All Publications


  • Split Green Fluorescent Proteins: Scope, Limitations, and Outlook. Annual review of biophysics Romei, M. G., Boxer, S. G. 2019

    Abstract

    Many proteins can be split into fragments that spontaneously reassemble, without covalent linkage, into a functional protein. For split green fluorescent proteins (GFPs), fragment reassembly leads to a fluorescent readout, which has been widely used to investigate protein-protein interactions. We review the scope and limitations of this approach as well as other diverse applications of split GFPs as versatile sensors, molecular glues, optogenetic tools, and platforms for photophysical studies. Expected final online publication date for the Annual Review of Biophysics Volume 48 is May 3, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

    View details for PubMedID 30786230

  • Split Green Fluorescent Proteins: Scope, Limitations, and Outlook ANNUAL REVIEW OF BIOPHYSICS, VOL 48 Romei, M. G., Boxer, S. G., Dill, K. A. 2019; 48: 19–44
  • A unified model for photophysical and electro-optical properties of Green Fluorescent Proteins. Journal of the American Chemical Society Lin, C. Y., Romei, M. G., Oltrogge, L. M., Mathews, I. I., Boxer, S. G. 2019

    Abstract

    Green fluorescent proteins (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all the observed strong correlations among photophysical properties; related subtopics are extensively discussed in Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue this model should also be generally applicable to both biological and non-biological polymethine dyes.

    View details for DOI 10.1021/jacs.9b07152

    View details for PubMedID 31450887

  • Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins. Journal of the American Chemical Society Chang, J., Romei, M. G., Boxer, S. G. 2019

    Abstract

    Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of cis and trans rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the trans state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas, in a tighter packing (7% smaller unit cell size), the hula-twist occurs.

    View details for DOI 10.1021/jacs.9b08356

    View details for PubMedID 31533429

  • Photoactive Split Green Fluorescent Protein: Engineering a New Optogenetic and Imaging System Romei, M. G., Longwell, C. K., Cochran, J. R., Boxer, S. G. CELL PRESS. 2018: 177A–178A