03325nas a2200145 4500008004100000245008500041210006900126260001200195520283400207100002103041700001903062700001303081700002003094856006503114 2021 eng d00aAnisotropic Terahertz Microscopy of Lysozyme in Different CrystalLattice Systems0 aAnisotropic Terahertz Microscopy of Lysozyme in Different Crysta c02/20213 a
Long-range vibrational modes of proteins at terahertz (THz) frequencies havebeen associated with protein function and allosteric control. The characteriza-tion of these motions has been challenging due to energy overlap with waterabsorption and a large vibrational density of states. Recently it has been demon-strated both experimentally and theoretically that vibrational bands can be iso-lated using stationary sample anisotropic terahertz microscopy (SSTAM) fororiented samples, typically realized using protein crystals [1, 2]. In those earlymeasurements, inhibitor binding contrast was demonstrated for high symmetrytetragonal crystals. While high symmetry crystals are ideal for structural deter-minations, they can limit the types of vibrations observable in the ATM mea-surements. Here we show a survey of ATM measurements of triclinic,monoclinic and tetragonal crystals, demonstrating the unique signaturesobservable for the different symmetry groups, leading to a more completedetermination of the vibrational hot spots that may contribute to enzymatic ef-ficiency. The SSATM spectra indicate the presence of conserved vibrationalmodes near 40 cm-1 and 55 cm-1 for CEWL in triclinic, monoclinic and tetrag-onal lattice systems respectively. For CEWL in the monoclinic lattice system, aprominent band at 20cm1was consistently observed in the SSATM spectrabut not in the triclinic or tetragonal systems. The conserved bands may repre-sent vibrational modes that are unperturbed by crystal contact forces while thedifferences may be related to unique molecular orientation in different crystalsystems.
1.Niessen, K., Y. Deng, and A.G. Markelz,Near-field THz micropo-larimetry.Opt Express, 2019.27(20): p. 28036-28047.
2.Romo, T.D., A.Grossfield, and A.G. MarkelzPersistent Protein Motions in a RuggedEnergy Landscape Revealed by Normal Mode Ensemble Analysis. AcceptedJournal of Chemical Information and Modeling, 2020.
1 aMcKinney, J., A.1 aGeorge, D., K.1 aDeng, Y.1 aMarkelz, A., G. uhttps://www.cell.com/biophysj/fulltext/S0006-3495(20)31879-802497nas a2200193 4500008004100000245012000041210006900161260001200230300001200242490000600254520187500260100001302135700002102148700001902169700002002188700001502208700001802223856006202241 2021 eng d00aNear-Field Stationary Sample Terahertz Spectroscopic Polarimetry for Biomolecular Structural Dynamics Determination0 aNearField Stationary Sample Terahertz Spectroscopic Polarimetry c02/2021 a658-6680 v83 aTHz polarimetry on environmentally sensitive and microscopic samples can provide unique insight into underlying mechanisms of complex phenomena. For example, near-field THz anisotropic absorption successfully isolated protein structural vibrations which are connected to biological function. However, to determine how these vibrations impact function requires high throughput measurements of these complex systems, which is challenged by the need for near field detection, sample environmental control and full polarization variation. Stationary sample anisotropic terahertz spectroscopy (SSATS) and near-field stationary sample anisotropic terahertz microscopy (SSATM) have been proposed using synchronous control of THz and electro optic probe polarizations along an iso-response curve. Here we realize these techniques through robust control and calibration of the THz and NIR polarization states. Both methods rapidly measure the linear dichroism in the far field and near field. Validation measurements using standard birefringent sucrose single crystals found the crystal orientation can be determined by scanning the reference polarization and the synchronous pump–probe polarization settings can be optimized to eliminate artifacts. SSATM is then used to determine spectral reproducibility and dehydration effects for a series of chicken egg white lysozyme samples. Reproducible anisotropic absorbance bands are found at about 30, 44, 55, and 62 cm–1. These bands initially sharpen with slow dehydration, similar to the increase in resolution achieved in X-ray crystallographic protein structure determination. The SSATM technique confirms the reliability of anisotropic absorption characterization of protein intramolecular vibrations and opens an avenue for rapid determination of how these long-range dynamics affect biological function.
