Full engagement of liganded maltose-binding protein stabilizes a semi-open ATP-binding cassette dimer in the maltose transporter.

MalFGK2 is an ATP-binding cassette (ABC) transporter that mediates the uptake of maltose/maltodextrins into Escherichia coli. A periplasmic maltose-binding protein (MBP) delivers maltose to the transmembrane subunits (MalFG) and stimulates the ATPase activity of the cytoplasmic nucleotide-binding subunits (MalK dimer). This MBP-stimulated ATPase activity is independent of maltose for purified transporter in detergent micelles. However, when the transporter is reconstituted in membrane bilayers, only the liganded form of MBP efficiently stimulates its activity. To investigate the mechanism of maltose stimulation, electron paramagnetic resonance spectroscopy was used to study the interactions between the transporter and MBP in nanodiscs and in detergent. We found that full engagement of both lobes of maltose-bound MBP unto MalFGK2 is facilitated by nucleotides and stabilizes a semi-open MalK dimer. Maltose-bound MBP promotes the transition to the semi-open state of MalK when the transporter is in the membrane, whereas such regulation does not require maltose in detergent. We suggest that stabilization of the semi-open MalK2 conformation by maltose-bound MBP is key to the coupling of maltose transport to ATP hydrolysis in vivo, because it facilitates the progression of the MalK dimer from the open to the semi-open conformation, from which it can proceed to hydrolyze ATP.

Guiding synchrotron X-ray diffraction by multimodal video-rate protein crystal imaging.

Synchronous digitization, in which an optical sensor is probed synchronously with the firing of an ultrafast laser, was integrated into an optical imaging station for macromolecular crystal positioning prior to synchrotron X-ray diffraction. Using the synchronous digitization instrument, second-harmonic generation, two-photon-excited fluorescence and bright field by laser transmittance were all acquired simultaneously with perfect image registry at up to video-rate (15 frames s(-1)). A simple change in the incident wavelength enabled simultaneous imaging by two-photon-excited ultraviolet fluorescence, one-photon-excited visible fluorescence and laser transmittance. Development of an analytical model for the signal-to-noise enhancement afforded by synchronous digitization suggests a 15.6-fold improvement over previous photon-counting techniques. This improvement in turn allowed acquisition on nearly an order of magnitude more pixels than the preceding generation of instrumentation and reductions of well over an order of magnitude in image acquisition times. These improvements have allowed detection of protein crystals on the order of 1 µm in thickness under cryogenic conditions in the beamline. These capabilities are well suited to support serial crystallography of crystals approaching 1 µm or less in dimension.

High frame-rate multichannel beam-scanning microscopy based on Lissajous trajectories.

A simple beam-scanning optical design based on Lissajous trajectory imaging is described for achieving up to kHz frame-rate optical imaging on multiple simultaneous data acquisition channels. In brief, two fast-scan resonant mirrors direct the optical beam on a circuitous trajectory through the field of view, with the trajectory repeat-time given by the least common multiplier of the mirror periods. Dicing the raw time-domain data into sub-trajectories combined with model-based image reconstruction (MBIR) 3D in-painting algorithms allows for effective frame-rates much higher than the repeat time of the Lissajous trajectory. Since sub-trajectory and full-trajectory imaging are simply different methods of analyzing the same data, both high-frame rate images with relatively low resolution and low frame rate images with high resolution are simultaneously acquired. The optical hardware required to perform Lissajous imaging represents only a minor modification to established beam-scanning hardware, combined with additional control and data acquisition electronics. Preliminary studies based on laser transmittance imaging and polarization-dependent second harmonic generation microscopy support the viability of the approach both for detection of subtle changes in large signals and for trace-light detection of transient fluctuations.

Selective formation of metastable ferrihydrite in the chiton tooth.

Metastable precursors are thought to play a major role in the ability of organisms to create mineralized tissues. Of particular interest are the hard and abrasion-resistant teeth formed by chitons, a class of rock-grazing mollusks. The formation of chiton teeth relies on the precipitation of metastable ferrihydrite (Fh) in an organic scaffold as a precursor to magnetite. In vitro synthesis of Fh under physiological conditions has been challenging. Using a combination of X-ray absorption and electron paramagnetic resonance spectroscopy, we show that, prior to Fh formation in the chiton tooth, iron ions are complexed by the organic matrix. In vitro experiments demonstrate that such complexes facilitate the formation of Fh under physiological conditions. These results indicate that acidic molecules may be integral to controlling Fh formation in the chiton tooth. This biological approach to polymorph selection is not limited to specialized proteins and can be expropriated using simple chemistry.

Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline.

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼10(3)-10(4)-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied.

Both maltose-binding protein and ATP are required for nucleotide-binding domain closure in the intact maltose ABC transporter.

The maltose transporter MalFGK(2) of Escherichia coli is a member of the ATP-binding cassette superfamily. A periplasmic maltose-binding protein (MBP) delivers maltose to MalFGK(2) and stimulates its ATPase activity. Site-directed spin labeling EPR spectroscopy was used to study the opening and closing of the nucleotide-binding interface of MalFGK(2) during the catalytic cycle. In the intact transporter, closure of the interface coincides not just with the binding of ATP, as seen with isolated nucleotide-binding domains, but requires both MBP and ATP, implying that MBP stimulates ATPase activity by promoting the closure of the nucleotide-binding interface. After ATP hydrolysis, with MgADP and MBP bound, the nucleotide-binding interface resides in a semi-open configuration distinct from the fully open configuration seen in the absence of any ligand. We propose that P(i) release coincides with the reorientation of transmembrane helices to an inward-facing conformation and the final step of maltose translocation into the cell.

Discrete retardance second harmonic generation ellipsometry.

A new instrument was constructed to perform discrete retardance nonlinear optical ellipsometry (DR-NOE). The focus of the design was to perform second harmonic generation NOE while maximizing sample and application flexibility and minimizing data acquisition time. The discrete retardance configuration results in relatively simple computational algorithms for performing nonlinear optical ellipsometric analysis. NOE analysis of a disperse red 19 monolayer yielded results that were consistent with previously reported values for the same surface system, but with significantly reduced acquisition times.

Influence of lipid chemistry on membrane fluidity: tail and headgroup interactions.

Membrane fluidity plays an important role in cell function and may, in many instances, be adjusted to facilitate specific cellular processes. To understand better the effect that lipid chemistry has on membrane fluidity the inclusion of three different lipids into egg phosphatidylcholine (eggPC) bilayers has been examined; the three lipids are egg phosphatidylethanolamine ((eggPE) made by transphosphatidylation of eggPC in the presence of ethanolamine), lyso-phosphatidylcholine (LPC), and lyso-phosphatidylethanolamine (LPE). The fluidity of the membranes was determined using fluorescence recovery after photobleaching and the intermolecular interactions were examined using attenuated total reflection Fourier transform infrared spectroscopy. It was observed that both headgroup and tail chemistry can significantly modulate lipid diffusion. Specifically, the inclusion of LPC and eggPE significantly altered the lipid diffusion, increased and decreased, respectively, whereas the inclusion of LPE had an intermediate effect, a slight decrease in diffusion. Strong evidence for the formation of hydrogen-bonds between the phosphate group and the amine group in eggPE and LPE was observed with infrared spectroscopy. The biological implications of these results are discussed.

Direct determination of effective interfacial optical constants by nonlinear optical null ellipsometry of chiral films.

Nonlinear optical null ellipsometry (NONE) measurements of chiral interfaces allowed direct experimental measurement of the linear interfacial optical constants in surface second harmonic generation (SHG) measurements. Since phase information is retained in NONE measurements, the real and imaginary components of the interfacial refractive index (n and k, respectively) were uniquely obtained from the measured chiral chi((2)) tensor elements of a fluorescein-labeled bovine serum albumin film. The sensitivity of the calculated chi((2)) tensor elements on the assumed values of the interfacial optical constants allowed measurements of n and k to four significant figures with no additional adjustable parameters and independent of molecular symmetry. The optical constants measured by SHG agreed within a relative error of 0.8% with values predicted independently using a simple effective medium approximation, also with no adjustable parameters. Additionally, those same optical constants produced relationships between the achiral chi((2)) tensor elements in excellent agreement with predictions for systems exhibiting weak orientational order. This study suggests that the far-field intensity and polarization state of the nonlinear optical beam may be largely independent of the near-field optical constants within the interfacial layer in the limit of a film thickness much less than the wavelength of light.