Global and target analysis of time-resolved spectra

AbstractIn biological/bioenergetics research the response of a complex system to an externally applied perturbation is often studied. Spectroscopic measurements at multiple wavelengths are used to monitor the kinetics. These time-resolved spectra are considered as an example of multiway data. In this paper, the methodology for global and target analysis of time-resolved spectra is reviewed. To fully extract the information from the overwhelming amount of data, a model-based analysis is mandatory. This analysis is based upon assumptions regarding the measurement process and upon a physicochemical model for the complex system. This model is composed of building blocks representing scientific knowledge and assumptions. Building blocks are the instrument response function (IRF), the components of the system connected in a kinetic scheme, and anisotropy properties of the components. The combination of a model for the kinetics and for the spectra of the components results in a more powerful spectrotemporal model. The model parameters, like rate constants and spectra, can be estimated from the data, thus providing a concise description of the complex system dynamics. This spectrotemporal modeling approach is illustrated with an elaborate case study of the ultrafast dynamics of the photoactive yellow protein

Verification of the Kcat Value of AAT via COPASI

PLP is a chromophoric cofactor required for catalytic activity by a wide variety of enzymes. While PLP enzymes are thermally activated in vivo, it has been reported that some PLP enzymes can be activated by UV light. One enzyme that uses PLP as a cofactor is apsartate aminotransferase (AAT). Previous studies with AAT suggest that the carbanionic quinonoid intermediate is photogenerated by UV laser excitation. AAT is central to nitrogen metabolism in all living systems and has a large body of literature. As such, it is a useful prototype for the fundamental studies on this class of enzymes which were previously conducted. In order to verify these experimental results, the computer program COPASI was used. Two models of AAT mechanisms were examined using COPASI one with the quinonoid intermediate on the productive path and one with the quinonoid off the productive path

Toward An Understanding Of The Retinal Chromophore In Rhodopsin Mimics

Recently, a rhodopsin protein mimic was constructed by combining mutants of the cellular retinoic acid binding protein II (CRABPII) with an all-trans retinal chromophore. Here, we present a combine computational quantum mechanics/molecular mechanics (QM/MM) and experimental ultrafast kinetic study of CRABPII. We employ the QM/MM models to study the absorption (lambda(a)(max)), fluorescence (lambda(f)(max)), and reactivity of a CRABPII triple mutant incorporating the all-trans protonated chromophore (PSB-KLE-CRABPII). We also study the spectroscopy of the same mutant incorporating the unprotonated chromophore and of another double mutant incorporating the neutral unbound retinal molecule held inside the pocket. Finally, for PSB-KLE-CRABPII, stationary fluorescence spectroscopy and ultrafast transient absorption spectroscopy resolved two different evolving excited state populations which were computationally assigned to distinct locally excited and charge-transfer species. This last species is shown to evolve along reaction paths describing a facile isomerization of the biologically relevant 11-cis and 13-cis double bonds. This work represents a first exploratory attempt to model and study these artificial protein systems. It also indicates directions for improving the QM/MM models so that they could be more effectively used to assist the bottom-up design of genetically encodable probes and actuators employing the retinal chromophore

Origin of line broadening in the electronic absorption spectra of conjugated polymers: Three-pulse-echo studies of MEH-PPV in toluene

Integrated three-pulse stimulated echo peak shift data are compared for N,N-bis-dimethylphenyl-1-2,4,6,8-perylenetetracarbonyl diamide and poly[2-(2'-ethylhexyloxy)-5-methoxy-1,4-phenylenevinylene] (MEH-PPV) in toluene solvent. These two molecules represent a model probe of solvation dynamics and a prototypical soluble, electroluminescent conjugated polymer, respectively. The results indicate that it is inappropriate to describe the linear absorption spectrum of MEH-PPV as being primarily inhomogeneously broadened. Conformational disorder along the polymer backbone gives rise to an ensemble of polyene electronic oscillators that are strongly coupled to each other. As a consequence, fluctuations in the electronic energy gap on a time-scale of 50-fs derive primarily from bath-mediated exciton scattering. The data reported here provide an explanation for the broad, structureless electronic absorption of MEH-PPV. This interpretation provides a valuable insight into the nature of the initial photoexcited state, and the efficient population of the emissive state

Tracking the secondary photodynamics of the green/red cyanobacteriochrome RcaE from Fremyella diplosiphon

Cyanobacteriochrome RcaE regulates Type III complementary chromatic adaption in the cyanobacterium Fremyella diplosiphon by photoswitching between a green-absorbing dark state (15ZPg) and red-absorbing photoproduct (15EPr). Ultrafast photodynamics of RcaE involve tautomerization of the bilin chromophore, inhomogeneity, and the generation of three primary photointermediates in the forward reaction (Lumi-Go, Lumi-Gr, and Lumi-Gf). The secondary photodynamics reported here show that only Lumi-Go evolves to 15EPr via spectrally similar Meta-Go1 and Meta-Go2 intermediates, with a protonation reaction occurring at the final step on the millisecond timescale. Reverse reaction dynamics were characterized and reveal an unusually long-lived Lumi-Rf photoproduct and a blue-shifted Meta-Ry intermediate

ADAPT and LibreStudio: Building the Textbook of the Future with Next Generation OER Homework System

Today, online homework systems are the most commonly required curricula materials, ADAPT is a new online homework system being developed by LibreTexts to serve the Open Education community. With the support of the California Educational Learning Lab, ADAPT is designed to combine adaptive learning incorporating learning trees with culturally responsive pedagogy at minimal cost. We will demonstrate how instructors can use ADAPT to augment existing and newly constructed OER textbooks with summative exercises and embed them in LMSs, LibreTexts textbooks, in a standalone application, and in-class clickers. Discussions will demonstrate how the ADAPT homework system empowers faculty to build and use existing questions in multiple modalities. Participants will learn how to build autograded questions based on four technologies – H5P, WebWork, IMathAS, and native (QTI) – that can be used interchangeably to allow for maximal impact. Open ended questions can be evaluated using a sophisticated checker to rapidly mark text, audio or other files for grading. We will also introduce the LibreStudio platform for construction, storage and distribution of H5P assessments. Participants will be able to join LibreStudio to create and share H5P assessments, review the H5P of other authors, and build/join a community within Studio. The workshop will conclude with an overview of the analytics infrastructure to provide real-time learning analytics to instructors on student progress with pre-defined learning objectives

Photoreceptors Take Charge. Emerging Principles for Light Sensing

Kottke T, Xie A, Larsen DS, Hoff WD. Photoreceptors Take Charge. Emerging Principles for Light Sensing. Annual Review of Biophysics. 2018;47(1):291-313.The first stage in biological signaling is based on changes in the functional state of a receptor protein triggered by interaction of the receptor with its ligand(s). The light-triggered nature of photoreceptors allows studies on the mechanism of such changes in receptor proteins using a wide range of biophysical methods and with superb time resolution. Here, we critically evaluate current understanding of proton and electron transfer in photosensory proteins and their involvement both in primary photochemistry and subsequent processes that lead to the formation of the signaling state. An insight emerging from multiple families of photoreceptors is that ultrafast primary photochemistry is followed by slower proton transfer steps that contribute to triggering large protein conformational changes during signaling state formation. We discuss themes and principles for light sensing shared by the six photoreceptor families: rhodopsins, phytochromes, photoactive yellow proteins, light-oxygen-voltage proteins, blue-light sensors using flavin, and cryptochromes