@article {49455, title = {moxMaple3: a photoswitchable fluorescent protein for PALM and protein highlighting in oxidizing cellular environments.}, journal = {Scientific Reports}, volume = {8}, year = {2018}, month = {2018 Oct 03}, pages = {14738}, abstract = {

The ability of fluorescent proteins (FPs) to fold robustly is fundamental to the autocatalytic formation of the chromophore. While the importance of the tertiary protein structure is well appreciated, the impact of individual amino acid mutations for FPs is often not intuitive and requires direct testing. In this study, we describe the engineering of a monomeric photoswitchable FP, moxMaple3, for use in oxidizing cellular environments, especially the eukaryotic secretory pathway. Surprisingly, a point mutation to replace a cysteine substantially improved the yield of correctly folded FP capable of chromophore formation, regardless of cellular environment. The improved folding of moxMaple3 increases the fraction of visibly tagged fusion proteins, as well as FP performance in PALM super-resolution microscopy, and thus makes moxMaple3 a robust monomeric FP choice for PALM and optical highlighting applications.

}, issn = {2045-2322}, doi = {10.1038/s41598-018-32955-5}, author = {Kaberniuk, Andrii A and Mohr, Manuel A and Verkhusha, Vladislav V and Snapp, Erik Lee} } @article {48223, title = {moxDendra2: an inert photoswitchable protein for oxidizing environments.}, journal = {Chemical Communications}, volume = {53}, year = {2017}, month = {2017 Jan 19}, pages = {2106-9}, abstract = {

Fluorescent proteins (FPs) that can be optically highlighted enable PALM super-resolution microscopy and pulse-chase experiments of cellular molecules. Most FPs evolved in cytoplasmic environments either in the original source organism or in the cytoplasm of bacteria during the course of optimization for research applications. Consequently, many FPs may fold incorrectly in the chemically distinct environments in subcellular organelles. Here, we describe the first monomeric photoswitchable (from green to bright red) FP adapted for oxidizing environments.

}, doi = {10.1039/C6CC09997A}, author = {Kaberniuk, Andrii A and Morano, Nicholas C and Verkhusha, Vladislav V and Snapp, Erik Lee} } @article {47628, title = {Photocontrollable fluorescent proteins for superresolution imaging.}, journal = {Annual review of biophysics}, volume = {43}, year = {2014}, month = {2014}, pages = {303-29}, abstract = {

Superresolution fluorescence microscopy permits the study of biological processes at scales small enough to visualize fine subcellular structures that are unresolvable by traditional diffraction-limited light microscopy. Many superresolution techniques, including those applicable to live cell imaging, utilize genetically encoded photocontrollable fluorescent proteins. The fluorescence of these proteins can be controlled by light of specific wavelengths. In this review, we discuss the biochemical and photophysical properties of photocontrollable fluorescent proteins that are relevant to their use in superresolution microscopy. We then describe the recently developed photoactivatable, photoswitchable, and reversibly photoswitchable fluorescent proteins, and we detail their particular usefulness in single-molecule localization-based and nonlinear ensemble-based superresolution techniques. Finally, we discuss recent applications of photocontrollable proteins in superresolution imaging, as well as how these applications help to clarify properties of intracellular structures and processes that are relevant to cell and developmental biology, neuroscience, cancer biology and biomedicine.

}, keywords = {Animals, Cells, Luminescent Proteins, Mammals, Microscopy, Fluorescence}, issn = {1936-1238}, doi = {10.1146/annurev-biophys-051013-022836}, author = {Shcherbakova, Daria M and Sengupta, Prabuddha and Lippincott-Schwartz, Jennifer and Verkhusha, Vladislav V} } @article {45699, title = {Photoswitchable red fluorescent protein with a large stokes shift.}, journal = {Chemistry \& Biology}, volume = {21}, year = {2014}, month = {2014 Oct 23}, pages = {1402-14}, abstract = {

A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622\ nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621\ nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in\ vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us\ to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

}, issn = {1879-1301}, doi = {10.1016/j.chembiol.2014.08.010}, author = {Piatkevich, Kiryl D and English, Brian P and Malashkevich, Vladimir N and Xiao, Hui and Almo, Steven C and Singer, Robert H and Verkhusha, Vladislav V} } @article {47685, title = {Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.}, journal = {Journal of the American Chemical Society}, volume = {132}, year = {2010}, month = {2010 May 12}, pages = {6481-91}, abstract = {

Rapidly emerging techniques of super-resolution single-molecule microscopy of living cells rely on the continued development of genetically encoded photoactivatable fluorescent proteins. On the basis of monomeric TagRFP, we have developed a photoactivatable TagRFP protein that is initially dark but becomes red fluorescent after violet light irradiation. Compared to other monomeric dark-to-red photoactivatable proteins including PAmCherry, PATagRFP has substantially higher molecular brightness, better pH stability, substantially less sensitivity to blue light, and better photostability in both ensemble and single-molecule modes. Spectroscopic analysis suggests that PATagRFP photoactivation is a two-step photochemical process involving sequential one-photon absorbance by two distinct chromophore forms. True monomeric behavior, absence of green fluorescence, and single-molecule performance in live cells make PATagRFP an excellent protein tag for two-color imaging techniques, including conventional diffraction-limited photoactivation microscopy, super-resolution photoactivated localization microscopy (PALM), and single particle tracking PALM (sptPALM) of living cells. Two-color sptPALM imaging was demonstrated using several PATagRFP tagged transmembrane proteins together with PAGFP-tagged clathrin light chain. Analysis of the resulting sptPALM images revealed that single-molecule transmembrane proteins, which are internalized into a cell via endocytosis, colocalize in space and time with plasma membrane domains enriched in clathrin light-chain molecules.

}, keywords = {Amino Acid Sequence, Animals, Cell Survival, Cercopithecus aethiops, Color, COS Cells, Hela Cells, Humans, Light, Luminescent Proteins, Membrane Proteins, Microscopy, Molecular Imaging, Molecular Sequence Data, Mutagenesis, Photochemical Processes, Temperature}, issn = {1520-5126}, doi = {10.1021/ja100906g}, author = {Subach, Fedor V and Patterson, George H and Renz, Malte and Lippincott-Schwartz, Jennifer and Verkhusha, Vladislav V} }