Fluorescent protein. Origin of fluorescent proteins. Structural and biophysical analysis of FPs. Application in molecular biology and biotechnology

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1 Origin of fluorescent proteins types of FPs mechanism of fluorescence Fluorescent protein Structural and biophysical analysis of FPs Application in molecular biology and biotechnology

2 Variety of organisms in nature generate and use light for survival, mating, signaling, etc bioluminescence in firefly, anglerfish, Vibrio harveyi (bacteria) Some proteins found in nature are intrinsically fluorescent no cofactor required need to synthesize chromophore Green fluorescent protein (GFP) from A. victoria is widely used in biology and biotechnology for tracking and visualization label protein in vivo by creating a chimera Aquorea victoria localization experiment high throughput assay based on fluorescence energy resonance transfer (FRET)

3 Make nice drawings Application of FP General requirements: express well, non-toxic, bright, photostable, monomeric, orthogonal colors, insensitive to environmental parameters San Diego beach painted with bacteria transformed with eight different fluorescent proteins--shaner (Tsien lab)

4 In vivo labeling of protein

5 Engineered FP Biophysical properties of fluorescent proteins require optimization extinction coefficient, quantum yield, folding kinetics and efficiency, brightness, bleaching, quaternary structure, spectral shape, etc multicolor imagine experiments require tagging different proteins simultaneously with distinct colors white light mhoneydew mbanana morange tdtomato mtangerin mstrawberry mcherry fluorescent light Shaner et al, Nat Biotech 22, 1567 (2004)

6 Mechanism of fluorescence GFP comprises mainly antiparallel beta strands Forms a barrel with an alpha helix in the middle Roughly ~230 amino acids SYG on central helix rearranges to form a chromophore p-hydroxybenzylidene-imidazolidinone Heim and Tsien, PNAS 91, (1994)

7 Different chromophore results in different color In DsRed from the Discosoma genus, QYG rearrange to form a chromophore Yarborough et al, PNAS 98, 462 (2001) Obligate tetramer Slow maturation time > 24 hrs

8 Folding kinetics Introduce random mutation by error-prone PCR Low cost high through screen slide projector with a bandpass filter ( nm) goggles covered with a Kodak filter for > 550 nm pulse chase by inducing with IPTG for 30 min and adding protein synthesis inhibitor Nine point mutant R2A, K5E, N6D, T21S, H41T, N42Q, V44A, A145P, T217A normalized fluorescence

9 Chromophore Interaction of a chromophore with light is quantitatively described by its absorption and emission spectra wavelength (nm)

10 FRET Fluorescence Resonance Energy Transfer If the emission spectrum of one chromophore overlaps with the absorption spectrum of another, there may be a distance dependent energy transfer between the two chromophores

11 FRET may be serve as a nano-scale yardstick

12 Designing Ca++ detector Calmodulin (CaM) is a naturally occurring signaling molecule that undergoes a large conformational change to bind peptide ligand in the presence of Ca++ Attach a FRET donor-acceptor pair at either end of CaM Miyawaki et al, Nature 388, 882 (1997)

13 Improved FRET pairs Absorption and emission spectra, and other biophysical properties, of fluorescent proteins are not always optimized for FRET studies commonly used FRET pair: CFP YFP limited dynamic range (several folds) overlap between the donor and acceptor emission spectra sensitivity Construct mutant ECFP and YFP libraries by a combination of error-prone PCR, site-directed mutagenesis, and DNA shuffling Optimize each protein (CFP or YFP) separately Express library in E.coli and enrich by FACS Nguyen and Daugherty, NSB 23, 355 (2005)

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15 Structural complementation assay Protein misfolding underlies many genetic diseases (e.g. cystic fibrosis) Heterologously expressed recombinant proteins often have limited stability and solubility High throughput screen of mutants can help identify optimize the sequence to avoid aggregation Structural complementation assay relies on reconstituting activity by coexpressing two constituent parts of a protein Preferably if the fragment attached to the protein of interest ( P ) is small in order to avoid perturbing the inherent biophysical properties of the protein Readout needs to be amenable to high throughput assay colorimetric or fluorescence-based Wigley et al, Nat Biotech 19, 101 (2001)

16 Fragmented GFP Developing a structural complementation assay with GFP requires splitting the protein into two soluble complementary pieces DNA shuffling was used to optimize the stability of GFP S30R, Y39N, N105T, Y145F, I171V, A206V Coexpression of strands 1 10 and strand 11 resulted in functional GFP Cabantous et al, NSB 23, 102 (2005)

17 soluble insoluble