You are on page 1of 6

Rotation of Plane-polarized Light by

an Optically Active Sample


Structural Analysis of Protein
Structure

Circular Dicroism
Fluorescence

• 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.

Instrumentation
Methods for Secondary Structural Analysis
• A number of experimental techniques can selectively
examine certain general aspects of macromolecular • The most common instruments around are the
structure with relatively little investment of time and currently produced JASCO, JobinYvon, OLIS,
sample. and AVIV models.
• Reasonable estimates of protein secondary structure
content and structure change can be determined • We have the Jasco 710 and 810 models with
empirically through the use of temperature controllers. The air cooled 150W
Circular dichroism (CD) spectroscopy Xenon lamp does not necessitate water cooling.
Fluorescence spectroscopy • You still need to purge with ample nitrogen to get
Nuclear Magnetic Resonance (NMR) spectroscopy to lower wavelengths (below 190 nm).
FT-infrared spectroscopy

Typical Initial Concentrations


Circular Dichroism • Protein Concentration: 0.5 mg/ml (The protein concentration
needs to be adjusted to produce the best data).
• Cell Path Length: 0.5-1.0 mm. If absorption poses a problem,
• Circular dichroism (CD) spectroscopy is a form of light cells with shorter path (0.1 mm) and a correspondingly increased
absorption spectroscopy that measures the difference in protein concentration and longer scan time can be employed.
absorbance of right- and left-circularly polarized light (rather • Stabilizers (Metal ions, etc.): minimum
than the commonly used absorbance of isotropic light) by a • Buffer Concentration: 5 mM or as low as possible, while
substance. maintaining protein stability. A typical buffer used in CD
• It is measured with a CD spectropolarimeter. The instrument experiments is 10 mM phosphate, although low concentrations
needs to be able to measure accurately in the far UV at of Tris, perchlorate or borate is also acceptable.
wavelengths down to 190 - 170 nm (170 - 260 nm). • As a general rule of thumb, one requires that the total
• The difference in left and right handed absorbance A(l)- A(r) absorbance of the cell, buffer, and protein be between 0.4 and
1.0 (theoretically, 0.87 is optimal).
is very small (usually in the range of 0.0001) corresponding to • A spectra for secondary structure determination (260 - 178 nm)
an ellipticity of a few 1/100th of a degree. will require 30-60 minutes to record (plus an equivalent amount
of time for a baseline as every CD spectrometer.

1
Sample Preparation and Measurement
• Additives, buffers and stabilizing compounds: Any compound,
which absorbs in the region of interest, (250 - 190 nm) should be CD Data Analysis
avoided. A buffer or detergent, imidazole or other chemical should
not be used unless it can be shown that the compound in question • The molar ellipticity [] is related to the difference in
will not mask the protein signal.
• Protein solution: The protein solution should contain only those extinction coefficients
chemicals necessary to maintain protein stability/solubility, and at [] = 3298 Δε.
the lowest concentrations possible. The protein itself should be as
pure as possible, any additional protein will contribute to the CD • Here [] has the standard units of degrees cm2 dmol -1
signal.
• Contaminants: Particulate matter (scattering particles), anything • The molar ellipticity has the units degrees deciliters
that adds significant noise (or artificial signal contributions) to the mol-1 decimeter-1.
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 0.5 - 1.0 mm path
length offer a good starting point.

CD Data Analysis Circular Dichroism of Proteins


• The difference in absorption to be measured is very small.
The differential absorption is usually a few 1/100ths to a • It has been shown that CD spectra between 260 and
few 1/10th of a percent, but it can be determined quite approximately 180 nm can be analyzed for the different
accurately. The raw data plotted on the chart recorder secondary structural types: alpha helix, parallel and anti-
represent the ellipticity of the sample in radians, which can parallel beta sheets, turns, and other.
• A number of excellent review articles are available
be easily converted into degrees describing the technique and its application (Woody, 1985
and Johnson, 1990).
(radians) • 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).
(degrees)

CD Signal of Proteins
CD Data Analysis • For proteins we will be mainly concerned with absorption
• To be able to compare these ellipticity values we need to in the ultraviolet region of the spectrum from the peptide
convert into a normalized value. The unit most commonly bonds (symmetric chromophores) and amino acid
used in protein and peptide work is the mean molar sidechains in proteins.
ellipticity per residue. We need to consider path length l, • Protein chromophores can be divided into three classes: the
concentration c, molecular weight M and the number of peptide bond, the amino acid sidechains, and any
residues. prosthetic groups.
in proper units (CD spectroscopists use decimol) • The lowest energy transition in the peptide chromophore is
an n → p* transition observed at 210 - 220 nm with very
which finally reduces to weak intensity (emax~100).

