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Late Abstracts

Late M.S. Student Poster Presentation

MPP12

Fourier Transform Infrared Microscopy and Chemometric Analysis for Characterization of Cyanobacterial Cells

Jodian Thomas, Keillor Witt, and Gary Hastings*

Department of Physical and Astronomy, Georgia State University, Atlanta, Georgia 30303 USA.

*E-mail: ghastings@gsu.edu

Fourier transform infrared (FTIR) microscopy is a rapid technique that allows classification and characterization of biomolecules in cyanobacterial cells. In this study, we focus on cyanobacterial cells from Synechocystis sp. PCC 6803 and Spirulina platensis in various stages of growth.  With appropriate chemometric analysis, we show that different cell types can be distinguished and cells at different stages of growth can be distinguished.  FTIR spectra of cells provide information on the composition of biomolecules within the cells which can be considered separately in five spectral regions.  Stretching vibrations can be considered for C-H, N-H, and O-H in 3300-2800 cm-1 for region I.  The 1750-1710 cm-1 and 1700- 1500 cm-1 are typically due to bending of the carbon skeleton vibrations of lipids and secondary protein structures, respectively, in region II.  While the 1450-700 cm-1 are fingerprint vibrations in regions III-V and are due to other proteins, nucleic acids, polysaccharides, and phospholipids. The IR spectra for 6803 in various stages of growth are compared to investigate changes in cell structure as well as a comparison of S. 6803 and S. platensis at similar stage of growth. The IR absorption and second derivative spectra are used with principal component analysis (PCA) and curve fitting to analyze spectra.

Figure 1. Characterization of cyanobacterial cells using FTIR microscopy

Late Abstracts

Late Ph.D./Postdoc Poster Presentations

PPP02

Vibrational Spectroscopy: Modeling Semi-quinones in the A1 Binding Site in Photosystem I

Leyla Rohani, Hiroki Makita, and Gary Hastings*

Department of Physics and Astronomy, Georgia State University, Atlanta, GA 30303. *Email: ghastings@gsu.edu

In photosystem I (PSI), a phylloquinone (PhQ) molecule occupies the A1 binding site. We optimized the native quinone (PhQ) in the A1 binding site using a three-layer ONIOM (Our own N-layered Integrated molecular Orbital and molecular Mechanics) model. The optimizations step covers protein-pigment interactions in the A1 binding site.  Meanwhile, environmental electrostatic effect and reduced state of quinone were simulated. Density functional theory (DFT) was employed to compute geometry of quinone and part of our selection of environment at two different levels of calculation. First, we calculated unlabeled (16O) and 18O-labeled spectra for the optimized and extracted PhQ from our ONIOM structure. The calculated (18O- 16O) vibrational frequency was compared to corresponding Fourier transform infrared (FTIR) results. The calculated isotope labeled band-shift agrees well with the observed band-shift. Then, the structure of PhQ in the model (2-methyl, 3-phytyl naphthoquinone) was replaced by a 2MNQ (2-methyl naphthoquinone) and DMNQ (2,3-dimethyl naphthoquinone). The resulting vibrational spectra of quinones have been used to construct the related double difference spectrum (DDS). The calculated DDS in conformity with FTIR results for different incorporated quinones into the binding site reproduced the shape of experimental DDS. Indeed, the designed three-layer ONIOM model simulates the semi-quinone and its coupled environment very well. The current model is the most accurate ONIOM model to describe the pigment-protein interaction in the A1 binding site in PSI.

PPP03

Time-Resolved FTIR Spectroscopy of A1, the Secondary Electron Acceptor in Photosystem I

Hiroki Makita and Gary Hastings*

Department of Physical and Astronomy, Georgia State University, Atlanta, Georgia 30303. *E-mail: ghastings@gsu.edu

Fourier transform infrared (FTIR) absorption difference spectroscopy was applied to study A1, the secondary electron acceptor in photosystem I (PSI).  As an intermediate electron transfer (ET) cofactor, a phylloquinone (PhQ) molecule, which serves as A1 in PSI, quickly accepts and donates electron to complete transmembrane ET.  ET through A1 occurs on a nanosecond timescale at room temperature, and on a sub-millisecond timescale at cryogenic temperature.  To investigate the molecular interactions of the quinone with its surrounding protein environment, FTIR absorption difference spectroscopy was applied in a time-resolved manner, using the step-scan technique.  Here, using a non-invasive method to replace PhQ with other quinone analogues in the A1 binding site of PSI, FTIR double-difference spectra for PhQ and a series of non-native quinones were constructed.  For identification of infrared absorption bands due to anionic phyllosemiquinone (PhQ–) in the A1 binding site, three quinone analogues were used: 2-methyl-1,4-naphthoquinone, which lacks a phytyl chain in PhQ, 2,3-dimethyl-1,4-naphthoquinone, which replaces the phytyl chain with a methyl group, and 18O-labeled PhQ, which contains isotope-labeled oxygen atoms.  From the double-difference spectra using these non-native quinones, two peaks are identified as bands due to PhQ–.  Identification of infrared absorption bands due to neutral state quinone in the A1 binding site is complicated due to its absorption range overlapping with the region of protein absorption.  To circumvent this problem and probe bands due to neutral state quinone, a quinone substituted with an ester carbonyl group is used.  Acequinocyl, which acetoxy substituent contains an ester carbonyl, gives rise to an infrared absorption at higher frequency than other quinones.  By incorporating this quinone into 13C-labeled PSI, a quinone band is effectively upshifted while the protein band is downshifted.  By displacing the absorption ranges of quinone and protein, the infrared absorption bands due to neutral state quinone was identified for the first time.