1 aDeng, Y.1 aMcKinney, J., A.1 aGeorge, D., K.1 aNiessen, K., A.1 aSharma, A.1 aMarkelz, A.G. uhttps://pubs.acs.org/doi/abs/10.1021/acsphotonics.0c0187601859nas a2200241 4500008004500000020001400045245011000059210006900169260000800238300001400246490000800260520114300268653001501411100002101426700001501447700001601462700001301478700001901491700002201510700001601532700002001548856004901568 2020 Engldsh a0006-349500aEvidence of Intramolecular Structural Stabilization in Light Activated State of Orange Carotenoid Protein0 aEvidence of Intramolecular Structural Stabilization in Light Act cFeb a208A-208A0 v1183 aOrange carotenoid protein (OCP) controls efficiency of the light harvesting antenna, the phycobilisome (PBS), in diverse cyanobacteria and prevents oxidative damage. It is the only known photoactive protein that uses a carotenoid, canthaxanthin, as its chromophore. The structure of OCP consists of two globular domains, connected by an unstructured loop, that forms a hydrophobic pocket for the carotenoid. In low light, canthaxanthin bound OCP is inactive and appears orange. Illumination by strong light results in an active state that interacts with the PBS to induce fluorescence quenching, a red appearance and conformational changes that include a 12Å shift by canthaxanthin into the N-terminal domain. Terahertz (THz) dynamical transition measurements and anisotropic terahertz microscopy are used to measure the intramolecular structural dynamics in the inactive and active states, which can be induced by photoexcitation or chaotropic salts. The measurements indicate that the active state has a decrease in structural flexibility, which may be related to enhanced interactions with the PBS.
10aBiophysics1 aMcKinney, J., A.1 aSharma, A.1 aCrossen, K.1 aDeng, Y.1 aGeorge, D., K.1 aLechno-Yossef, S.1 aKerfeld, C.1 aMarkelz, A., G. uhttps://markelz.physics.buffalo.edu/node/25301739nas a2200205 4500008004500000020001400045245008900059210006900148260000800217300001400225490000800239520112900247653001501376100002101391700001701412700001901429700001601448700002001464856004901484 2020 Engldsh a0006-349500aLong Range Correlated Motions of TIM and their Possible Influence on Enzyme Function0 aLong Range Correlated Motions of TIM and their Possible Influenc cFeb a207A-207A0 v1183 aThe alpha-beta barrel structure of triosephosphate isomerase (TIM) is possibly the most common among enzymes. In the case of TIM, structural dynamics are known to be essential to function. In particular the stabilization of the binding pocket by a phosphodianion “handle” of the substrate and the closing of catalytic site loops 6 and 7 over the substrate. Loop 6 moves by as much as 7 Angstroms with binding. Recently a mutant survey for human TIM (hsTIM) found kcat can change significantly for a single mutation distant from the catalytic site. Crystallographic measurements find no structural change with the mutation, suggesting a dynamical mechanism for the allosteric effect. Here we use Stationary Sample Anisotropic Terahertz Microscopy (SSATM) to measure the long-range intramolecular vibrations and determine if specific vibrations couple the allosteric and catalytic sites. SSATM isolated protein long-range structural vibrations based on the dominant displacement direction [1-4]. We examine if specific vibrational bands are associate with loop 6 and loop 7 flexibility.
10aBiophysics1 aMcKinney, J., A.1 aDeng, Y., T.1 aGeorge, D., K.1 aRichard, J.1 aMarkelz, A., G. uhttps://markelz.physics.buffalo.edu/node/25200542nas a2200181 4500008004500000020001400045245007100059210006700130260000800197300001400205490000800219653001500227100002100242700001700263700001500280700001600295856004900311 2019 Engldsh a0006-349500aThe Effect of Crystal Contact Forces on the Protein Global Motions0 aEffect of Crystal Contact Forces on the Protein Global Motions cFeb a489A-489A0 v11610aBiophysics1 aMcKinney, J., A.1 aDeng, Y., T.1 aGeorge, D.1 aMarkelz, A. uhttps://markelz.physics.buffalo.edu/node/251