1. Structural Analysis of Protein Structure
Circular Dicroism
Fluorescence

2. 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.
Reasonable estimates of protein secondary structure content and structure change can be determined empirically through the use of
Circular dichroism (CD) spectroscopy
Fluorescence spectroscopy
Nuclear Magnetic Resonance (NMR) spectroscopy
FT-infrared spectroscopy

3. Circular Dichroism
Circular dichroism (CD) spectroscopy is a form of light absorption spectroscopy that measures the difference in absorbance of right- and left-circularly polarized light (rather than the commonly used absorbance of isotropic light) by a substance.
It is measured with a CD spectropolarimeter. The instrument needs to be able to measure accurately in the far UV at wavelengths down to nm ( nm).
The difference in left and right handed absorbance A(l)- A(r) is very small (usually in the range of ) corresponding to an ellipticity of a few 1/100th of a degree.

4. Rotation of Plane-polarized Light by an Optically Active Sample
Pockels cell produces a beam that is alternately switched between L and R. The beam then passes through the sample to a photomultiplier. The detected signal can then be processed as ΔA vs λ.

5. Instrumentation
The most common instruments around are the currently produced JASCO, JobinYvon, OLIS, and AVIV models.
We have the Jasco 710 and 810 models with temperature controllers. The air cooled 150W Xenon lamp does not necessitate water cooling.
You still need to purge with ample nitrogen to get to lower wavelengths (below 190 nm).

6. Typical Initial Concentrations
Protein Concentration: 0.5 mg/ml (The protein concentration needs to be adjusted to produce the best data).
Cell Path Length: mm. If absorption poses a problem, cells with shorter path (0.1 mm) and a correspondingly increased protein concentration and longer scan time can be employed.
Stabilizers (Metal ions, etc.): minimum
Buffer Concentration: 5 mM or as low as possible, while maintaining protein stability. A typical buffer used in CD experiments is 10 mM phosphate, although low concentrations of Tris, perchlorate or borate is also acceptable.
As a general rule of thumb, one requires that the total absorbance of the cell, buffer, and protein be between 0.4 and 1.0 (theoretically, 0.87 is optimal).
A spectra for secondary structure determination ( nm) will require minutes to record (plus an equivalent amount of time for a baseline as every CD spectrometer.

7. Sample Preparation and Measurement
Additives, buffers and stabilizing compounds: Any compound, which absorbs in the region of interest, ( nm) should be avoided. A buffer or detergent, imidazole or other chemical should not be used unless it can be shown that the compound in question will not mask the protein signal.
Protein solution: The protein solution should contain only those chemicals necessary to maintain protein stability/solubility, and at the lowest concentrations possible. The protein itself should be as pure as possible, any additional protein will contribute to the CD signal.
Contaminants: Particulate matter (scattering particles), anything that adds significant noise (or artificial signal contributions) to the CD spectrum must be avoided. Filtering of the solutions (0.02 m syringe filters) may improve signal to noise ratio.
Data collection: Initial experiments are useful to establish the best conditions for the "real" experiment. Cells of mm path length offer a good starting point.

8. CD Data Analysis
The difference in absorption to be measured is very small. The differential absorption is usually a few 1/100ths to a few 1/10th of a percent, but it can be determined quite accurately. The raw data plotted on the chart recorder represent the ellipticity of the sample in radians, which can be easily converted into degrees
(radians)
(degrees)

9. CD Data Analysis
To be able to compare these ellipticity values we need to convert into a normalized value. The unit most commonly used in protein and peptide work is the mean molar ellipticity per residue. We need to consider path length l, concentration c, molecular weight M and the number of residues.
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

10. CD Data Analysis
The molar ellipticity [] is related to the difference in extinction coefficients
[] = 3298 Δε.
Here [] has the standard units of degrees cm2 dmol -1
The molar ellipticity has the units degrees deciliters mol-1 decimeter-1.

11. Circular Dichroism of Proteins
It has been shown that CD spectra between 260 and approximately 180 nm can be analyzed for the different secondary structural types: alpha helix, parallel and anti-parallel beta sheets, turns, and other.
A number of excellent review articles are available describing the technique and its application (Woody, 1985 and Johnson, 1990).
Modern secondary structure determination by CD are reported to achieve accuracies of 0.97 for helices, 0.75 for beta sheet, 0.50 for turns, and 0.89 for other structure types (Manavalan & Johnson, 1987).

