Showing papers by "Brian R. Crane published in 2011"
TL;DR: A 2.3-Å resolution crystal structure of Drosophila CRY with an intact C terminus is reported, demonstrating how conserved protein architecture and photochemistry can be elaborated into a range of light-driven functions.
Abstract: The cryptochrome/photolyase family of photoreceptors mediates cellular responses to ultraviolet and blue light exposure in all kingdoms of life: cryptochromes transduce signals important for growth, development, magnetosensitivity and circadian clocks, and photolyases repair photolesions in DNA. Zoltowski et al. have now solved the X-ray crystal structure of full-length cryptochrome from Drosophila. They find that a C-terminal helix docks in a groove that is known to bind DNA substrates in photolyases, and a conserved tryptophan protrudes into the catalytic centre of the cryptochrome, mimicking how DNA-repair photolyases recognize lesions in DNA. The cryptochrome/photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to ultraviolet and blue light exposure in all kingdoms of life1,2,3,4,5. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs) and 6-4 photolesions caused by ultraviolet radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks1,2,3,4,5. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism3,6. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalysed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable carboxy-terminal tail that appends the conserved PL homology domain (PHD) and is important for function7,8,9,10,11,12. Here, we report a 2.3-A resolution crystal structure of Drosophila CRY with an intact C terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp 536 juts into the CRY catalytic centre to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture and photochemistry can be elaborated into a range of light-driven functions.
144 citations
TL;DR: The mechanism of dimerization of VVD provides molecular details that explain how members of a large family of photoreceptors convert light responses to alterations in protein-protein interactions and may inform mechanisms by which LOV-containing proteins interact.
Abstract: Light, oxygen, or voltage (LOV) protein domains are present in many signaling proteins in bacteria, archaea, protists, plants, and fungi. The LOV protein VIVID (VVD) of the filamentous fungus Neurospora crassa enables the organism to adapt to constant or increasing amounts of light and facilitates proper entrainment of circadian rhythms. Here, we determined the crystal structure of the fully light-adapted VVD dimer and reveal the mechanism by which light-driven conformational change alters the oligomeric state of the protein. Light-induced formation of a cysteinyl-flavin adduct generated a new hydrogen bond network that released the amino (N) terminus from the protein core and restructured an acceptor pocket for binding of the N terminus on the opposite subunit of the dimer. Substitution of residues critical for the switch between the monomeric and the dimeric states of the protein had profound effects on light adaptation in Neurospora. The mechanism of dimerization of VVD provides molecular details that explain how members of a large family of photoreceptors convert light responses to alterations in protein-protein interactions.
111 citations
TL;DR: In this paper, a stoichiometric mismatch between FliG and FliM (26 subunits) was explained in terms of two distinct positions for FlmM: one where it binds the FlmG central domain and another where it binding the C-terminal domain.
Abstract: Rotation and switching of the bacterial flagellum depends on a large rotor-mounted protein assembly composed of the proteins FliG, FliM and FliN, with FliG most directly involved in rotation. The crystal structure of a complex between the central domains of FliG and FliM, in conjunction with several biochemical and molecular-genetic experiments, reveals the arrangement of the FliG and FliM proteins in the rotor. A stoichiometric mismatch between FliG (26 subunits) and FliM (34 subunits) is explained in terms of two distinct positions for FliM: one where it binds the FliG central domain and another where it binds the FliG C-terminal domain. This architecture provides a structural framework for addressing the mechanisms of motor rotation and direction switching and for unifying the large body of data on motor performance. Recently proposed alternative models of rotor assembly, based on a subunit contact observed in crystals, are not supported by experiment.
95 citations
TL;DR: The [Re(I)(CO)(3)(4,7-dimethyl-1,10-phenanthroline)(histidine-124)(tryptophan-122)] complex of Pseudomonas aeruginosa azurin behaves as a single photoactive unit that triggers very fast electron transfer (ET) from a distant (2 nm) Cu(I) center in the protein.
