Table 1

Focus areas for fluorescent protein (FP) optimization

FP property

Examples for relevance

Reference


Reduce or increase sensitivity to environmental changes (for example, pH, ionic conditions)

Reduce sensitivity to gain specificity of response as in metabolite sensors; or increase as in high pH sensitivity of Keima tracer for compartmentalization and membrane topology

[a]


Monomer versus oligomerization

Especially important for environments in which diffusion is constrained like membranes

[b-e]


Folding time

Important for time-sensitive expression studies and some cell biology applications like protein trafficking

[f]


Brightness (quantum yield and extinction coefficient)

Generally, brighter FPs are important for getting signal above high background or when imaging biomolecules or processes that have low abundance (for example, when measuring expression from weak promoters)

[g-i]


Absorption and emission spectra (color change, stokes shift, narrowing of spectra)

Important for avoiding autofluorescence, multicolor imaging and/or specialized imaging modalities such as multiphoton and Stimulated emission depletion microscopy (STED)

[a, j, k-p]


Monoexponential decay

Important for Fluorescence-lifetime imaging microscopy (FLIM)

[g, q]


Photoswitching, photoactivation and photoconversion

Important for monitoring dynamics of proteins and protein populations

[r, s]


Photostability

Important for extended, high-resolution and/or single molecule imaging

[e, t, u]


Footnote: references for the Table:

a. Tantama M, Hung YP, Yellen G: Imaging intracellular pH in live cells with a genetically encoded red fluorescent protein sensor. J Am Chem Soc 2011, 133:10034-10037.

b. Baird GS, Zacharias DA, Tsien RY: Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A 2000, 97:11984-11989.

c. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY: A monomeric red fluorescent protein. Proc Natl Acad Sci U S A 2002, 99:7877-7882.

d. Espagne A, Erard M, Madiona K, Derrien V, Jonasson G, Levy B, Pasquier H, Melki R, Merola F: Cyan fluorescent protein carries a constitutive mutation that prevents its dimerization. Biochemistry 2011, 50:437-439.

e. Shu X, Lev-Ram V, Olson ES, Aguilera TA, Jiang T, Whitney M, Crisp JL, Steinbach P, Deerinck T, Ellisman MH, Ellies LG, Nguyen QT, Tsien RY: Spiers Memorial Lecture. Breeding and building molecular spies. Faraday Discuss 2011, 149:9; discussion 63-77.

f. Andrews BT, Gosavi S, Finke JM, Onuchic JN, Jennings PA: The dual-basin landscape in GFP folding. Proc Natl Acad Sci U S A 2008, 105:12283-12288.

g. Subach OM, Cranfill PJ, Davidson MW, Verkhusha VV: An enhanced monomeric blue fluorescent protein with the high chemical stability of the chromophore. PLoS One 2011, 6:e28674.

h. Hunt ME, Scherrer MP, Ferrari FD, Matz MV: Very bright green fluorescent proteins from the Pontellid copepod Pontella mimocerami. PLoS One 2010, 5:e11517.

i. Goedhart J, von Stetten D, Noirclerc-Savoye M, Lelimousin M, Joosen L, Hink MA, van Weeren L, Gadella TW Jr, Royant A: Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat Commun 2012, 3:751.

j. Olenych SG, Claxton NS, Ottenberg GK, Davidson MW: The fluorescent protein color palette. In Current Protocols in Cell Biology. 2008/01/30 edition. Edited by Bonifacino JS, Dasso M, Harford JB, Lippincott-Schwartz J, Yamada KM. Hoboken, NJ: John Wiley & Sons, Inc.; 2007: 21.25.21-21.25.34.

k. Day RN, Davidson MW: Fluorescent proteins for FRET microscopy: Monitoring protein interactions in living cells. Bioessays 2012, 34:341-350.

l. Mathur J: The illuminated plant cell. Trends Plant Sci 2007, 12:506-513.

m. Shcherbo D, Murphy CS, Ermakova GV, Solovieva EA, Chepurnykh TV, Shcheglov AS, Verkhusha VV, Pletnev VZ, Hazelwood KL, Roche PM, Lukyanov S, Zaraisky AG, Davidson MW, Chudakov DM: Far-red fluorescent tags for protein imaging in living tissues. Biochem J 2009, 418:567-574.

n. Zhao Y, Araki S, Wu J, Teramoto T, Chang YF, Nakano M, Abdelfattah AS, Fujiwara M, Ishihara T, Nagai T, Campbell RE: An expanded palette of genetically encoded Ca(2) indicators. Science 2011, 333:1888-1891.

o. Shcherbakova DM, Hink M, Joosen L, Gadella TW, Verkhusha VV: An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging. J Am Chem Soc 2012 [Epub ahead of print].

p. Filonov GS, Piatkevich KD, Ting LM, Zhang J, Kim K, Verkhusha VV: Bright and stable near-infrared fluorescent protein for in vivo imaging. Nat Biotechnol 2011, 29:757-761.

q. Ha T, Tinnefeld P: Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. Annu Rev Phys Chem 2012, 63:595-617.

r. Markwardt ML, Kremers GJ, Kraft CA, Ray K, Cranfill PJ, Wilson KA, Day RN, Wachter RM, Davidson MW, Rizzo MA: An improved cerulean fluorescent protein with enhanced brightness and reduced reversible photoswitching. PLoS One 2011, 6:e17896.

s. Mathur J, Griffiths S, Barton K, Schattat MH: Green-to-red photoconvertible mEosFP-aided live imaging in plants. Methods Enzymol 2012, 504:163-181.

t. Bogdanov AM, Bogdanova EA, Chudakov DM, Gorodnicheva TV, Lukyanov S, Lukyanov KA: Cell culture medium affects GFP photostability: a solution. Nat Methods 2009, 6:859-860.

u. Shaner NC, Lin MZ, McKeown MR, Steinbach PA, Hazelwood KL, Davidson MW, Tsien RY: Improving the photostability of bright monomeric orange and red fluorescent proteins. Nat Methods 2008, 5:545-551.

Jones et al. BMC Biology 2012 10:39   doi:10.1186/1741-7007-10-39

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