Coupled Motions Direct Electrons along Human Microsomal P450 Chains

Directional electron transfer through biological redox chains can be achieved by coupling reaction chemistry to conformational changes in individual redox enzymes

Production of Propane and Other Short-Chain Alkanes by Structure-Based Engineering of Ligand Specificity in Aldehyde-Deformylating Oxygenase

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98800/1/cbic_201300307_sm_miscellaneous_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/98800/2/1204_ftp.pd

Ultrafast Red Light Activation of Synechocystis Phytochrome Cph1 Triggers Major Structural Change to Form the Pfr Signalling-Competent State

Phytochromes are dimeric photoreceptors that regulate a range of responses in plants and microorganisms through interconversion of red light-absorbing (Pr) and far-red light-absorbing (Pfr) states. Photoconversion between these states is initiated by light-driven isomerization of a bilin cofactor, which triggers protein structural change. The extent of this change, and how light-driven structural changes in the N-terminal photosensory region are transmitted to the C-terminal regulatory domain to initiate the signalling cascade, is unknown. We have used pulsed electron-electron double resonance (PELDOR) spectroscopy to identify multiple structural transitions in a phytochrome from Synechocystis sp. PCC6803 (Cph1) by measuring distances between nitroxide labels introduced into the protein. We show that monomers in the Cph1 dimer are aligned in a parallel 'head-to-head' arrangement and that photoconversion between the Pr and Pfr forms involves conformational change in both the N- and C-terminal domains of the protein. Cryo-trapping and kinetic measurements were used to probe the extent and temporal properties of protein motions for individual steps during photoconversion of Cph1. Formation of the primary photoproduct Lumi-R is not affected by changes in solvent viscosity and dielectric constant. Lumi-R formation occurs at cryogenic temperatures, consistent with their being no major structural reorganization of Cph1 during primary photoproduct formation. All remaining steps in the formation of the Pfr state are affected by solvent viscosity and dielectric constant and occur only at elevated temperatures, implying involvement of a series of long-range solvent-coupled conformational changes in Cph1. We show that signalling is achieved through ultrafast photoisomerization where localized structural change in the GAF domain is transmitted and amplified to cause larger-scale and slower conformational change in the PHY and histidine kinase domains. This hierarchy of timescales and extent of structural change orientates the histidine kinase domain to elicit the desired light-activated biological response

Characterization of the initial photoisomerization dynamics of the Pr state of Cph1.

<p>Cph1 was contained in a 5% sucrose solution and absorbance spectra recorded after photoexcitation with a laser pulse centred at ∼590 nm. (A) Transient absorption difference spectra at delay times of 1, 5, 10, 29, 60, 299, and 988 ps after excitation. (B) Kinetic transient at 673 nm (black squares) with a fit of the data to 2 exponentials shown in red.</p

Characterization of the Pr → Pfr conversion by cryogenic absorbance measurements.

<p>(A). 77 K absorbance difference spectra of samples containing 15 µM Cph1 after illumination for 10 mins at different temperatures ranging from 77 K to 180 K. The difference spectra were obtained by using a non-illuminated sample as a blank. The formation of the absorbance peak at 684 nm and simultaneous disappearance of the absorbance band at 668 nm at higher temperatures are indicated by the arrows. (B). 77 K absorbance difference spectra of samples containing 15 µM Cph1 after illumination at 180 K for 10 mins and incubation in the dark for 10 mins at increasing temperatures. The difference spectra were obtained by using the sample that was illuminated at 180 K as a blank. The arrows indicate the formation and disappearance of the different absorbance bands at higher temperatures. The raw absorbance spectra are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052418#pone.0052418.s004" target="_blank">figure S4</a>.</p

The effect of solvent viscosity (η) on the lifetimes associated with the initial photoisomerization dynamics of the Pr state of Cph1.

<p>The lifetime values were obtained by fitting the decay at 673 nm, recorded over 350 ps (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052418#pone-0052418-g005" target="_blank">Figure 5B</a>), to 2 exponentials.</p

The viscosity-dependence of the slower steps in the Pr → Pfr photoconversion.

<p>The viscosity dependence of the rate constant for the 1^st (<b>A</b>), 2^nd (<b>B</b>), 3^rd (<b>C</b>) and 4^th steps (<b>D</b>) of the increase in absorbance at 720 nm are shown. All measurements were recorded over a range of timescales and the data are fitted to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052418#pone.0052418.e002" target="_blank">equation 2</a> as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052418#s4" target="_blank">Materials and Methods</a>. The error bars were calculated from the average of at least 3 traces.</p

The temperature dependence of the steps involved in the Pr → Pfr photoconversion.

<p>The temperature dependence of the initial formation of the Lumi-R state (•) and the remaining step(s) to form the Pfr state (○) were obtained by analysis of the cryogenic absorbance measurements (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052418#pone.0052418.s005" target="_blank">Figure S5</a>) and are shown together with the kT value for each step, where k is the Boltzmann constant (0.695 cm⁻¹) and T is the mid-point temperature of each process. The ‘glass transition’ temperature of proteins is also shown as a reference (dashed line).</p

PELDOR analysis of Cph1 conformations.

<p>PELDOR data obtained from spin-labeled <i>Synechocystis</i> PCC 6803 Cph1. Raw four-pulse ELDOR traces together with the third order polynomial function used to baseline the data (in red) are shown in the left hand panel, while the right hand panel shows the conjugate Fourier transforms of these data. (A) and (G). Pr form of the N-terminal photosensory region with spin-label at C371. (B) and (H). Pfr form of the N-terminal photosensory region with spin-label at C371. (C) and (I). Pr form of full-length Cph1 with spin-label at C371. (D) and (J). Pfr form of full-length Cph1 with spin-label at C371. (E) and (K). Pr form of full-length Cph1 with spin-label at C371 and N733C. (F) and (L). Pfr form of full-length Cph1 with spin-label at C371 and N733C. Pulse sequences and data processing are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052418#s4" target="_blank">Materials and Methods</a>. Numbers in the right hand panel indicate ν_DD and distances referred to in the text.</p