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.
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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
Comparison of the UV absorbance (left) and the circular dichroism (right) of poly-L-lysine in different secondary structure conformations as a function of pH.
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
CD data is converted into the unit of mean residue ellipticity before parameter estimation with the program
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.
Melting point measurement of EGFP-wt with its CD spectra at different temperatures
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
Fluorescence spectrum of proteins different temperatures
1 different temperaturesst exited singlet state
vex > vem
lex < lem
Ground singlet state
Electronic mechanism of fluorescence
Fluorescence measurement different temperatures
Trp different temperatures
85ºCTrp Fluorescence Emission Spectra of CD2 under Different Conditions
Interaction between calmodulin and Ca different temperatures2+
λex = 254 nm
λex = 277 nm
Fluorescence resonance energy transfer (FRET) between EMOC-N85 and Tb3+
[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.
GFP becomes one of the most popular and exciting new technologies in biochemistry and cell biology
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 Ca technologies in biochemistry and cell biology2+ sensors with different Ca2+-binding affinities are developed by two different approaches
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.
Ca technologies in biochemistry and cell biology2+ binding response
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)