopic evidence for the three-dimensional global folding process has been rarely reported. By measuring the TG signal of CO-bound Cytc (Cytc-CO) in the presence of a denaturant, we clearly detected the change of diffusion coefficient that reflects the size change of Cytc upon photodissociation of the CO ligand from unfolded Cytc-CO. The quantitative analysis of TG signals supports that the optically triggered folding reaction of Cytc in the presence of a denaturant takes place through a detectable intermediate (three-state folding kinetics). This is in contrast with the two-state folding dynamics of Cytc under a denaturant-free environment without any detectable intermediate. (1) From the quantitative global analysis of the TG signals, the rate constants for the U → I and I → N transitions in a CAPS buffer solution (pH 7) at room temperature in the presence of a denaturant at various concentrations are determined to be 1065 ± 17 to 3476 ± 103 s(-1) and 101 ± 6 to 589 ± 21 s(-1), respectively. In addition, the activation energies (E(a)) for the U → I and I → N transitions are determined to be 8.7 ± 1.0 kcal/mol and 7.1 ± 1.3 kcal/mol, respectively. The folding dynamics of Cytc initiated by the CO photolysis is discussed based in terms of the protein size change.

Folding dynamics of ferrocytochrome C in a denaturant-free environment probed by transient grating spectroscopy.

There have been numerous experimental and theoretical studies that aim to clarify the folding process occurring in biological systems, but many unsolved important questions still remain for protein folding. Generally, the folding processes of most proteins have been interpreted by two-state or sequential mechanisms. The two-state mechanism involves a transition from the unfolded state to the folded state without any detectable intermediates, whereas the sequential mechanism comprises multistate transitions through intermediates. To illuminate the detailed mechanism of protein folding processes, it is important to detect and characterize the species and intermediates involved in the folding process. Folding intermediates are short-lived, often heterogeneous, and cannot be studied by the usual crystallographic or NMR methods. Therefore, faster spectroscopic methods, such as time-resolved infrared, time-resolved circular dichroism (CD), 6,13, 14] transient absorption, and stopped-flow optical spectroscopic methods, have been utilized. These diverse methods can provide reaction rates, signal the accumulation of intermediates, and give local information on the role of particular amino acids or averaged parameters of the main chain. However, they do not provide global structural information in general. In this respect, the diffusion coefficient (D), which represents molecular migration in the liquid phase, is certainly a useful quantity for monitoring a protein-folding process because it is a fundamental physical parameter directly linked to the macromolecular size and shape. Indeed, Terazima and co-workers have shown that the change in diffusion coefficient reflects the conformational change of a biomolecule that occurs in the process of various biological reactions, such as protein folding. They used laser-induced transient grating (TG) spectroscopy to measure the change in D followed by a protein folding process. The TG technique can detect a spatial concentration modulation of chemical species induced by laser irradiation. From the temporal profile of the TG signal intensity, the D values of the parent molecule and transient species involved in a photoreaction can be determined directly from the decay rate of the signal measured. In summary, TG spectroscopy provides information about global structural change, whereas transient absorption is more sensitive to local structure and CD is informative for secondary structures. In the study reported herein, we investigate the optically triggered folding dynamics of CO-bound ferrocytochrome c (CytC-CO) under highly basic conditions (pH 13) by using timeresolved TG spectroscopy. The folding dynamics of CytC with a denaturant has been previously studied by using a combination of the electron transfer of nicotinamide adenine dinucleotide (NADH) and TG spectroscopy. Our study differs in that no denaturant is used so that the effect of the denaturant can be estimated, and the photodissociation of CO is a much faster reaction-triggering method than electron transfer of NADH ( ms), thereby greatly improving the time resolution. Indeed, we captured a process of about 700 ns, which cannot be studied by the latter reaction-triggering method. CytC-CO molecules in strongly basic solution are unfolded without a denaturant, such as guanidine hydrochloride (Gd-HCl) and urea. By contrast, CytC in the absence of the CO ligand has a nativelike structure in terms of secondary and tertiary structural content in strongly basic solutions (see Figure 1). There-

Cooperative protein structural dynamics of homodimeric hemoglobin linked to water cluster at subunit interface revealed by time-resolved X-ray solution scattering

