This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Establish concurrent UV, visible, and Raman spectroscopy and x-ray diffraction. A. M. Orville, A. H?roux , D. Stoner-Ma New Personnel ?The administrative supplement to this grant (3P41RR012408-13S1) provides funds for a full-time scientist to help accelerate the development of the facility at beamline X26-C. In the fall of 2009, we recruited a new Assistant Scientist in the Biology Department at BNL. This 24 month appointment requires that the qualified scientist must have earned PhD and have completed several years of post-doctoral training in Raman spectroscopy. Fifteen applications were received and letters of support were collected. Two persons were identified for further consideration. We interviewed Deborah Stoner-Ma, Ph.D. because she has nearly all the qualifications and has very strong recommendation letters. She has Raman experience in her post-doc training. She works well in collaborative groups. She has R&D projects that could help develop the strengths of the PXRR group and the lab in general. Her skill-set complements, but does not overlap directly with others in the PXRR group. The PXRR scientists expressed their enthusiastic and unanimous support to recruit her. She was offered the job, accepted, and started on Dec 14, 2009. Deborah Stoner-Ma, Ph.D. has been working with Dr. Orville and the PXRR staff to commission the new single-crystal Raman spectrometer purchased from Horiba-JY. Objectives ?The objectives stated in the recent renewal proposal are these: we will improve our current single crystal UV/Vis microspectrophotometry facility to provide optical spectra from single crystals in an essentially seamless and routine fashion at the X-ray beamline, and develop both single crystal fluorescence spectroscopy and single crystal Raman spectroscopy. Results ?Our current capability at beamline X26-C (Figure 1) consists of a Crystal Logic diffractometer with an ADSC Q210 area detector (on loan from the NSLS), a 4DX-ray Systems AB optical system, a Newport 75W Xe research arc lamp, an Ocean Optics USB 4000 CCD-based spectrophotometer running through the beamline controls on a LINUX operating systems, and an Horiba Jobin-Yvon Inc., Raman system consisting of two diode lasers (532 and 785 nm), a Raman probe head specific for each laser, an IHR 550 spectrometer and Synapse CCD detector. X-ray diffraction and optical absorption data collection are fully integrated and controlled by the beamline-control software, allowing temporal coordination of data collection. The results are linked directly to our database tracking system, the PXdb. The Raman system is operational;protein vibrational spectra before and after x-ray exposure can now be collected. Currently, the Raman system is controlled by the Windows-based LabSpec software provided by Horiba-JY. The optical absorption spectra collection utilized visible light (350 - 850 nm) from a Xe arc lamp. The light is brought to the sample and the transmitted light brought to the spectrophotometer via through quartz optical fibers. The 15x microscope objectives are based upon the Schwarzschild parabolic mirror design, which uses an all-reflecting principle and are, therefore, free from chromatic aberration. The light is focused to a spot size that depends upon objective and the diameter of the optical fiber to which it is connected. For example, the incident photons are focused to 25 ?m diameter spot through a 50 ?m optical fiber, whereas photons are collected through a 75 ?m diameter region focused by a 400 ?m optical fiber. This arrangement yields full range electronic absorption spectra typically in less than one second. The standard experiments involve the following steps, which are integrated into our beamline control software (CBASS): 1) Mount and center a crystal with the goniometer on the diffractomer. 2) Record 72 digital images of the loop/crystal, one every 5? around a full 360? rotation, first with illumination lamp used for visual inspection on, and again with the lamp off. Comparison of the resultant rotation angle dependent spectra allows one to inspect and choose an optimal angle at which the subsequent collection of absorption data will occur during the x-ray diffraction sweep(s). This some-what cumbersome method has the advantage of removing artifacts originating from the illumination lamp;it will be automated in the near future. From this point forward, two mutually exclusive types of data can be collected: time-resolved X-ray dose dependent (Step 3) or diffraction data-dependent (skip to Steps 4). 3) Dose dependent data collection: Optical spectra between 350 - 850 nm are collected from a stationary crystal at the optimal angle in a time-dependent mode, during which the X-ray shutter is opened at a selected time and for a designated exposure time. This provides a data set from which reaction kinetics can be determined from the analysis of changes in optical spectra at one or more wavelengths. 