Biological Fluorescence Subgroup
Biological Fluorescence Subgroup Meeting: March 5th
1:00 PM Keith Weninger, NC State Synaptic protein conformations reveled with single molecule fluorescence
SNARE Proteins, involved in vesicle trapping at the target membrane, smFRET reveals dynamics and structure
Single molecule FRET slide E = 1/[1+(r/5nm)6] = Red/(Red+Green)
Total internal reflectance --membrane on glass slide with SNARE protein embedded at surface of membrane
Syntaxin open closed transitions, PDB3C98 shows a four helix bundle that can unwind to have low FRET (open complex) or high FRET (closed complex). 16% open structure in wild type and 59% open in Interface mutant L165A.
with other protein
+ Munc18, all closed, important in neuroscience but not much known. Last 19 amino acids of SNARE that must snap open that is N terminus, but important for stabilizing Munc.
+ SNAP-25, all closed --- now donor acceptor pair put on SNAP-25, intrinsically disordered protein...further apart along contour, average FRET goes down, but if a complex formed, you can watch FRET go up or down depending on secondary and tertiary structure, this can be done in vivo in cells and watch trapped SNAP-25, dynamic, stochastic switching. Two regions of the SNAP switch between 2 helix and 3 helix state
+ final peptide to form a ternary SNARE complex, crystal solution known (not very interesting
+ SNARE + complexin a smaller molecules/proteins ---
+ SNARE + Synaptotagmin (no crystal structure)....made six mutants at many sites to add dyes, simulate dype position, empirical R0, correct for quantum and detection efficiencies, 34 FRET derived distances
1:30 Ashok Deniz, Scripps Research Institute Multicolor Single Molecule FRET: a novel tool to probe biological folding and assembly
Complexity in the folded state, the case of Rop, homodimeric 4 helix bundle, modulates plasmid copy #, wt is in anti geometry which is active, but can be switched over to syn geometry w. mutants which are less active (FRET changes from little to lots), dual minimum energy landscape. A2L2; A2I2 How does it switch from syn to anti
freely diffusing in solution avoids surface artifacts (confocal detection), 3 color smFRET - w 2 channel detection, a simple experiment, Alexa 488-Alexa 647 (larger Ro) and Alexa 499 - Alexa594 (better overlap - smaller R0, more FRET). Evidence that switching from syn to anti occurs.
Three color smFRET with 3 channel detection, so can look at multiple sites, on RNA, different structures with protein binding to RNA.
Three Challenges and Emerging Solutions
1. Dye Photobleaching, increase # of dyes, more problematic, improving dye photostability by microfluidic device with gas exchanger (JACS 2009 13610) ---- gets rid of oxygen
2. getting good donor and acceptor in absence of each other
3. Dissociation of Complexes, need to work at low complexes, at <100 nM Kd, low level of complexed matrial at 1 nM (Nature Methods 2011 8:239)
2:00 History of Resolution in Optical Spectroscopy, Andreas Schonle
a long history...eye, 750 bc lens were made (Greeks and Romans) 1000, glasses; 1590ability to resolve small things
eye: 20 cm distance, 100 um resolution,
one lens magnifiers, 30 um;
two lens, twice as much; magnify 30x eye
1: 1600s: Leewnhoek aberration limited spectroscopy; have a microsope
(microbiology) but 2 microns (aberration limited), red blood cells, sperm cells, bacteria, magnification so large it was limited by chromatic aberrations
2: late 1800s Zeiss, Abbe, Schott Diffraction Limited Spectroscopy, understand your microscope
Team: Zeiss -->vision, Abbe -->industrializing lens, Schott -->produced glasses ~ 200 nm
one step for person rather than one lens per person, , aperture smaller but instead of making this better, it was smaller because of diffraction. delta x = lambda/2nsinalpha, homogeneous oil immersion,
confocal is a big deal in 1957, but still diffraction limited, d>lambda; so decrease wavelength? so UV and electron microscope (but really just still diffraction limited
scanning probe microsope Synge 1928) ---1980s near field scanning microscopes really developed
3. Breaking the diffraction barrier: diffraction limit (below 200 nm cannot be resolved), if inhibit fluorescence of one group from within a larger subset of fluorophores, then you can see a subpopulation, fluorescence inhibition STED microsope, stimulated emission within sample
4. another seminal transition ahead??? near-sighted superlens????