Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging
Reads0
Chats0
TLDR
The engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral is reported, named mTFP1 [monomeric TFP1 (teal FP 1], one of the brightest and most photostable FPs reported to date.Abstract:
The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.read more
Citations
More filters
Journal ArticleDOI
Fluorescence Lifetime Measurements and Biological Imaging
TL;DR: The lifetime of a photophysical process is the time required by a population of N electronically excited molecules to be reduced by a factor of e via the loss of energy through fluorescence and other non-radiative processes and the average length of time τ is called the mean lifetime, or simply lifetime.
Journal ArticleDOI
Fluorescent Proteins and Their Applications in Imaging Living Cells and Tissues
TL;DR: The structure, evolution, and function of GFP-like proteins and their numerous applications for in vivo imaging are focused on, with particular attention to recent techniques.
Journal ArticleDOI
Improving the photostability of bright monomeric orange and red fluorescent proteins.
Nathan C. Shaner,Michael Z. Lin,Michael R. McKeown,Paul Steinbach,Kristin L. Hazelwood,Michael W. Davidson,Roger Y. Tsien +6 more
TL;DR: This work developed highly photostable variants of mOrange and TagRFP that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.
Journal ArticleDOI
Fluorescent protein FRET: the good, the bad and the ugly
David W. Piston,Gert-Jan Kremers +1 more
TL;DR: The physical basis of FRET and the fluorescent proteins appropriate for these experiments are described and the approaches that can be used to measure FRET are reviewed, with particular emphasis on the potential artifacts associated with each approach.
Journal ArticleDOI
Advances in fluorescent protein technology
TL;DR: The relentless search for a bright, monomeric and fast-maturing red FP has yielded a host of excellent candidates, although none is yet optimal for all applications, and photoactivatable FPs are emerging as a powerful class of probes for intracellular dynamics and as useful tools for the development of superresolution microscopy applications.
References
More filters
Book ChapterDOI
Processing of X-ray diffraction data collected in oscillation mode
Zbyszek Otwinowski,Wladek Minor +1 more
TL;DR: The methods presented in the chapter have been applied to solve a large variety of problems, from inorganic molecules with 5 A unit cell to rotavirus of 700 A diameters crystallized in 700 × 1000 × 1400 A cell.
Journal ArticleDOI
Coot: model-building tools for molecular graphics.
Paul Emsley,Kevin Cowtan +1 more
TL;DR: CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for X-ray structure solution, structure comparison and analysis, and publication-quality graphics.
Journal ArticleDOI
PROCHECK: a program to check the stereochemical quality of protein structures
TL;DR: The PROCHECK suite of programs as mentioned in this paper provides a detailed check on the stereochemistry of a protein structure and provides an assessment of the overall quality of the structure as compared with well refined structures of the same resolution.
Journal ArticleDOI
MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures
TL;DR: The MOLSCRIPT program as discussed by the authors produces plots of protein structures using several different kinds of representations, including simple wire models, ball-and-stick models, CPK models and text labels.
Journal ArticleDOI
NMRPipe: a multidimensional spectral processing system based on UNIX pipes
TL;DR: The asynchronous pipeline scheme provides other substantial advantages, including high flexibility, favorable processing speeds, choice of both all-in-memory and disk-bound processing, easy adaptation to different data formats, simpler software development and maintenance, and the ability to distribute processing tasks on multi-CPU computers and computer networks.