Papers by Robert Blankenship
Analytical Chemistry, Jun 22, 2011
The high sensitivity, extended mass range, and fast data acquisition/processing of mass spectrome... more The high sensitivity, extended mass range, and fast data acquisition/processing of mass spectrometry and its coupling with native electrospray ionization (ESI) make the combination complementary to other biophysical methods of protein analysis. Protein assemblies with molecular masses up to MDa are now accessible by this approach. Most current approaches have used quadrupole/time-of-flight tandem mass spectrometry, sometimes coupled with ion mobility, to reveal stoichiometry, shape, and dissociation of protein assemblies. The amino-acid sequence of the subunits, however, still relies heavily on independent bottom-up proteomics. We describe here an approach to study protein assemblies that integrates electron-capture dissociation (ECD), native ESI, and FTICR mass spectrometry (12 Tesla). Flexible regions of assembly subunits of yeast alcohol dehydrogenase (147 kDa), concanavalin A (103 kDa), and photosynthetic Fenna-Matthews-Olson antenna protein complex (140 kDa) can be sequenced by ECD or "activated-ion" ECD. Furthermore, non-covalent metal-binding sites can also be determined for the ConA assembly. Most importantly, the regions that undergo fragmentation, either from one of the termini by ECD or from the middle of a protein, as initiated by CID, correlate well with the Bfactor from X-ray crystallography of that protein. This factor is a measure of the extent an atom can move from its coordinated position as a function of temperature or crystal imperfections. The approach provides not only top-down proteomics information of the complex subunits but also structural insights complementary to those obtained by ion mobility.
Biochemistry, Apr 11, 2011
The nature and stoichiometry of pigments in the Fenna-Matthews-Olson (FMO) photosynthetic antenna... more The nature and stoichiometry of pigments in the Fenna-Matthews-Olson (FMO) photosynthetic antenna protein complex were determined by native electrospray mass spectrometry. The FMO antenna complex was the first chlorophyll-containing protein that was crystallized. Previous results indicate that the FMO protein forms a trimer with seven bacteriochlorophyll a in each monomer. This model has long been a working basis to understand the molecular mechanism of energy transfer through pigment/pigment and pigment/protein coupling. Recent results have suggested, however, that an eighth bacteriochlorophyll is present in some subunits. In this report, a direct mass-spectrometry measurement of the molecular weight of the intact FMO protein complex clearly indicates the existence of an 8 th pigment, which is assigned as a bacteriochlorophyll a by mass analysis of the complex and HPLC analysis of the pigment. The 8 th pigment is found to be easily lost during purification, which results in its partial occupancy in the mass spectra of the intact complex prepared by different procedures. The results are consistent with the recent X-ray structural models. The existence of the 8 th bacteriochlorophyll a in this model antenna protein gives new insights into the functional role of the FMO protein and motivates the need for new theoretical and spectroscopic assignments of spectral features of the FMO protein.