Homodimeric hemoglobin (HbI) consisting of two subunits is a good model system for investigating the allosteric structural transition as it exhibits cooperativity in ligand binding. In this work, as an effort to extend our previous study on wild-type and F97Y mutant HbI, we investigate structural dynamics of a mutant HbI in solution to examine the role of well-organized interfacial water cluster, which has been known to mediate intersubunit communication in HbI. In the T72V mutant of HbI, the interfacial water cluster in the T state is perturbed due to the lack of Thr72, resulting in two less interfacial water molecules than in wild-type HbI. By performing picosecond time-resolved X-ray solution scattering experiment and kinetic analysis on the T72V mutant, we identify three structurally distinct intermediates (I1, I2, and I3) and show that the kinetics of the T72V mutant are well described by the same kinetic model used for wild-type and F97Y HbI, which involves biphasic kinetics, geminate recombination, and bimolecular CO recombination. The optimized kinetic model shows that the R-T transition and bimolecular CO recombination are faster in the T72V mutant than in the wild type. From structural analysis using species-associated difference scattering curves for the intermediates, we find that the T-like deoxy I3 intermediate in solution has a different structure from deoxy HbI in crystal. In addition, we extract detailed structural parameters of the intermediates such as E-F distance, intersubunit rotation angle, and heme-heme distance. By comparing the structures of protein intermediates in wild-type HbI and the T72V mutant, we reveal how the perturbation in the interfacial water cluster affects the kinetics and structures of reaction intermediates of HbI.

SVD-aided pseudo principal-component analysis: A new method to speed up and improve determination of the optimum kinetic model from time-resolved data

Determination of the optimum kinetic model is an essential prerequisite for characterizing dynamics and mechanism of a reaction. Here, we propose a simple method, termed as singular value decomposition-aided pseudo principal-component analysis (SAPPA), to facilitate determination of the optimum kinetic model from time-resolved data by bypassing any need to examine candidate kinetic models. We demonstrate the wide applicability of SAPPA by examining three different sets of experimental time-resolved data and show that SAPPA can efficiently determine the optimum kinetic model. In addition, the results of SAPPA for both time-resolved X-ray solution scattering (TRXSS) and transient absorption (TA) data of the same protein reveal that global structural changes of protein, which is probed by TRXSS, may occur more slowly than local structural changes around the chromophore, which is probed by TA spectroscopy.

Reply to “Comment on ‘Proton Transfer of Guanine Radical Cations Studied by Time-Resolved Resonance Raman Spectroscopy Combined with Pulse Radiolysis’”

Studied by Time-Resolved Resonance Raman Spectroscopy Combined with Pulse Radiolysis’” Jungkweon Choi,*,†,‡ Cheolhee Yang,‡,§ Mamoru Fujitsuka,† Sachiko Tojo,† Hyotcherl Ihee,‡,§ and Tetsuro Majima*,† †The Institute of Scientific and Industrial Research (SANKEN), Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan ‡Center for Nanomaterials and Chemical Reactions, Institute for Basic Science (IBS), Daejeon 305-701, Republic of Korea Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea

Photocycle of Photoactive Yellow Protein in Cell-Mimetic Environments: Molecular Volume Changes and Kinetics

Using various spectroscopic techniques such as UV-visible spectroscopy, circular dichroism spectroscopy, NMR spectroscopy, small-angle X-ray scattering, transient grating, and transient absorption techniques, we investigated how cell-mimetic environments made by crowding influence the photocycle of photoactive yellow protein (PYP) in terms of the molecular volume change and kinetics. Upon addition of molecular crowding agents, the ratio of the diffusion coefficient of the blue-shifted intermediate (pB) to that of the ground species (pG) significantly changes from 0.92 and approaches 1.0. This result indicates that the molecular volume change accompanied by the photocycle of PYP in molecularly crowded environments is much smaller than that which occurs in vitro and that the pB intermediate under crowded environments favors a compact conformation due to the excluded volume effect. The kinetics of the photocycle of PYP in cell-mimetic environments is greatly decelerated by the dehydration, owing to the interaction between the protein and small crowding agents, but is barely affected by the excluded volume effect. The results lead to the inference that the signaling transducer of PYP may not necessarily utilize the conformational change of PYP to sense the signaling state.

Correction to “Photocycle of Photoactive Yellow Protein in Cell-Mimetic Environments: Molecular Volume Changes and Kinetics”

I page 775 and Supporting Information (the legends of Figures S12 and S13), the wavelengths 380, 465, and 494 nm for temporal profiles of the TA signal of PYP were misreported as 362, 446, and 477 nm, respectively. As shown in Figure S14, the fitting results using the temporal profiles measured at 380, 465, and 494 nm are consistent with those using the temporal profiles measured at 362, 446, and 477 nm. This means that these errata do not affect rate constants and conclusions reported in the paper. The first sentence on page 775 should be corrected as follows: “The temporal profiles of the TA signal of PYP in the absence and presence of molecular crowding agents were measured at 380, 465, and 494 nm.” The corrected Supporting Information is provided here.