lecture_18 - Protein Structure Determination Protein...

Protein Structure Determination
Protein Folding
Molecular Chaperones
Prions
Alzyheimer’s

Tertiary Structure of Proteins
Two methods: 1. X-RAY diffraction crystal structure
2. NMR solution structure
This is a crystal X-ray diffraction pattern of sperm whale myoglobin

Electron density map
6 Å 2.0 Å 1.5 Å 1.1 Å
From the diffraction pattern (spots and intensity) one can get a
mathematical description of the electron density of a molecule. With
proper model construction a 3-D image of the protein is constructed.

NMR

By using chemical shifts of backbone hydrogens and their
chemical splitting bond angles can be determined. COSY NMR
or Correlated Spectroscopy. By manipulating parameters
protons that are close to each other in space but not linked
through bonds can be determined by NOSY NMR or Nuclear
Overhauser spectroscopy. Growing the protein in bacteria where
the carbon source can be substituted by
13
C and the nitrogen by
15
N (stable isotope substitution) more restraints can be achieved.
The liquid structure(s) can be determined as a group that fit a
certain structure space.

Quaternary Structure and
Symmetry
Subunits can associate noncovalently, subunits are
protomers if identical .
Protomer subunits are symmetrically arranged
Only rotational symmetry allowed.
i.e. cyclic symmetry C2, C3, C6 etc.
Dihedral symmetry N-fold intersects a two-fold
rotational symmetry at right angles
Other higher order types, octahedral or tetrahedral

Protein folding is
“one of the great unsolved problems of science”
Alan Fersht

protein folding can be seen as a connection
between the genome (sequence) and what the
proteins actually do (their function).

Protein folding problem
•
Prediction of three dimensional structure from its
amino acid sequence
•
Translate “Linear” DNA Sequence data to spatial
information

Why solve the folding problem?
•
Acquisition of sequence data relatively quick
•
Acquisition of experimental structural information
slow
•
Limited to proteins that crystallize or stable in
solution for NMR

Protein folding dynamics
Electrostatics, hydrogen bonds and van der Waals forces hold a
protein together.
Hydrophobic effects force global protein conformation.
Peptide chains can be cross-linked by disulfides, Zinc, heme or
other liganding compounds. Zinc has a complete d orbital , one
stable oxidation state and forms ligands with sulfur, nitrogen and
oxygen.
Proteins refold very rapidly and generally in only one stable
conformation.

The sequence contains all the information to
specify 3-D structure

Random search and the
Levinthal paradox
•
The initial stages of folding must be nearly random, but if the entire process
was a random search it would require too much time. Consider a 100 residue
protein. If each residue is considered to have just 3 possible conformations the