Trans?Cis Isomerization is Responsible for the Red-Shifted Fluorescence in Variants of the Red Fluorescent Protein eqFP611

An important class of red fluorescent proteins (RFPs) feature a 2-iminomethyl-5-(4-hydroxybenzylidene)imidazolinone chromophore. Among these proteins, eqFP611 has the chromophore in a coplanar trans orientation, whereas the cis isomer is preferred by other RFPs such as DsRed and its variants. In the photoactivatable protein asFP595, the chromophore can even be switched from the nonfluorescent trans to the fluorescent cis state by light. By using X-ray crystallography, we have determined the structure of dimeric eqFP611 at high resolution (up to 1.1 Å). In the far-red emitting eqFP611 variant d2RFP630, which carries an additional Asn143Ser mutation, the chromophore resides predominantly (?80%) in the cis isomeric state, and in RFP639, which has Asn143Ser and Ser158Cys mutations, the chromophore is found completely in the cis form. The pronounced red shift of excitation and emission maxima of RFP639 can thus unambiguously be assigned to trans?cis isomerization of the chromophore. Among RFPs, eqFP611 is thus unique because its chromophore is highly fluorescent in both the cis and trans isomeric forms.<br/

The power of line-narrowing techniques: Applications to photosynthetic chromoprotein complexes and autofluorescent proteins

Laser spectroscopic techniques at low temperature, such as fluorescence line-narrowing and hole burning, enable an increase of spectral resolution by a factor of 1

Structural basis of photoswitching in fluorescent proteins.

International audienceFluorescent proteins have revolutionized life sciences because they allow noninvasive and highly specific labeling of biological samples. The subset of "phototransformable" fluorescent proteins recently attracted a widespread interest, as their fluorescence state can be modified upon excitation at defined wavelengths. The fluorescence emission of Reversibly Switchable Fluorescent Proteins (RSFPs), in particular, can be repeatedly switched on and off. RSFPs enable many new exciting modalities in fluorescence microscopy and biotechnology, including protein tracking, photochromic Förster Resonance Energy Transfer, super-resolution microscopy, optogenetics, and ultra-high-density optical data storage. Photoswitching in RSFPs typically results from chromophore cis-trans isomerization accompanied by a protonation change, but other switching schemes based on, e.g., chromophore hydration/dehydration have also been discovered. In this chapter, we review the main structural features at the basis of photoswitching in RSFPs

Effect of computational methodology on the conformational dynamics of the protein photosensor LOV1 from Chlamydomonas reinhardtii

LOV domains are the light-sensitive protein domains of plant phototropins and bacteria. They photochemically form a covalent bond between a flavin mononucleotide (FMN) chromophore and a cysteine, attached to the apo-protein, upon irradiation with blue light, which triggers a signal in the adjacent kinase. Although their signaling state has been well characterized through experimental means, their signal transduction pathway as well as dark-state activity are generally only poorly understood. Here we show results from molecular dynamics simulations where we investigated the effect of thermostating and long-range electrostatics on the solution structure and dynamical behavior of the wild-type LOV1 domain from the green algae Chlamydomonas reinhardtii in the dark. We demonstrate that these computational issues can dramatically affect the conformational fluctuations of such protein domains by suppressing configurations far from equilibrium or destabilizing local configurations, leading to artificial changes of the protein secondary structure as well as the H-bond network formed by the amino acids and the FMN. By comparing our calculation results with recent experimental data, we show that the non-invasive thermostating strategy, where the protein solute is only indirectly coupled to the thermostat via the solvent, in conjunction with the particle-mesh Ewald technique, provides dark-state conformers, which are in consistency with experimental observations. Moreover, our calculations indicate that the LOV1 domains can alter the intersystem crossing rate and rate of adduct formation by adjusting the population distribution of these dark-state conformers. This might permit them to function as a modulator of the signal intensity under low light conditions

Fluorescence lifetime of fluorescent proteins

Jung G, Brockhinke A, Gensch T, Hötzer B, Schwedler S, Veettil SK. Fluorescence lifetime of fluorescent proteins. In: Jung G, ed. Fluorescent Proteins I. From Understanding to Design. Springer Series on Fluorescence 11. Berlin, Heidelberg: Springer Verlag; 2012: 69-97