No abstract

A variety of organisms have evolved mechanisms to detect and respond to light, in which the response is mediated by protein structural changes following photon absorption. The initial step is often the photo-isomerization of a conjugated chromophore. Isomerization occurs on ultrafast timescales, and is substantially influenced by the chromophore environment. Here we identify structural changes associated with the earliest steps in the trans to cis isomerization of the chromophore in photoactive yellow protein. Femtosecond, hard X-ray pulses emitted by the Linac Coherent Light Source were used to conduct time-resolved serial femtosecond crystallography on PYP microcrystals over the time range from 100 femtoseconds to 3 picoseconds to determine the structural dynamics of the photoisomerization reaction.

Density functional theory has become a popular method for studying the electronic structure and potential energy surface properties of large molecules. Its accuracy has been extensively validated for organic and organometallic systems. However, this is not yet the case for classical inorganic compounds with biological importance. This study presents a systematic evaluation of modern DFT calculations using the spectroscopically well understood molecule [CuCl4]2-. The BP86 and B3LYP functionals with saturated basis sets give a ground-state bonding description that is too covalent, and the calculated ligand-field and ligand-to-metal charge transitions are shifted to higher and lower energies, respectively, relative to experiment. A spectroscopically adjusted hybrid DFT functional (B(38HF)P86) was optimized to match the ground-state experimental Cu spin density (0.62 ± 0.02e). This adjusted hybrid functional also gives an improved excited-state description with a rms error in transition energies of 1000 cm-1. The potential energy surface of the [CuCl4]2- was studied in gas and condensed phases. In the gas phase, the tetragonal (D 4 h ) geometry was found to be a transition state along the b2u distortion mode connecting distorted tetrahedral (D 2 d ) structures. The replacement of 38% local + nonlocal DF exchange with HF exchange improves the calculated Cu−Cl bond lengths by 0.03 Å, increases the frequency of the a1g mode by 30 cm-1 and changes the energetics by 3 kcal mol-1 relative to the BP86 method. It is found that the crystal lattice stabilizes the D 4 h [CuCl4]2- structure through van der Waals and hydrogen bonding interactions worth about 10 kcal mol-1 demonstrating the role of the environment in determining the geometric and electronic structure of the Cu site. The importance of the type and the amount of DF correlation has been investigated and alternative nonhybrid methods of adjusting the ground-state description have been evaluated.

CXC chemokine receptor (CXCR)4 is an HIV coreceptor and a chemokine receptor that plays an important role in several physiological and pathological processes, including hematopoiesis, leukocyte homing and trafficking, metastasis, and angiogenesis. This receptor belongs to the class A family of G protein-coupled receptors and is a validated target for the development of a new class of antiretroviral therapeutics. This study compares the interactions of three structurally diverse small-molecule CXCR4 inhibitors with the receptor and is the first report of the molecular interactions of the nonmacrocyclic CXCR4 inhibitor (S)-NЈ-(1H-benzimidazol-2-ylmethyl)-NЈ- (5,6,7,8-tetrahydroquinolin-8-yl)butene-1,4-diamine (AMD11070). Fourteen CXCR4 single-site mutants representing amino acid residues that span the entire putative ligand binding pocket were used in this study. These mutants were used in binding studies to examine how each single-site mutation affected the ability of the inhibitors to compete with 125 I-stromal-derived factor-1␣ binding. Our data suggest that these CXCR4 inhibitors bind to overlapping but not identical amino acid residues in the transmembrane regions of the receptor. In addition, our results identified amino acid residues that are involved in unique interactions with two of the CXCR4 inhibitors studied. These data suggest an extended binding pocket in the transmembrane regions close to the second extracellular loop of the receptor. Based on site-directed mutagenesis and molecular modeling, several potential binding modes were proposed for each inhibitor. These mechanistic studies might prove to be useful for the development of future generations of CXCR4 inhibitors with improved clinical pharmacology and safety profiles.CXCR4 is a chemokine receptor and a coreceptor for T-tropic (CXCR4-using) HIV viruses. The developments of drug resistance and toxicities in the current drug classes have prompted the investigation into novel antiretroviral agents with unique targets of action. Inhibition of viral entry by coreceptor blockade has been a target under active investigation (Moore and Doms, 2003). CXCR4 belongs to the class A family of seven transmembrane G protein-coupled receptors (GPCRs), which also includes a significant portion of proven drug targets (Klabunde and Hessler, 2002). CXCR4 is expressed on a multitude of tissues and cell types and has been shown to be involved in the homing and trafficking of leukocytes and hematopoietic progenitor cells, brain development, vascularization, neonatal development, T-cell activation and migration at sites of inflammation, and hematopoiesis (Murdoch, 2000). SDF-1 (also known as CXCL12) is the only known ligand that binds to CXCR4. In addition to its physiological roles, the SDF-1␣-CXCR4 axis has been suggested to play an important role in the progression of different types of cancer, including breast cancer, small cell lung cancer, chronic lymphocytic leukemia, and neuroblastoma (Burger and Kipps, 2006),. The effects of the SDF-1␣-CXCR4 axis on t...

