Structural Organization and Dynamic Processes in Protein Complexes Determined by Multiparameter Imagingстатья

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1. NanoPhotoBioSciences_Structural_Organization_and_Dynamic_... NanoPhotoBioSciences_Structural_Organization_and_Dynamic_... 2,5 МБ 30 июля 2013 [emaksimoff]

[1] Structural organization and dynamic processes in protein complexes determined by multiparameter imaging / F. J. Schmitt, E. G. Maksimov, C. Junghans et al. // Signpost Open Access Journal of NanoPhotoBioSciences. — 2013. — Vol. 1, no. 1. — P. 1–47. A combination of complementary spectroscopic techniques was used to determine certain structural parameters and excitation energy transfer (EET) processes in biological pigment-protein complexes and fluorescent proteins. While a single spectroscopic technique does not provide unambiguous data related to the structural organization, and since only dynamic processes in a restricted temporal domain can be gathered, we present examples in which the combination of complementary techniques is suitable to deliver a complete picture of a certain parameter set. First, EET processes in light harvesting complexes of the cyanobacterium A. marina were determined. Structural details of the macroscopic organization are visualized by transmission electron microscopy (TEM) while the migration of excited states is analyzed with time resolved ps-fluorescence spectroscopy and compared with published results from fs-pump-probe spectroscopy. Second, the theory of Förster Resonance Energy Transfer (FRET) was used to correlate the distance between transition dipole moments with dynamical processes that are accessible with highly time-resolved studies. With FRET, the average distance between semiconductor nanocrystals attached to photosynthetic light harvesting complexes was determined. Furthermore, the configuration of a strongly excitonically coupled Chl dimer in the water soluble Chl binding protein WSCP of class IIa from cauliflower genetically expressed in E. coli and reconstituted with Chl a, Chl b or mixtures of Chl a and Chl b was analyzed. Details on the pigment-protein interaction between Chl and the protein backbone in WSCP represent structural details that are accessible by the thorough theoretical analysis of ultrafast exciton relaxation processes, time-resolved fluorescence spectroscopic and site-selective hole-burning studies. Third, the combination of time-resolved and time-integrated fluorescence spectroscopy with fluorescence correlation spectroscopy (FCS) allowed the estimation of the distance between dipole moments of donor and acceptor in FRET pairs containing enhanced green fluorescent protein (eGFP) as donor and red fluorescent (tagRFP) protein as acceptor. Simultaneously, the hydrodynamic radius of the eGFP-tagRFP-FRET construct was determined. This hydrodynamic radius is by a factor of four bigger than the dipole distance determied by FRET, and also about four times as big as the size of the construct from available crystal structures. Therefore, internal degrees of freedom seem to hamper the diffusion of the flexible FRET construct, which can not be decribed as a homogeneous sphere as done by the simplified calculation of the hydrodynamic radius from diffusion. The given examples summarize typical strategies of a combined analysis of multiple parameters by complementary techniques that allow the determination of the structural organization of complex biological macromolecules and the dynamics of bioenergetic processes in such structures such as excitation energy transfer.

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