The Journal of Physical Chemistry B, 2019
Modeling of various optical spectra. The modeling approach is described in ref 1; in brief, the d... more Modeling of various optical spectra. The modeling approach is described in ref 1; in brief, the disorder is introduced into the diagonal matrix elements (i.e., 𝐸 0 𝑛 ) by a Monte Carlo approach with normal distributions centered at 𝐸 0 𝑛 (n labeling various pigments) and with fwhm representing the inhomogeneous broadenings (Γinh), which are site-dependent. Eigen decomposition of the interaction matrix provides eigen coefficients (𝑐 𝑛 𝑀 ) and eigenvalues (𝜔 𝑀 ). Phonon and vibrational Huang-Rhys factors (S) are used as free or fixed parameters and are optimized simultaneously against the experimental spectra. Moreover, it is assumed that the phonon spectral density (weighted phonon profile) can be described by a continuous function, which is chosen to be a lognormal distribution, 2 𝐽 𝑝ℎ (𝜔) = 𝑆 𝑝ℎ 𝜔𝜎√2𝜋 𝑒 -[ln( 𝜔 𝜔 𝑐 )] 2 /2𝜎 2
The journal of physical chemistry. B, Apr 12, 2018
We report high-resolution (low-temperature) absorption, emission, and nonresonant/resonant hole-b... more We report high-resolution (low-temperature) absorption, emission, and nonresonant/resonant hole-burned (HB) spectra and results of excitonic calculations using a non-Markovian reduced density matrix theory (with an improved algorithm for parameter optimization in heterogeneous samples) obtained for the Y16F mutant of the Fenna-Matthews-Olson (FMO) trimer from the green sulfur bacterium Chlorobium tepidum. We show that the Y16F mutant is a mixture of FMO complexes with three independent low-energy traps (located near 817, 821, and 826 nm), in agreement with measured composite emission and HB spectra. Two of these traps belong to mutated FMO subpopulations characterized by significantly modified low-energy excitonic states. Hamiltonians for the two major subpopulations (Sub and Sub) provide new insight into extensive changes induced by the single-point mutation in the vicinity of BChl 3 (where tyrosine Y16 was replaced with phenylalanine F16). The average decay time(s) from the higher...
The Journal of Physical Chemistry B, 2016
Carotenoids are a class of natural pigments present in all phototrophic organisms, mainly in thei... more Carotenoids are a class of natural pigments present in all phototrophic organisms, mainly in their light-harvesting proteins in which they play roles of accessory light absorbers and photoprotectors. Extensive time-resolved spectroscopic studies of these pigments have revealed unexpectedly complex photophysical properties, particularly for carotenoids in light-harvesting LH2 complexes from purple bacteria. An ambiguous, optically forbidden electronic excited state designated as S* has been postulated to be involved in carotenoid excitation relaxation and in an alternative carotenoid-tobacteriochlorophyll energy transfer pathway, as well as being a precursor of the carotenoid triplet state. However, no definitive and satisfactory origin of the carotenoid S* state in these complexes has been established, despite a wide-ranging series of studies. Here, we resolve the ambiguous origin of the carotenoid S* state in LH2 complex from Rba. sphaeroides by showing that the S* feature can be seen as a combination of ground state absorption bleaching of the carotenoid pool converted to cations and the Stark spectrum of neighbor neutral carotenoids, induced by temporal electric field brought by the carotenoid cationbacteriochlorophyll anion pair. These findings remove the need to assign an S* state, and thereby significantly simplify the photochemistry of carotenoids in these photosynthetic antenna complexes.
The journal of physical chemistry. A, Jan 11, 2016
The Fenna-Matthews-Olson (FMO) trimer (composed of identical subunits) from the green sulfur bact... more The Fenna-Matthews-Olson (FMO) trimer (composed of identical subunits) from the green sulfur bacterium Chlorobaculum tepidum is an important protein model system to study exciton dynamics and excitation energy transfer (EET) in photosynthetic complexes. In addition, FMO is a popular model for excitonic calculations, with many theoretical parameter sets reported describing different linear and nonlinear optical spectra. Due to fast exciton relaxation within each subunit, intermonomer EET results predominantly from the lowest energy exciton states (contributed to by BChl a 3 and 4). Using experimentally determined shapes for the spectral densities, simulated optical spectra are obtained for the entire FMO trimer. Simultaneous fits of low-temperature absorption, fluorescence, and hole-burned spectra place constraints on the determined pigment site energies, providing a new Hamiltonian that should be further tested to improve modeling of 2D electronic spectroscopy data and our understan...