This article describes the structure determination of a membrane protein by serial injection of microcrystals in lipidic cubic phases into a synchrotron microfocus beam. The method is discussed with respect to serial femtosecond crystallography at free-electron lasers.

Spectroscopically calibrated DFT is used to investigate the reaction coordinate of O(2) binding to Hemocyanin (Hc). A reaction path is calculated in which O(2) approaches the binuclear copper site with increasing metal-ligand overlap, which switches the coordination mode from end-on eta(1)-eta(1), to mu-eta(1):eta(2), then to butterfly, and finally to the planar [Cu(2)(mu-eta(2):eta(2)O(2))] structure. Analysis of the electronic structures during O(2) binding reveals that simultaneous two-electron transfer (ET) takes place. At early stages of O(2) binding the energy difference between the triplet and the singlet state is reduced by charge transfer (CT), which delocalizes the unpaired electrons and thus lowers the exchange stabilization onto the separated copper centers. The electron spins on the copper(II) ions are initially ferromagnetically coupled due to close to orthogonal magnetic orbital pathways through the dioxygen bridging ligand, and a change in the structure of the Cu(2)O(2) core turns on the superexchange coupling between the coppers. This favors the singlet state over the triplet state enabling intersystem crossing. Comparison with mononuclear model complexes indicates that the protein matrix holds the two copper(I) centers in close proximity, which enthalpically and entropically favors O(2) binding due to destabilization of the reduced binuclear site. This also allows regulation of the enthalpy by the change of the Cu--Cu distance in deoxyHc, which provides an explanation for the O(2) binding cooperativity in Hc. These results are compared to our earlier studies of Hemerythrin (Hr) and a common theme emerges where the spin forbiddeness of O(2) binding is overcome through delocalization of unpaired electrons onto the metal centers and the superexchange coupling of the metal centers via a ligand bridge.

Bi-functional μ- and δ- opioid receptor (OR) ligands are potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects. We solved the structure of human δ-OR bound to the bi-functional δ-OR antagonist and μ-OR agonist tetrapeptide H-Dmt(1)-Tic(2)-Phe(3)-Phe(4)-NH2 (DIPP-NH2) by serial femtosecond crystallography, revealing a cis-peptide bond between H-Dmt(1) and Tic(2). The observed receptor-peptide interactions are critical to understand the pharmacological profiles of opioid peptides, and to develop improved analgesics.

The three-dimensional structures of macromolecules and their complexes are predominantly elucidated by X-ray protein crystallography. A major limitation is access to high-quality crystals, to ensure X-ray diffraction extends to sufficiently large scattering angles and hence yields sufficiently high-resolution information that the crystal structure can be solved. The observation that crystals with shrunken unit-cell volumes and tighter macromolecular packing often produce higher-resolution Bragg peaks1,2 hints that crystallographic resolution for some macromolecules may be limited not by their heterogeneity but rather by a deviation of strict positional ordering of the crystalline lattice. Such displacements of molecules from the ideal lattice give rise to a continuous diffraction pattern, equal to the incoherent sum of diffraction from rigid single molecular complexes aligned along several discrete crystallographic orientations and hence with an increased information content3. Although such continuous diffraction patterns have long been observed—and are of interest as a source of information about the dynamics of proteins4 —they have not been used for structure determination. Here we show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5 Å limit of measurable Bragg peaks, which allows us to directly phase5 the pattern. With the molecular envelope conventionally determined at 4.5 Å as a constraint, we then obtain a static image of the photosystem II dimer at 3.5 Å resolution. This result shows that continuous diffraction can be used to overcome long-supposed resolution limits of macromolecular crystallography, with a method that puts great value in commonly encountered imperfect crystals and opens up the possibility for model-free phasing6,7.