Structural Analysis of Protein Structure. Circular Dicroism Fluorescence X-ray NMR. Methods for Secondary Structural Analysis.
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
Δε  = 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
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
Drug design information Conditions
In order to obtain a crystal, molecules must assemble into a periodic lattice.
Each unit cell can contain several molecules that are related by symmetry.
The diagram shows identical blocks, each containing two objects packed against each other.
When the X-ray goes through the crystal, beams is diffracted and diffraction pattern is recorded on a detector. The crystal is rotated a certain degree while this pattern is recorded. A series of frames are collected.
Determine the size of the unit cell by Bragg's law:
2dsinq = λ d= λ/(2* sinq).
phase, which is related to its interference, positive or negative, with other beams, and
wavelength, which is set by the x-ray source for monochromatic radiation.
(d) 1.1 Å
High Resolution Crystal Structures other in 3D.
In the refinement process, the model is changed to minimize the difference between the experimentally observed diffraction amplitudes and those calculated for a hypothetical crystal containing the model instead of the real molecules.
The difference is called the R factor, with 0.0 being exact agreement and 0.59 total disagreement.
0.15 < R < 0.20 = well determined structure
R ~ 0.30 = medium structure
R > 0.30 = bad structure
ATOM 1 N PRO A 190 -0.567 24.363 16.753 49.28
ATOM 2 CA PRO A 190 -0.399 23.026 17.339 49.21
ATOM 3 C PRO A 190 -1.288 21.990 16.644 49.61
ATOM 4 O PRO A 190 -2.520 22.007 16.772 49.44
3J = A cos (θ) +B cos2 (θ) + C
where A, B, and C are empirically derived constants for each type of coupling constant (e.g., 3JHAHN or 3JHAHB).
Since hydrogen atoms in two adjacent residues are covalently
connected through at least three other atoms (for instance,
HCa-C'-NH), all COSY signals reveal interactions within the same
amino acid residue. These interactions are different for different
types of side chains. The NMR signals therefore give a "fingerprint"
of each amino acid. The diagram illustrates fingerprints (red) of
residues Ala and Ser.
Shown above, amide proton exchange rates with solvent water (filled diamonds) kNH < 0.02 min-1, coupling constants: 3JHNa (filled circles) < 6.0 Hz and (open circles) > 7.0 Hz, and sequential backbone dNN and daN NOE connectivities are classified as strong, weak, or absent and are represented by the thickness (or absence) of a bar connecting the residues in question. Medium range NOE connectivities daN (i, i+3) and (i, i+4) are drawn as line segments connecting the residues contributing to the observed cross peak if present.