4) Diffraction dependent data collection: The researcher screens the crystal for X-ray diffraction, indexes the unit cell, and determines the data collection strategy. The rotation angle for optical data collection, x-ray exposure time, and rotation sweep angle are set within CBASS. Data acquisition begins with the collection of an absorption spectrum at the optimal angle, then switched to X-ray diffraction data collection. After each of the first 20 x-ray exposures, the crystal is rotated back to the best orientation for optical spectrum. This occurs while the X-ray shutter is closed and during the readout of the x-ray detector. Diffraction data collection then resumes and the cycle repeats. Recently collected optical data during our RapiData 2010 course are shown in Figure 1. The absorption spectra of Zn2+ Insulin crystals is shown as a function of rotation angle and illustrates that this particular sample is essentially spectroscopically """"""""silent."""""""" In contrast excitation with the 532nm Raman laser yields rich Raman stretching features from within the protein molecules of the crystal. This includes aromatic residues and information related to the disulfide bond, the latter of which is susceptible to degradation by x-ray dose. Raman data collection is currently done manually prior to and following the collection of correlated diffraction and visible absorption date. The selection of excitation wavelength used depends on the protein sample. The 785 nm laser is used for proteins which fluorescence or which may suffer from photo-damage. The 532 nm laser is useful for resonance-Raman spectroscopy in which the vibrational modes of a ligand, cofactor or intrinsic metal group can be enhanced and the protein modes diminished. Following calibration of the laser and the CCD detector, the laser beam is focused on a mesh, then fine-focused on the crystal sample. Spectra are collected at several different crystal rotation angles to determine the best angle that optimizes the signal and reduces or eliminates potential florescence contamination of the Raman spectrum. The particular collection parameters are optimized for each type of protein;these conditions include spectral range, time per acquisition, and total number of acquisitions. All monitors and lights are shut off, then the data is collected. Shown in Figure 1 is the Raman spectrum obtained from an insulin crystal during RapiData 2010 with only 50 seconds of accumulation time. The vibrational modes match well with published spectra. Many of the samples of interest to beamline X26-C contain chromophores that are sensitive to x-ray exposure. As illustrated in Figure 2, this includes the classics, myoglobin and heomoglobins isolated form a wide range of organisms. The single-crystal spectroscopy indicates a rapid change in the oxidation state for the iron that depends upon x-ray dose. An implication of this type of data is that any structure that results from the entire dataset does not reflect the """"""""true"""""""" starting material, but rather is transformed during the measurements necessary to yield the atomic structure. Plans ?Absorption spectroscopy ?We are expanding our capabilities further into the UV region of the spectrum such that absorption changes down to 250 nm can be probed. This will allow evaluation of possible changes involving tryptophans and tyrosines. Non-resonance and Resonance Raman Spectroscopy - We plan to expand the excitation wavelengths offered to include 633 and 472 nm. To simplify the switching of laser sources, we are working with Horiba to design a single probe head which will incorporate the optics required for all four lasers. LabSpec control of Raman collection will be incorporated into the CBASS software and the operations will be automated. Tools will be developed to allow for the remote, computerized operation of the laser shutters, etc. Off-line single-crystal spectroscopy ?An enclosure for off-line spectroscopy has been constructed adjacent to the X26-C hutch. We are in the process of procuring the required equipment. This was accomplished in large part, with the ARRA supplement to the P41 grant. Single Crystal, Fluorescence-Emission Spectroscopy ?Fluorescence spectroscopy complements the electronic absorption and Raman spectroscopy capabilities. The various options for light sources and detection instruments are under investigation. Significance ?The technologies we currently provide are unique in the United States, and will be enhanced further in the coming year. The scientific problems that we and our users will address are central to the progress of macromolecular sciences in the United States. The national resource we envision will support unprecedented, highly correlated studies. The results will provide much needed data on the complex relationships among macromolecular atomic structure, electronic structure and chemistry. These data will be used by the large number of national and international researchers in the field. Our plans will place the United States in a leadership position in this area. Publications ? H?roux, A., Bozinovski, D.M., Valley, M.P., Fitzpatrick, P.F. and Orville, A.M. Crystal Structures of Intermediates in the Nitroalkane Oxidase Reaction, Biochemistry 48, 3407-3416 (2009). Orville, A.M., Lountos, G.T., Finnegan, S., Gadda, G.,Prabhakar R. Crystallographic, Spectroscopic, and Computational Analysis of a Flavin-C4a-Oxygen Adduct in Choline Oxidase, Biochemistry 48, 720-728 (2009). Funds are provided to attend relevant meetings. Dr. Orville participated in the Metals in Biology Gordon Research Conference in January 2010 in Ventura, CA. He also presented a talk entitled, """"""""Correlated Single-Crystal Spectroscopy and X-ray Crystallography at Beamline X26-C of the NSLS,"""""""" at the 6th International Workshop on X-ray Radiation Damage to Biological Crystalline Samples (March 11-13, 2010) at the National Accelerator Laboratory, Menlo Park, CA. During that trip Dr. Orville also spent a full day presenting four Science demonstrations to two 5th grade and two 7th grade classes at the Barry Elementary School in the Yuba City unified school district.
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