12. CD Signal of Proteins
For proteins we will be mainly concerned with absorption in the ultraviolet region of the spectrum from the peptide bonds (symmetric chromophores) and amino acid sidechains in proteins.
Protein chromophores can be divided into three classes: the peptide bond, the amino acid sidechains, and any prosthetic groups.
The lowest energy transition in the peptide chromophore is an n → p* transition observed at nm with very weak intensity (emax~100).
----p* p → p* ~`190 nm emax~7000
----n n → p* , nm emax~100
----p

14. 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.
The n → p* transition appears in the a-helical form of the polymer as a small shoulder near 220 nm on the tail of a much stronger absorption band centered at 190 nm. This intense band, responsible for the majority of the peptide bond absorbance, is a
p → p* transition (emax ~ 7000).
Using CD, these different transitions are more clearly evident. Exciton splitting of the p → p* transition results in the negative band at 208 and positive band at 192 nm.

15. CD Spectra of Proteins
Different secondary structures of peptide bonds have different relative intensity of n → p* transitions, resulting in different CD spectra at far UV region ( nm).
CD is very sensitive to the change in secondary structures of proteins. CD is commonly used in monitoring the conformational change of proteins.
The CD spectrum is additive. The amplitude of CD curve is a measure of the degree of asymmetry.
The helical content in peptides and proteins can be estimated using CD signal at 222 nm
e222= 33,000 degrees cm2 dmol -1 res-1
Several curve fitting algorithms can be used to deconvolute relative secondary structures of proteins using the CD spectra of proteins with known structures.

16. Protein CD Signal
The three aromatic side chains that occur in proteins (phenyl group of Phe, phenolic group of Tyr, and indole group of Trp) also have absorption bands in the ultraviolet spectrum. However, in proteins, the contributions to the CD spectra in the far UV (where secondary structural information is located) is usually negligible. Aromatic residues, if unusually abundant, can have significant effects on the CD spectra in the region < 230 nm, complicating analysis.
The disulfide group is an inherently asymmetric chromophore as it prefers a gauche conformation with a broad CD absorption around 250 nm.

17. Far UV CD Spectra of Proteins
[] x10-3 degrees cm2 dmol -1

18. CD Spectra of Protein
Each of the three basic secondary structures of a polypeptide chain (helix, sheet, coil) show a characteristic CD spectrum. A protein consisting of these elements should therefore display a spectrum that can be deconvoluted into the three individual contributions.

19. CD Spectra Fit
In a first approximation, a CD spectrum of a protein or polypeptide can be treated as a sum of three components: a-helical, b-sheet, and random coil contributions to the spectrum.
At each wavelength, the ellipticity (θ) of the spectrum will contain a linear combination of these components:
(1)
θT is the total measured ellipticity, θh the contribution from helix, θs for sheet, θc for coil, and the corresponding χ the fraction of this contribution.

20. CD Spectra Fit
As we have three unknowns in this equation, a measurement at 3 points (different wavelengths) would suffice to solve the problem for χ, the fraction of each contribution to the total measured signal.
We usually have many more data points available from our measurement (e.g., a whole CD spectrum, sampled at 1 nm intervals from 190 to 250 nm). In this case, we can try to minimize the total deviation between all data points and calculated model values. This is done by a minimization of the sum of residuals squared (s.r.s.), which looks as follows in our case :

21. DICHROWEB

22. Conformational Change of CD2
6M GuHCl
25 ºC
85 ºC

23. CD2 Becomes Significantly Helical in TFE

24. Melting point measurement of EGFP-wt with its CD spectra at different temperaturesB A
Tm = 65.7 °C

25. Fluorescence spectrum of proteins

27. Electronic mechanism of fluorescence
Ground singlet state
1st exited singlet state
Absorption
fluorescence
Phosphorescence
hvex
hvem
Intersystem crossing
Triplet state
vex > vem or
lex < lem
Vibration levels

28. Fluorescence measurement
Xe lamp
Fluorescence measurement

29. lex (nm)
lem (nm)
e (M-1 cm-1) Y
Trp (W)
280
348
5600
0.2
Tyr (Y)
274
303
1490
0.1
Phe (F)
257
282
240
0.04

30. Trp Fluorescence Emission Spectra of CD2 under Different Conditions
In a hydrophobic environment (inside of a folded protein), Trp emission occurs at shorter wavelength. When it is exposed to solvent, its emission is very similar to that of the free Trp amino acid (red shift occurs).
Trp
25ºC
6M GuHCl
85ºC

31. Summary of fluorescence
Fluorescence is the emission of radiation that occurs when a molecule in an excited electronic state returns to the ground state.
Application: Fluorescence has an important role in the structural determinants of proteins, DNA or RNA, etc.
Advantages:
– 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
Disadvantages:
– Large Stoke’s Shift
– Background fluorescence (Impurities in buffers and autofluorescence in cells)
– Scattered light (problem with cloudy samples)