Structural Analysis of Protein Structure. Circular Dicroism Fluorescence. Methods for Secondary Structural Analysis. A number of experimental techniques can selectively examine certain general aspects of macromolecular structure with relatively little investment of time and sample.
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
Circular dichroism (CD) spectroscopy
Nuclear Magnetic Resonance (NMR) spectroscopy
in proper units (CD spectroscopists use decimol)
which finally reduces to
The values for mean molar ellipticity
per residue are usually in the 10,000's
 = 3298 Δε.
----p* p → p* ~`190 nm emax~7000
----n n →p* 208-210, 191-193 nm emax~100
p→p* transition (emax ~ 7000).
e222= 33,000 degrees cm2 dmol -1 res-1
 x10-3 degrees cm2 dmol -1
CD2 is a cell adhesion molecules.
Domain 1 of CD2 has a IgG fold. Nine b-strands form a beta-sandwich structure.
Two Trp residues, W-7 and W-32 (green) are located at the exposed and buried region of the protein, respectively.
Our lab has used CD2 as a model system to understand conformation flexibility of proteins
If [protein] = 10 mM and cell width = 1 mm,
Mean residue ellipticity (q) = CD data x 1.0x105/residue number
Residue number of EGFP-wt: 38 + 239.
Tm = 65.7 °C
Relative changes due to influence of environment on sample (pH, denaturants, temperature, etc.) can be monitored accurately.
Only very dilute, non-absorbing buffers allow measurements below 200 nm
Absolute measurements subject to a number of experimental errors
Average accuracy of fits about +/- 10%
CD spectropolarimeter is relatively expensive
vex > vem
lex < lem
Ground singlet state
Electronic mechanism of fluorescence
85ºCTrp Fluorescence Emission Spectra of CD2 under Different Conditions
λex = 254 nm
λex = 277 nm
[P] = 1 mM
25 mM PIPES
100 mM KCl
lex = 278 nm
FRET fluorescence spectra between EMOC-N85 and Tb3+ (A) and its curve fitting (B) of a Tb3+ titration. The interaction between Tb3+ and EMOC-N85 variants was indicated with different Tb3+-binding affinities (Kd) 12.1 mM (EMOC-N85): 8.2 mM (EMOC-N85m1); 29.2 mM (EMOC-N85m2), respectively.
A. Ribbon diagram of the WT GFP structure taken from Proc. Natl. Acad. Sci. USA, (1997), 94, 2306-2311. The a-helices are shown in red, the b-strands are shown in green, and the chromophore is shown as a ball-and-stick model.
B. An example of GFP in the natural environment of jellyfish in the ocean, taken from http://www.plantscicam.ac.uk/haseloff /GFP/GFPbackgrnd.html/
EGFP-based Ca2+ sensors with different Ca2+ affinities in the range of 0.1 mM - 5 mM can be developed by creating a Ca2+ binding site into the chromophore sensitive locations of fluorescent proteins.
Spectroscopic characterization of Ca2+ sensor Ca-G1-37. (A) Visible absorption spectrum for sensor Ca-G1-37 with increasing Ca2+ concentrations. Ca2+ dependence of fluorescence emission spectra with excitation of lex = 398 nm (B) and lex = 490 nm (C). Symbols of different Ca2+ concentrations in (B) and (C) are same as that in (A). The measurements were performed at 17 mM Ca-G1-37 for visible absorption and 1.7 mM Ca-G1-37 for fluorescence experiments with 10 mM Tris and 1 mM DTT (pH 7.4), respectively. The arrows indicate the direction of signal change resulting from an increase in the Ca2+ concentration. (D) Normalized F(398nm)/F(490nm) ratio curve-fitting of the Ca2+ titration data.
Ex 398 nm
Ex 490 nm
– Small sample volumes (800μL – 3mL)
– Low concentration (0.1 – 5 mM)
– Short experiment time (10-60 minute)
– Short data analysis time (5-30 minute)
– Recovery of sample
– Large Stoke’s Shift
– Background fluorescence (Impurities in buffers and autofluorescence in cells)
– Scattered light (problem with cloudy samples)