Carotenoid-to-Bacteriochlorophyll Energy Transfer in the LH1-RC Core Complex of a Bacteriochlorophyll b-Containing Purple Photosynthetic Bacterium Blastochloris viridis
The journal of physical chemistry. B, Jun 24, 2016
Carotenoid-to-bacteriochlorophyll energy transfer has been widely investigated in bacteriochlorop... more Carotenoid-to-bacteriochlorophyll energy transfer has been widely investigated in bacteriochlorophyll (BChl) a-containing light harvesting complexes. Blastochloris (B.) viridis utilizes BChl b, whose absorption spectrum is more red-shifted than that of BChl a. This has implications on the efficiency and pathways of carotenoid-to-BChl energy transfer in this organism. The carotenoids that comprise the light-harvesting reaction center core complex (LH1-RC) of B. viridis are 1,2-dihydroneurosporene and 1,2-dihydrolycopene, which are derivatives of carotenoids found in the light harvesting complexes of several BChl a-containing purple photosynthetic bacteria. Steady-state and ultrafast time-resolved optical spectroscopic measurements were performed on the LH1-RC complex of B. viridis at room and cryogenic temperatures. The overall efficiency of carotenoid-to-bacteriochlorophyll energy transfer obtained from steady-state absorption and fluorescence measurements were determined to be ~27%...
Frontiers in Microbiology, 2011
Photosynthesis is the biological process that converts solar energy to biomass, bioproducts, and ... more Photosynthesis is the biological process that converts solar energy to biomass, bioproducts, and biofuel. It is the only major natural solar energy storage mechanism on Earth. To satisfy the increased demand for sustainable energy sources and identify the mechanism of photosynthetic carbon assimilation, which is one of the bottlenecks in photosynthesis, it is essential to understand the process of solar energy storage and associated carbon metabolism in photosynthetic organisms. Researchers have employed physiological studies, microbiological chemistry, enzyme assays, genome sequencing, transcriptomics, and 13 C-based metabolomics/fluxomics to investigate central carbon metabolism and enzymes that operate in phototrophs. In this report, we review diverse CO 2 assimilation pathways, acetate assimilation, carbohydrate catabolism, the tricarboxylic acid cycle and some key, and/or unconventional enzymes in central carbon metabolism of phototrophic microorganisms. We also discuss the reducing equivalent flow during photoautotrophic and photoheterotrophic growth, evolutionary links in the central carbon metabolic network, and correlations between photosynthetic and non-photosynthetic organisms. Considering the metabolic versatility in these fascinating and diverse photosynthetic bacteria, many essential questions in their central carbon metabolism still remain to be addressed. Keywords: acetate assimilation, autotrophic and anaplerotic CO 2 assimilation, biomass and biofuel, 13 C-based metabolomics, citrate metabolism, photosynthesis, unconventional pathways and enzymes www.frontiersin.org
Proceedings of the National Academy of Sciences, 2009
The high excitation energy-transfer efficiency demanded in photosynthetic organisms relies on the... more The high excitation energy-transfer efficiency demanded in photosynthetic organisms relies on the optimal pigment-protein binding orientation in the individual protein complexes and also on the overall architecture of the photosystem. In green sulfur bacteria, the membrane-attached Fenna-Matthews-Olson (FMO) antenna protein functions as a “wire” to connect the large peripheral chlorosome antenna complex with the reaction center (RC), which is embedded in the cytoplasmic membrane (CM). Energy collected by the chlorosome is funneled through the FMO to the RC. Although there has been considerable effort to understand the relationships between structure and function of the individual isolated complexes, the specific architecture for in vivo interactions of the FMO protein, the CM, and the chlorosome, ensuring highly efficient energy transfer, is still not established experimentally. Here, we describe a mass spectrometry-based method that probes solvent-exposed surfaces of the FMO by lab...
Photosynthesis Research, 2011
Your article is protected by copyright and all rights are held exclusively by Springer Science+Bu... more Your article is protected by copyright and all rights are held exclusively by Springer Science+Business Media B.V.. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your work, please use the accepted author's version for posting to your own website or your institution's repository. You may further deposit the accepted author's version on a funder's repository at a funder's request, provided it is not made publicly available until 12 months after publication.