----p* p → p* ~`190 nm emax~7000


The values for mean molar ellipticity
per residue are usually in the 10,000's ----n n → p* 208-210, 191-193 nm emax~100
----p

2
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.

Comparison of the
UV absorbance
(left) and the
Far UV CD Spectra of Proteins
circular dichroism
(right) of poly-L-
[] x10-3 degrees cm2 dmol -1

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.

CD Spectra of Proteins CD Spectra of


• Different secondary structures of peptide bonds have
different relative intensity of n → p* transitions, resulting
• Each of the three
basic secondary Protein
in different CD spectra at far UV region (180 - 260 nm). structures of a
• CD is very sensitive to the change in secondary structures polypeptide chain
of proteins. CD is commonly used in monitoring the (helix, sheet, coil)
conformational change of proteins. show a characteristic
CD spectrum. A
• The CD spectrum is additive. The amplitude of CD curve protein consisting of
is a measure of the degree of asymmetry. these elements should
• The helical content in peptides and proteins can be therefore display a
estimated using CD signal at 222 nm spectrum that can be
e222= 33,000 degrees cm2 dmol -1 res-1 deconvoluted into the
• Several curve fitting algorithms can be used to deconvolute three individual
relative secondary structures of proteins using the CD contributions.
spectra of proteins with known structures.

3
CD Spectra Fit Conformational Change of CD2
c
• In a first approximation, a CD spectrum of a protein or
polypeptide can be treated as a sum of three components: 0

a-helical, b-sheet, and random coil contributions to the


6M GuHCl
spectrum.
• At each wavelength, the ellipticity (θ) of the spectrum will

[ ] (deg cm2 dmol )


-1
contain a linear combination of these components: -1000

25 ºC
(1)
-2000
• θT is the total measured ellipticity, θh the contribution from
helix, θs for sheet, θc for coil, and the corresponding χ the 85 ºC
fraction of this contribution.
-3000
200 210 220 230 240 250 260
Wavelength (nm)

CD2 Becomes Significantly Helical in TFE


CD Spectra Fit
• As we have three unknowns in this equation, a 5000

measurement at 3 points (different wavelengths) would


suffice to solve the problem for χ, the fraction of each 0
[] (deg cm2 dmol-1 res -1)

contribution to the total measured signal.


• We usually have many more data points available from our -5000
measurement (e.g., a whole CD spectrum, sampled at 1 nm 0% TFE
10% TFE
intervals from 190 to 250 nm). In this case, we can try to -1 10 4 17% TFE
minimize the total deviation between all data points and 19% TFE
30% TFE
calculated model values. This is done by a minimization of 4
80% TFE
-1.5 10
the sum of residuals squared (s.r.s.), which looks as
follows in our case : 4
-2 10
200 210 220 230 240 250 260

Wavelength (nm)

DICHROWEB Melting point measurement of EGFP-wt with its CD


spectra at different temperatures
http://www.cryst.bbk.ac.uk/cdweb/html/home.html

EGFP-wt Tm measurement Curve fitting of EGFP-wt


20 -1
A y = M1+M2/(1+ exp(-(M0-M3)/M... B
35 -2 Value Error
45
15 55 m1 -8.614 0.024757
65 -3 m2 7.2129 0.047864
CD (218 nm), mdeg

75 m3 65.718 0.11006
10 85
-4 m4 2.644 0.095431
95
CD, mdeg

Chisq 2.7395 NA
R 0.99849 NA
5 -5
B
-6
0
-7
Tm = 65.7 °C
-5
-8

-10 -9
200 210 220 230 240 250 260 0 20 40 60 80 100
Wavelength, nm Temperature, C

4
Fluorescence measurement

Fluorescence spectrum of proteins Xe lamp

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

Electronic mechanism of fluorescence Trp Fluorescence Emission Spectra of CD2


under Different Conditions
1st exited singlet state Triplet state
Intersystem crossing
• In a hydrophobic
environment (inside of
4 10 4

Trp
c
a folded protein), Trp
25ºC
emission occurs at
shorter wavelength.
Fluorescence intensity

4
3 10

Absorption fluorescence Phosphorescence


6M GuHCl When it is exposed to
hvex
4
2 10

hvem
85ºC
solvent, its emission is
vex > vem 1 10 4

very similar to that of


Vibration levels or the free Trp amino acid
lex < lem 0
300 320 340 360
Wavelength (nm)
380 400
(red shift occurs).
Ground singlet state

5
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 M)
– 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)