Naranbaatar Dashdorj

Protein structural dynamics in solution unveiled via 100-ps time-resolved x-ray scattering

We have developed a time-resolved x-ray scattering diffractometer capable of probing structural dynamics of proteins in solution with 100-ps time resolution. This diffractometer, developed on the ID14B BioCARS (Consortium for Advanced Radiation Sources) beamline at the Advanced Photon Source, records x-ray scattering snapshots over a broad range of q spanning 0.02–2.5 Å-1, thereby providing simultaneous coverage of the small-angle x-ray scattering (SAXS) and wide-angle x-ray scattering (WAXS) regions. To demonstrate its capabilities, we have tracked structural changes in myoglobin as it undergoes a photolysis-induced transition from its carbon monoxy form (MbCO) to its deoxy form (Mb). Though the differences between the MbCO and Mb crystal structures are small (rmsd < 0.2 Å), time-resolved x-ray scattering differences recorded over 8 decades of time from 100 ps to 10 ms are rich in structure, illustrating the sensitivity of this technique. A strong, negative-going feature in the SAXS region appears promptly and corresponds to a sudden > 22 Å3 volume expansion of the protein. The ensuing conformational relaxation causes the protein to contract to a volume ∼2 Å3 larger than MbCO within ∼10 ns. On the timescale for CO escape from the primary docking site, another change in the SAXS/WAXS fingerprint appears, demonstrating sensitivity to the location of the dissociated CO. Global analysis of the SAXS/WAXS patterns recovered time-independent scattering fingerprints for four intermediate states of Mb. These SAXS/WAXS fingerprints provide stringent constraints for putative models of conformational states and structural transitions between them.

Electrochromic Shift of Chlorophyll Absorption in Photosystem I from Synechocystis sp. PCC 6803: A Probe of Optical and Dielectric Properties around the Secondary Electron Acceptor

Nanosecond absorption dynamics at approximately 685 nm after excitation of photosystem I (PS I) from Synechocystis sp. PCC 6803 is consistent with electrochromic shift of absorption bands of the Chl a pigments in the vicinity of the secondary electron acceptor A(1). Based on experimental optical data and structure-based simulations, the effective local dielectric constant has been estimated to be between 3 and 20, which suggests that electron transfer in PS I is accompanied by considerable protein relaxation. Similar effective dielectric constant values have been previously observed for the bacterial photosynthetic reaction center and indicate that protein reorganization leading to effective charge screening may be a necessary structural property of proteins that facilitate the charge transfer function. The data presented here also argue against attributing redmost absorption in PS I to closely spaced antenna chlorophylls (Chls) A38 and A39, and suggest that optical transitions of these Chls, along with that of connecting chlorophyll (A40) lie in the range 680-695 nm.

On the structural role of the aromatic residue environment of the chlorophyll a in the cytochrome b6f complex

Because light is not required for catalytic turnover of the cytochrome b 6 f complex, the role of the single chlorophyll a in the structure and function of the complex is enigmatic. Photodamage from this pigment is minimized by its short singlet excited-state lifetime ( approximately 200 ps), which has been attributed to quenching by nearby aromatic residues ( Dashdorj et al., 2005). The crystal structure of the complex shows that the fifth ligand of the chlorophyll a contains two water molecules. On the basis of this structure, the properties of the bound chlorophyll and the complex were studied in the cyanobacterium, Synechococcus sp. PCC 7002, through site-directed mutagenesis of aromatic amino acids in the binding niche of the chlorophyll. The b 6 f complex was purified from three mutant strains, a double mutant Phe133Leu/Phe135Leu in subunit IV and single mutants Tyr112Phe and Trp125Leu in the cytochrome b 6 subunit. The purified b 6 f complex from Tyr112Phe or Phe133Leu/Phe135Leu mutants was characterized by (i) a loss of bound Chl and b heme, (ii) a shift in the absorbance peak and increase in bandwidth, (iii) multiple lifetime components, including one of 1.35 ns, and (iv) relatively small time-resolved absorbance anisotropy values of the Chl Q y band. A change in these properties was minimal in the Trp125Leu mutant. In vivo, no decrease in electron-transport efficiency was detected in any of the mutants. It was concluded that (a) perturbation of its aromatic residue niche influences the stability of the Chl a and one or both b hemes in the monomer of the b 6 f complex, and (b) Phe residues (Phe133/Phe135) of subunit IV are important in maintaining the short lifetime of the Chl a singlet excited state, thereby decreasing the probability of singlet oxygen formation.

Probing anisotropic structure changes in proteins with picosecond time-resolved small-angle X-ray scattering.

We have exploited the principle of photoselection and the method of time-resolved small-angle X-ray scattering (SAXS) to investigate protein size and shape changes following photoactivation of photoactive yellow protein (PYP) in solution with ∼150 ps time resolution. This study partially overcomes the orientational average intrinsic to solution scattering methods and provides structural information at a higher level of detail. Photoactivation of the p-coumaric acid (pCA) chromophore in PYP produces a highly contorted, short-lived, red-shifted intermediate (pR0), and triggers prompt, protein compaction of approximately 0.3% along the direction defined by the electronic transition dipole moment of the chromophore. Contraction along this dimension is accompanied by expansion along the orthogonal directions, with the net protein volume change being approximately -0.25%. More than half the strain arising from formation of pR0 is relieved by the pR0 to pR1 structure transition (1.8 ± 0.2 ns), with the persistent strain presumably contributing to the driving force needed to generate the spectroscopically blue-shifted pB signaling state. The results reported here are consistent with the near-atomic resolution structural dynamics reported in a recent time-resolved Laue crystallography study of PYP crystals and suggest that the early time structural dynamics in the crystalline state carry over to proteins in solution.