Journal of Materials Chemistry, 2012
The chlorosome is a highly specialized supramolecular light-harvesting antenna complex found in g... more The chlorosome is a highly specialized supramolecular light-harvesting antenna complex found in green photosynthetic bacteria and is composed of self-assembled bacteriochlorophyll (BChl) pigments entrapped in a lipid vesicle. These organelles are of interest for development of synthetic devices for solar harvesting and conversion because the organization and packing of BChls in the chlorosome provides a highly efficient light collection and energy funneling mechanism with properties that are superior to similar artificial systems based on self-assembled BChl pigment analogues. In this study, we investigated sol-gel chemistry as an approach to entrap and stabilize chlorosomes isolated from Chloroflexus aurantiacus. Two distinct synthesis approaches that differed in the H 2 O/Si ratio in the gels were investigated. Spectrophotometric analysis showed that the chlorosomes were intact when encapsulated in sol-gels and did not suffer any deleterious effects during the entrapment process. In addition, the integrity of the chlorosomes was unaffected by methanol levels that can result during the formation of sol-gels. Using small-angle neutron scattering it was not only possible to characterize the properties of the sol-gel matrix but also the size, shape and aggregation state of the entrapped chlorosomes. The sol-gels formed at a higher H 2 O/Si ratio (FH gels) resulted in a more branched gel structure with a larger pore size compared to the gels formed at lower H 2 O/Si ratio (PH gels). The chlorosomes entrapped in FH gels had dimensions of $16.0 Â 51.1 Â 180.1 nm which agrees well with the size of chlorosomes previously determined using cryo-transmission electron microscopy, while the chlorosomes in the PH gels appear to be aggregated. The approach described here offers new possibilities for the development of artificial solar-harvesting and energy conversion devices based on naturally occurring photosynthetic systems.
Journal of Bacteriology, 2007
“ Candidatus Chlorothrix halophila” is a recently described halophilic, filamentous, anoxygenic p... more “ Candidatus Chlorothrix halophila” is a recently described halophilic, filamentous, anoxygenic photoautotroph (J. A. Klappenbach and B. K. Pierson, Arch. Microbiol. 181: 17-25, 2004) that was enriched from the hypersaline microbial mats at Guerrero Negro, Mexico. Analysis of the photosynthetic apparatus by negative staining, spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the photosynthetic apparatus in this organism has similarities to the photosynthetic apparatus in both the Chloroflexi and Chlorobi phyla of green photosynthetic bacteria. The chlorosomes were found to be ellipsoidal and of various sizes, characteristics that are comparable to characteristics of chlorosomes in other species of green photosynthetic bacteria. The absorption spectrum of whole cells was dominated by the chlorosome bacteriochlorophyll c (BChl c ) peak at 759 nm, with fluorescence emission at 760 nm. A second fluorescence emission band was observed at 870 nm an...
Journal of Bacteriology, 2013
Chlorosomes are large light-harvesting complexes found in three phyla of anoxygenic photosyntheti... more Chlorosomes are large light-harvesting complexes found in three phyla of anoxygenic photosynthetic bacteria. Chlorosomes are primarily composed of self-assembling pigment aggregates. In addition to the main pigment, bacteriochlorophyll c , d , or e , chlorosomes also contain variable amounts of carotenoids. Here, we use X-ray scattering and electron cryomicroscopy, complemented with absorption spectroscopy and pigment analysis, to compare the morphologies, structures, and pigment compositions of chlorosomes from Chloroflexus aurantiacus grown under two different light conditions and Chlorobaculum tepidum . High-purity chlorosomes from C. aurantiacus contain about 20% more carotenoid per bacteriochlorophyll c molecule when grown under low light than when grown under high light. This accentuates the light-harvesting function of carotenoids, in addition to their photoprotective role. The low-light chlorosomes are thicker due to the overall greater content of pigments and contain domain...