Abstract: The [Re^I(CO)_3(4,7-dimethyl-1,10-phenanthroline)(histidine-124)(tryptophan-122)] complex, denoted [Re^I(dmp)(W122)], of Pseudomonas aeruginosa azurin behaves as a single photoactive unit that triggers very fast electron transfer (ET) from a distant (2 nm) Cu^I center in the protein. Analysis of time-resolved (ps–μs) IR spectroscopic and kinetics data collected on [Re^I(dmp)(W122)AzM] (in which M=Zn^(II), Cu^(II), Cu^I; Az=azurin) and position-122 tyrosine (Y), phenylalanine (F), and lysine (K) mutants, together with excited-state DFT/time-dependent (TD)DFT calculations and X-ray structural characterization, reveal the character, energetics, and dynamics of the relevant electronic states of the [Re^I(dmp)(W122)] unit and a cascade of photoinduced ET and relaxation steps in the corresponding Re–azurins. Optical population of [Re^I(imidazole-H124)(CO)_3]→dmp ^1CT states (CT=charge transfer) is followed by around 110 fs intersystem crossing and about 600 ps structural relaxation to a ^3CT state. The IR spectrum indicates a mixed Re^I(CO)_3,A→dmp/π→π^*(dmp) character for aromatic amino acids A122 (A=W, Y, F) and Re^I(CO)_3→dmp metal–ligand charge transfer (MLCT) for [Re^I(dmp)(K122)AzCu^(II)]. In a few ns, the ^3CT state of [Re^I(dmp)(W122)AzM] establishes an equilibrium with the [Re^I(dmp.^−)(W122.^+)AzM] charge-separated state, ^3CS, whereas the ^3CT state of the other Y, F, and K122 proteins decays to the ground state. In addition to this main pathway, ^3CS is populated by fs- and ps-W(indole)→Re^(II) ET from ^1CT and the initially “hot” ^3CT states, respectively. The ^3CS state undergoes a tens-of-ns dmp.^−→W122.^+ ET recombination leading to the ground state or, in the case of the Cu^I azurin, a competitively fast (≈30 ns over 1.12 nm) Cu^I→W.^+ ET, to give [Re^I(dmp.^−)(W122)AzCu^(II)]. The overall photoinduced CuI→Re(dmp) ET through [Re^I(dmp)(W122)AzCu^I] occurs over a 2 nm distance in <50 ns after excitation, with the intervening fast ^3CT–^3CS equilibrium being the principal accelerating factor. No reaction was observed for the three Y, F, and K122 analogues. Although the presence of [Re(dmp)(W122)AzCu^(II)] oligomers in solution was documented by mass spectrometry and phosphorescence anisotropy, the kinetics data do not indicate any significant interference from the intermolecular ET steps. The ground-state dmp–indole π–π interaction together with well-matched W/W.^+ and excited-state [Re^II(CO)_3(dmp.^−)]/[Re^I(CO)_3(dmp.^−)] potentials that result in very rapid electron interchange and ^3CT–^3CS energetic proximity, are the main factors responsible for the unique ET behavior of [Re^I(dmp)(W122)]-containing azurins.
49 citations
TL;DR: Data indicate that αF unlatches to switch association of PER with itself to its partner Timeless, and Conservation of PER interaction residues among a family of PAS-AB-containing transcription factors suggests that contacts mediating closed PAS -AB dimers serve a general function.
24 citations
Patent•
23 May 2011Abstract: The present invention is directed to methods of producing recombinant functional heme-binding proteins with complete heme incorporation and purified preparations of the same. The present invention is further directed to methods of identifying agents that modulate the activity of heme-binding proteins.
6 citations
TL;DR: 2.2 Å resolution crystal structure of Thermotoga maritima CheB methylesterase domain is determined to provide insight into the interaction mode between CheB and chemoreceptors, and a CheB:chemoreceptor interaction mode is proposed.
5 citations
TL;DR: It is proposed that the two consecutive glutamates in the β4/α4 loop of T. maritima CheB that is absent in T. Marina CheY and in E. coli/S.
2 citations
TL;DR: CheW from Thermotoga maritima has been overexpressed in Escherichia coli and crystallized at 298 K using ammonium sulfate as a salt precipitant, and belonged to space group P6(3), with unit-cell parameters a = b = 61.265, c = 361.045 Å.
Abstract: The CheW protein plays a key role in bacterial chemotaxis signal transduction by coupling CheA to chemotaxis receptors. CheW from Thermotoga maritima has been overexpressed in Escherichia coli and crystallized at 298 K using ammonium sulfate as a salt precipitant. X-ray diffraction data have been collected to 3.10 A resolution at 100 K using synchrotron radiation. The crystal belonged to space group P63, with unit-cell parameters a = b = 61.265, c = 361.045 A. The asymmetric unit may contain four to six CheW molecules.
2 citations
TL;DR: The author’s development of new ICAT reagents based on the glucamine backbone that are more water soluble, but have varying degrees of electrophilicity is a big advantage in avoiding background labeling in the complex mixture of native bacterial membranes.
1 citations
TL;DR: The chemotaxis histidine kinase CheA assembles into a dimer in which the P3 dimerization domain forms a four-helix bundle by the parallel association of two α-helical hairpins from each subunit as discussed by the authors.
Abstract: The chemotaxis histidine kinase CheA assembles into a dimer in which the P3 dimerization domain forms a four-helix bundle by the parallel association of two α-helical hairpins from each subunit. Ligand occupancy of the chemoreceptor regulates signal transduction by controlling the autophosphorylation activity of CheA. Autophosphorylation of CheA occurs in trans, i.e. one subunit phosphorylates the other. The P3 domain of CheA from Escherichia coli has been overexpressed in E. coli and crystallized at 298 K using PEG as a precipitant. X-ray diffraction data to 2.80 A resolution have been collected at 100 K using synchrotron radiation. The crystal belonged to space group P1, with unit-cell parameters a = 59.271, b = 67.674, c = 82.815 A, α = 77.568, β = 86.073, γ = 64.436°. The asymmetric unit may contain up to ten dimeric units of P3 four-helix bundles.