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 a

THz 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 a

Orange 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/25301881nas a2200181 4500008004100000245009900041210006900140260001600209520131100225100001701536700001501553700001301568700001501581700002201596700001601618700001601634856004901650 2020 eng d00aStabilization of Terahertz Vibrational Modes in Illuminated Orange Carotenoid Protein Crystals0 aStabilization of Terahertz Vibrational Modes in Illuminated Oran aBuffalo, NY3 a

Orange carotenoid protein (OCP) controls efficiency of the phycobilisome (PBS), the light harvesting antenna in cyanobacteria, to prevent oxidative damage. The OCP switches from resting state to photo protective state with intense blue light illumination. Questions persist as to why OCPR interaction increases with the PBS over that with the OCPO. Here we examine the role of long-range intramolecular vibrations within OCP. Using Stationary Sample Anisotropic Terahertz Microscopy (SSATM) we measure changes in the intramolecular vibrations with photo switching. We report the first observation of switching in the intramolecular vibrations with photoexcitation. Results suggest that there is a stiffening of the molecule in the photo protective state. This increase in structural stability may enhance the interaction with the PBS change in OCP interaction with PBS. In low light, carotenoid bound OCP appears orange (OCP o ) and is inactive. Illumination by strong light converts OCP to the active, red (OCPR) state, which interacts with the PBS. A comparison of anisotropic THz microscopy measurements of dark adapted (OCP o ) and illuminated OCP crystals indicate differences in their vibrational modes that may be important for OCP-PBS interactions.

1 aMcKinney, J.1 aSharma, A.1 aDeng, Y.1 aGeorge, D.1 aLechno-Yossef, S.1 aKerfeld, C.1 aMarkelz, A. uhttps://markelz.physics.buffalo.edu/node/54400492nas a2200145 4500008004100000245008500041210006900126260001200195100001300207700001700220700001300237700001900250700002000269856005700289 2019 eng d00aProtein Intramolecular Motions with Deuteration and Inhibitor Binding Dependence0 aProtein Intramolecular Motions with Deuteration and Inhibitor Bi c03/20191 aDeng, Y.1 aMcKinney, J.1 aRomo, T.1 aGrossfield, A.1 aMarkelz, A., G. uhttps://meetings.aps.org/Meeting/MAR19/Session/R63.300501nas a2200145 4500008004100000245011200041210006900153490001200222100001100234700001300245700001300258700001500271700001600286856005300302 2018 eng d00aMeasuring Protein Intramolecular Dynamics with Terahertz Light: Functional Changes and Relevance to Biology0 aMeasuring Protein Intramolecular Dynamics with Terahertz Light F0 vH50.0011 aXu, M.1 aDeng, Y.1 aLuck, C.1 aSharma, A.1 aMarkelz, A. uhttp://meetings.aps.org/link/BAPS.2018.MAR.H50.100451nas a2200121 4500008004100000245010900041210006900150490001200219100001300231700001600244700001600260856005300276 2018 eng d00aRapid Terahertz Dichroism Near Field Microscopy for Biomolecular Intramolecular Vibrational Spectroscopy0 aRapid Terahertz Dichroism Near Field Microscopy for Biomolecular0 vA50.0081 aDeng, Y.1 aNiessen, K.1 aMarkelz, A. uhttp://meetings.aps.org/link/BAPS.2018.MAR.A50.800508nas a2200145 4500008004100000245011600041210006900157260001200226100001300238700001100251700001200262700002000274700001600294856005200310 2017 eng d00aGlobal Picosecond Structural Dynamics of Orange Carotenoid Protein in Photo/Chemical Activated Signaling States0 aGlobal Picosecond Structural Dynamics of Orange Carotenoid Prote c03/20171 aDeng, Y.1 aXu, M.1 aLiu, H.1 aBlankenship, R.1 aMarkelz, A. uhttp://meetings.aps.org/link/BAPS.2017.MAR.S4.2