Journal of Bacteriology, 2009
The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic an... more The green filamentous bacterium Chloroflexus aurantiacus employs chlorosomes as photosynthetic antennae. Chlorosomes contain bacteriochlorophyll aggregates and are attached to the inner side of a plasma membrane via a protein baseplate. The structure of chlorosomes from C. aurantiacus was investigated by using a combination of cryo-electron microscopy and X-ray diffraction and compared with that of Chlorobi species. Cryo-electron tomography revealed thin chlorosomes for which a distinct crystalline baseplate lattice was visualized in high-resolution projections. The baseplate is present only on one side of the chlorosome, and the lattice dimensions suggest that a dimer of the CsmA protein is the building block. The bacteriochlorophyll aggregates inside the chlorosome are arranged in lamellae, but the spacing is much greater than that in Chlorobi species. A comparison of chlorosomes from different species suggested that the lamellar spacing is proportional to the chain length of the ...
Journal of Bacteriology, 2009
The green phototrophic bacteria contain a unique complement of chlorophyll pigments, which self-a... more The green phototrophic bacteria contain a unique complement of chlorophyll pigments, which self-assemble efficiently into antenna structures known as chlorosomes with little involvement of protein. The few proteins found in chlorosomes have previously been thought to have a primarily structural function. The biosynthetic pathway of the chlorosome pigments, bacteriochlorophylls c , d , and e , is not well understood. In this report, we used spectroscopic, proteomic, and gene expression approaches to investigate the chlorosome proteins of the green filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus . Surprisingly, Mg-protoporphyrin IX monomethyl ester (oxidative) cyclase, AcsF, was identified under anaerobic growth conditions. The AcsF protein was found in the isolated chlorosome fractions, and the proteomics analysis suggested that significant portions of the AcsF proteins are not accessible to protease digestion. Additionally, quantitative real-time PCR studies s...
BMC Microbiology, 2010
Background Heliobacterium modesticaldum is a gram-positive nitrogen-fixing phototrophic bacterium... more Background Heliobacterium modesticaldum is a gram-positive nitrogen-fixing phototrophic bacterium that can grow either photoheterotrophically or chemotrophically but not photoautotrophically. Surprisingly, this organism is lacking only one gene for the complete reverse tricarboxylic acid (rTCA) cycle required for autotrophic carbon fixation. Along with the genomic information reported recently, we use multiple experimental approaches in this report to address questions regarding energy metabolic pathways in darkness, CO2 fixation, sugar assimilation and acetate metabolism. Results We present the first experimental evidence that D-ribose, D-fructose and D-glucose can be photoassimilated by H. modesticaldum as sole carbon sources in newly developed defined growth medium. Also, we confirm two non-autotrophic CO2-fixation pathways utilized by H. modesticaldum: reactions catalyzed by pyruvate:ferredoxin oxidoreductase and phosphoenolpyruvate carboxykinase, and report acetate excretion du...
Biophysical Journal, 2001
Ultrafast transient absorption spectroscopy was used to probe excitation energy transfer and trap... more Ultrafast transient absorption spectroscopy was used to probe excitation energy transfer and trapping at 77 K in the photosystem I (PSI) core antenna from the cyanobacterium Synechocystis sp. PCC 6803. Excitation of the bulk antenna at 670 and 680 nm induces a subpicosecond energy transfer process that populates the Chl a spectral form at 685-687 nm within few transfer steps (300 -400 fs). On a picosecond time scale equilibration with the longest-wavelength absorbing pigments occurs within 4 -6 ps, slightly slower than at room temperature. At low temperatures in the absence of uphill energy transfer the energy equilibration processes involve low-energy shifted chlorophyll spectral forms of the bulk antenna participating in a 30 -50-ps process of photochemical trapping of the excitation by P 700 . These spectral forms might originate from clustered pigments in the core antenna and coupled chlorophylls of the reaction center. Part of the excitation is trapped on a pool of the longest-wavelength absorbing pigments serving as deep traps at 77 K. Transient hole burning of the ground-state absorption of the PSI with excitation at 710 and 720 nm indicates heterogeneity of the red pigment absorption band with two broad homogeneous transitions at 708 nm and 714 nm (full-width at half-maximum (fwhm) ϳ 200 -300 cm Ϫ1 ). The origin of these two bands is attributed to the presence of two chlorophyll dimers, while the appearance of the early time bleaching bands at 683 nm and 678 nm under excitation into the red side of the absorption spectrum (Ͼ690 nm) can be explained by borrowing of the dipole strength by the ground-state absorption of the chlorophyll a monomers from the excited-state absorption of the dimeric red pigments.