Spectral Resolution of the Primary Electron Acceptor A0 in Photosystem I

The reduced state of the primary electron acceptor of Photosystem I, A(0), was resolved spectroscopically in its lowest energy Q(y) region for the first time without the addition of chemical reducing agents and without extensive data manipulation. To carry this out, we used the menB mutant of Synechocystis sp. PCC 6803 in which phylloquinone is replaced by plastoquinone-9 in the A(1) sites of Photosystem I. The presence of plastoquinone-9 slows electron transfer from A(0) to A(1), leading to a long-lived A(0)(-) state. This allows its spectral signature to be readily detected in a time-resolved optical pump-probe experiment. The maximum bleaching (A(0)(-) - A(0)) was found to occur at 684 nm with a corresponding extinction coefficient of 43 mM(-1) cm(-1). The data show evidence for an electrochromic shift of an accessory chlorophyll pigment, suggesting that the latter Q(y) absorption band is centered around 682 nm.

Chapter 20:Structure–Function of the Cytochrome b6f Complex: A Design that has Worked for Three Billion Years

The 3.0–3.1 A X-ray structures of the cytochrome b6 f complex from the thermophilic cyanobacterium Mastigocladus laminosus and from the green alga Chlamydomonas reinhardtii are very similar. Eight natural prosthetic groups, four hemes, one [2Fe-2S] cluster, one Chl, one β-carotene, and one n-side plastoquinone are embedded in the eight polypeptide subunits of the complex, four large (18–33 kDa) and four small (∼4 kDa). The complex is organized as a dimer with a molecular weight of 217 kDa in M. laminosus. Other subunits such as ferredoxin: NADP+ reductase may bind transiently and more weakly to the n-side of the complex. Major features of the structure are: (i) a large inter-monomer lipophilic “quinone exchange cavity” that exchanges plastoquinone/quinol with the quinone pool in the lipid bilayer membrane; (ii) a labyrinthine pathway of plastoquinone movement between n- and p-electron exchange sites through the 11 × 12 A portal at the roof of the cavity; (iii) three prosthetic groups with unknown function, a novel high-spin heme (cn) close to heme bn, a chlorophyll a, and a β-carotene; (iv) a proposed function of heme cn is in PS I-linked cyclic electron transport, although the presumed binding site of a “sometime” inhibitor of cyclic ET, antimycin A, is occluded by heme cn; (v) the single Chl a molecule in the monomer is characterized by a short (200 ps) fluorescence lifetime and large anisotropy of fluorescence; and (vi) transfer of energy from the Chl triplet state to the β-carotene occurs despite the 14 A separation of the pigments – it is proposed that this transfer operates through an intraprotein, interpigment O2 channel.

Picosecond Photobiology: Watching a Signaling Protein Function in Real Time via Time-Resolved Small- and Wide-Angle X-ray Scattering

The capacity to respond to environmental changes is crucial to an organisms survival. Halorhodospira halophila is a photosynthetic bacterium that swims away from blue light, presumably in an effort to evade photons energetic enough to be genetically harmful. The protein responsible for this response is believed to be photoactive yellow protein (PYP), whose chromophore photoisomerizes from trans to cis in the presence of blue light. We investigated the complete PYP photocycle by acquiring time-resolved small and wide-angle X-ray scattering patterns (SAXS/WAXS) over 10 decades of time spanning from 100 ps to 1 s. Using a sequential model, global analysis of the time-dependent scattering differences recovered four intermediates (pR0/pR1, pR2, pB0, pB1), the first three of which can be assigned to prior time-resolved crystal structures. The 1.8 ms pB0 to pB1 transition produces the PYP signaling state, whose radius of gyration (Rg = 16.6 Å) is significantly larger than that for the ground state (Rg = 14.7 Å) and is therefore inaccessible to time-resolved protein crystallography. The shape of the signaling state, reconstructed using GASBOR, is highly anisotropic and entails significant elongation of the long axis of the protein. This structural change is consistent with unfolding of the 25 residue N-terminal domain, which exposes the β-scaffold of this sensory protein to a potential binding partner. This mechanistically detailed description of the complete PYP photocycle, made possible by time-resolved crystal and solution studies, provides a framework for understanding signal transduction in proteins and for assessing and validating theoretical/computational approaches in protein biophysics.

Ultrafast Optical Studies of the Cytochrome b6f Complex in Solution and Crystalline States

The cytochrome b 6 f complex of oxygenic photosynthesis contains a single chlorophyll a molecule. The singlet excited state of the Chl a. molecule is quenched by the surrounding protein matrix, and thus the lifetime of this state may serve as a probe of the proteins structure. In this work, singlet excited state dynamics were measured in well-diffracting crystals using femtosecond time-resolved optical pump-probe methodology. Lifetimes of the Chl a molecule in crystals of the cytochrome b 6 f complex having different space groups were 3–6 times longer than those determined in detergent solution of the b 6 f. The observed differences in excited state dynamics may arise from small (1–1.5 A) changes in local protein structure caused by crystal packing. The Chl a excited state lifetimes measured in dissolved cytochrome b 6 f complexes from several different species are essentially the same, in spite of differences in the local amino acid sequences around the Chl a. This supports an earlier hypothesis that the short excited state lifetime of Chl a is critical for the function of the b 6 f complex.