Matching Items (9)

130284-Thumbnail Image.png

Expression, purification and crystallization of CTB-MPR, a candidate mucosal vaccine component against HIV-1

Description

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1),

CTB-MPR is a fusion protein between the B subunit of cholera toxin (CTB) and the membrane-proximal region of gp41 (MPR), the transmembrane envelope protein of Human immunodeficiency virus 1 (HIV-1), and has previously been shown to induce the production of anti-HIV-1 antibodies with antiviral functions. To further improve the design of this candidate vaccine, X-ray crystallography experiments were performed to obtain structural information about this fusion protein. Several variants of CTB-MPR were designed, constructed and recombinantly expressed in Escherichia coli. The first variant contained a flexible GPGP linker between CTB and MPR, and yielded crystals that diffracted to a resolution of 2.3 Å, but only the CTB region was detected in the electron-density map. A second variant, in which the CTB was directly attached to MPR, was shown to destabilize pentamer formation. A third construct containing a polyalanine linker between CTB and MPR proved to stabilize the pentameric form of the protein during purification. The purification procedure was shown to produce a homogeneously pure and monodisperse sample for crystallization. Initial crystallization experiments led to pseudo-crystals which were ordered in only two dimensions and were disordered in the third dimension. Nanocrystals obtained using the same precipitant showed promising X-ray diffraction to 5 Å resolution in femtosecond nanocrystallography experiments at the Linac Coherent Light Source at the SLAC National Accelerator Laboratory. The results demonstrate the utility of femtosecond X-ray crystallography to enable structural analysis based on nano/microcrystals of a protein for which no macroscopic crystals ordered in three dimensions have been observed before.

Contributors

Created

Date Created
  • 2014-08-20

130302-Thumbnail Image.png

Structural enzymology using X-ray free electron lasers

Description

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an

Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8 Å and 2.4 Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.

Contributors

Agent

Created

Date Created
  • 2016-12-15

130306-Thumbnail Image.png

Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

Description

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is demonstrated that LCP can also be used as a suitable carrier medium for microcrystals of soluble proteins, enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.

Contributors

Agent

Created

Date Created
  • 2015-08-04

130308-Thumbnail Image.png

A novel inert crystal delivery medium for serial femtosecond crystallography

Description

Serial femtosecond crystallography (SFX) has opened a new era in crystallo­graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron

Serial femtosecond crystallography (SFX) has opened a new era in crystallo­graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

Contributors

Created

Date Created
  • 2015-06-30

130313-Thumbnail Image.png

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

Description

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The γ-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergent evolution.

Contributors

Created

Date Created
  • 2015-12-17

129270-Thumbnail Image.png

A Hinge Migration Mechanism Unlocks the Evolution of Green-to-Red Photoconversion in GFP-like Proteins

Description

In proteins, functional divergence involves mutations that modify structure and dynamics. Here we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments,

In proteins, functional divergence involves mutations that modify structure and dynamics. Here we provide experimental evidence for an evolutionary mechanism driven solely by long-range dynamic motions without significant backbone adjustments, catalytic group rearrangements, or changes in subunit assembly. Crystallographic structures were determined for several reconstructed ancestral proteins belonging to a GFP class frequently employed in superresolution microscopy. Their chain flexibility was analyzed using molecular dynamics and perturbation response scanning. The green-to-red photoconvertible phenotype appears to have arisen from a common green ancestor by migration of a knob-like anchoring region away from the active site diagonally across the β barrel fold. The allosterically coupled mutational sites provide active site conformational mobility via epistasis. We propose that light-induced chromophore twisting is enhanced in a reverse-protonated subpopulation, activating internal acid-base chemistry and backbone cleavage to enlarge the chromophore. Dynamics-driven hinge migration may represent a more general platform for the evolution of novel enzyme activities.

Contributors

Agent

Created

Date Created
  • 2015-01-06

Serial time-resolved crystallography of photosystem II using a femtosecond X-ray laser

Description

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S[subscript 0] to S[subscript 4], in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S[subscript 1] state and after double laser excitation (putative S[subscript 3] state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn[subscript 4]CaO[subscript 5] core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn[subscript 3]CaO[subscript x] cubane in the S[subscript 2] to S[subscript 3] transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

Contributors

Agent

Created

Date Created
  • 2014-09-11

130320-Thumbnail Image.png

Diffraction Data of Core-shell Nanoparticles from an X-ray Free Electron Laser

Description

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory,

X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.

Contributors

Created

Date Created
  • 2017-04-11

154121-Thumbnail Image.png

Time-resolved crystallography using X-ray free-electron laser

Description

Photosystem II (PSII) is a large protein-cofactor complex. The first step in

photosynthesis involves the harvesting of light energy from the sun by the antenna (made

of pigments) of the PSII trans-membrane

Photosystem II (PSII) is a large protein-cofactor complex. The first step in

photosynthesis involves the harvesting of light energy from the sun by the antenna (made

of pigments) of the PSII trans-membrane complex. The harvested excitation energy is

transferred from the antenna complex to the reaction center of the PSII, which leads to a

light-driven charge separation event, from water to plastoquinone. This phenomenal

process has been producing the oxygen that maintains the oxygenic environment of our

planet for the past 2.5 billion years.

The oxygen molecule formation involves the light-driven extraction of 4 electrons

and protons from two water molecules through a multistep reaction, in which the Oxygen

Evolving Center (OEC) of PSII cycles through 5 different oxidation states, S0 to S4.

Unraveling the water-splitting mechanism remains as a grant challenge in the field of

photosynthesis research. This requires the development of an entirely new capability, the

ability to produce molecular movies. This dissertation advances a novel technique, Serial

Femtosecond X-ray crystallography (SFX), into a new realm whereby such time-resolved

molecular movies may be attained. The ultimate goal is to make a “molecular movie” that

reveals the dynamics of the water splitting mechanism using time-resolved SFX (TRSFX)

experiments and the uniquely enabling features of X-ray Free-Electron Laser

(XFEL) for the study of biological processes.

This thesis presents the development of SFX techniques, including development of

new methods to analyze millions of diffraction patterns (~100 terabytes of data per XFEL

experiment) with the goal of solving the X-ray structures in different transition states.

ii

The research comprises significant advancements to XFEL software packages (e.g.,

Cheetah and CrystFEL). Initially these programs could evaluate only 8-10% of all the

data acquired successfully. This research demonstrates that with manual optimizations,

the evaluation success rate was enhanced to 40-50%. These improvements have enabled

TR-SFX, for the first time, to examine the double excited state (S3) of PSII at 5.5-Å. This

breakthrough demonstrated the first indication of conformational changes between the

ground (S1) and the double-excited (S3) states, a result fully consistent with theoretical

predictions.

The power of the TR-SFX technique was further demonstrated with proof-of principle

experiments on Photoactive Yellow Protein (PYP) micro-crystals that high

temporal (10-ns) and spatial (1.5-Å) resolution structures could be achieved.

In summary, this dissertation research heralds the development of the TR-SFX

technique, protocols, and associated data analysis methods that will usher into practice a

new era in structural biology for the recording of ‘molecular movies’ of any biomolecular

process.

Contributors

Agent

Created

Date Created
  • 2015