Biophysical Journal, 2000
Results from high-pressure and Stark hole-burning experiments on isolated chlorosomes from the gr... more Results from high-pressure and Stark hole-burning experiments on isolated chlorosomes from the green sulfur bacterium Chlorobium tepidum are presented, as well as Stark hole-burning data for bacteriochlorophyll c (BChl c) monomers in a poly(vinyl butyral) copolymer film. Large linear pressure shift rates of Ϫ0.44 and Ϫ0.54 cm Ϫ1 /MPa were observed for the chlorosome BChl c Q y -band at 100 K and the lowest Q y -exciton level at 12 K, respectively. It is argued that approximately half of the latter shift rate is due to electron exchange coupling between BChl c molecules. The similarity between the above shift rates and those observed for the B875 and B850 BChl a rings of the light-harvesting complexes of purple bacteria is emphasized. For BChl c monomer, ƒ⌬ ϭ 0.35 D, where ⌬ is the dipole moment change for the Q y transition and ƒ is the local field correction factor. The data establish that ⌬ is dominated by the matrix-induced contribution. The change in polarizability (⌬␣) for the Q y transition of the BChl c monomer is estimated at 19 Å 3 , which is essentially identical to that of the Chl a monomer. Interestingly, no Stark effects were observed for the lowest exciton level of the chlorosomes (maximum Stark field of 10 5 V/cm). Possible explanations for this are given, and these include consideration of structural models for the chlorosome BChl c aggregates.
Biochemistry, 2003
Excitation energy transfer and trapping processes in an iron stress-induced supercomplex of photo... more Excitation energy transfer and trapping processes in an iron stress-induced supercomplex of photosystem I from the cyanobacterium Synechocystis sp. PCC6803 were studied by time-resolved absorption and fluorescence spectroscopy on femtosecond and picosecond time scales. The data provide evidence that the energy transfer dynamics of the CP43′-PSI supercomplex are consistent with energy transfer processes that occur in the Chl a network of the PSI trimer antenna. The most significant absorbance changes in the CP43′-PSI supercomplex are observed within the first several picoseconds after the excitation into the spectral region of CP43′ absorption (665 nm). The difference time-resolved spectra (∆∆A) resulting from subtraction of the PSI trimer kinetic data from the CP43′-PSI supercomplex data indicate three energy transfer processes with time constants of 0.2, 1.7, and 10 ps. The 0.2 ps kinetic phase is tentatively interpreted as arising from energy transfer processes originating within or between the CP43′ complexes. The 1.7 ps phase is interpreted as possibly arising from energy transfer from the CP43′ ring to the PSI trimer via closely located clusters of Chl a in CP43′ and the PSI core, while the slower 10 ps process might reflect the overall excitation transfer from the CP43′ ring to the PSI trimer. These three fast kinetic phases are followed by a 40 ps overall excitation decay in the supercomplex, in contrast to a 25 ps overall decay observed in the trimer complex without CP43′. Excitation of Chl a in both the CP43′-PSI antenna supercomplex and the PSI trimer completely decays within 100 ps, resulting in the formation of P700 + . The data indicate that there is a rapid and efficient energy transfer between the outer antenna ring and the PSI reaction center complex.
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Papers by Robert Blankenship