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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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Structural Analysis of Protein Structure

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Structural analysis of protein structure l.jpg

Structural Analysis of Protein Structure

Circular Dicroism

Fluorescence


Methods for secondary structural analysis l.jpg

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


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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 190 - 170 nm (170 - 260 nm).

  • The difference in left and right handed absorbance A(l)- A(r) is very small (usually in the range of 0.0001) corresponding to an ellipticity of a few 1/100th of a degree.


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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.


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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).


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Typical Initial Concentrations

  • 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, 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 (260 - 178 nm) will require 30-60 minutes to record (plus an equivalent amount of time for a baseline as every CD spectrometer.


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Sample Preparation and Measurement

  • Additives, buffers and stabilizing compounds: Any compound, which absorbs in the region of interest, (250 - 190 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 0.5 - 1.0 mm path length offer a good starting point.


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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)


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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


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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.


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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).


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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 210 - 220 nm with very weak intensity (emax~100).

----p*p → p* ~`190 nm emax~7000

----nn →p* 208-210, 191-193 nm emax~100

----p


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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.


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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 (180 - 260 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.


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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.


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Far UV CD Spectra of Proteins

[] x10-3 degrees cm2 dmol -1


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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.


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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.


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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 :


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DICHROWEB

http://www.cryst.bbk.ac.uk/cdweb/html/home.html


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Using CD to Monitor 3º Structure of Proteins

  • CD bands in the near UV region (260 – 350 nm) are observed in a folded protein where aromatic sidechains are immobilized in an asymmetric environment.

  • The CD of aromatic residues is very small in the absence of ordered structure (e.g. short peptides).

  • The signs, magnitudes, and wavelengths of aromatic CD bands cannot be calculated; they depend on the immediate structural and electronic environment of the immobilized chromophores.

  • The near-UV CD spectrum has very high sensitivity for the native state of a protein. It can be used as a finger-print of the correctly folded conformation.


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Domain 1of CD2

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


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CD2 is Stable from pH 1 to 10


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Conformational Change of CD2

6M GuHCl

25 ºC

85 ºC


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CD2 Becomes Significantly Helical in TFE


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Near UV CD Spectra of CD2

  • CD2 losses its native well packed tertiary structure at high temperature and in 6M GuHCl

6 MGuHCl

85 ºC

25 ºC


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CD2 losses its Tertiary Structure in TFE


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Secondary Structure Prediction of CD2


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CD2 vs. Helical Propensity

  • Residues on strands C, C’, C” and G have strong helical propensity.


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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.


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Melting point measurement of EGFP-wt with its CD spectra at different temperatures

B

A

Tm = 65.7 °C


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Summary of CD

  • Circular dichroism spectroscopy is used to gain information about the secondary structure and folded state of proteins and polypeptides in solution.

  • Benefits: Uses very little sample (200 ul of 0.5 mg/ml solution in standard cells)

    Non-destructive

    Relative changes due to influence of environment on sample (pH, denaturants, temperature, etc.) can be monitored accurately.

  • Drawbacks: Interference with solvent absorption in the UV region

    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


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Fluorescence spectrum of proteins


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1st exited singlet state

Triplet state

Intersystem crossing

Absorption

fluorescence

Phosphorescence

hvex

hvem

vex > vem

or

lex < lem

Vibration levels

Ground singlet state

Electronic mechanism of fluorescence


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Fluorescence measurement

Xe lamp


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Trp

25ºC

6M GuHCl

85ºC

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).


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Interaction between calmodulin and Ca2+

Phe

λex = 254 nm

Tyr

λex = 277 nm

By Jasmine


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Fluorescence resonance energy transfer (FRET) between donor and acceptor


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Fluorescence resonance energy transfer (FRET) between EMOC-N85 and Tb3+

B

A

[P] = 1 mM

25 mM PIPES

100 mM KCl

pH 6.8

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.


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GFP becomes one of the most popular and exciting new technologies in biochemistry and cell biology

  • 11 stranded antiparallel b-barrel, a single central a-helix

  • Several loops and short helices capping the barrel on each end

  • A chromophore formed by the residues 65-67 buried in the center of the barrel

  • Single-chain, 238 residues, ~27 kDa, a soluble globular protein

  • Residues of 7-229 are essential for the fluorescent property of GFP

A

B

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/


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EGFP-based Ca2+ sensors with different Ca2+-binding affinities are developed by two different approaches

Hypothesis

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.

Design

Grafting

3

2

5

1

4

6

EGFP

9

8

N-terminal

7

10

11

C-terminal


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Ca2+ 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


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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)


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