| Chem 431 |
Biochemistry |
Fall 2001 |
| Lecture Notes:: 21 September |
© R. Paselk 2001 |
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3D Structure of Proteins IV
Hierarchy of protein structure diagram
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- Aside: The reality of X-ray diffraction structures.
- Trouble is that most of our detailed knowledge of protein
3-D structure due to X-ray diffraction. Problem: Non-solution,
look at very concentrated, crystal structures for proteins.
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- Why do we think they represent reality?
- - Crystals very hydrated, in fact some enzymes maintain activities
in crystal form!
- - Chemical exchange studies, such as deuterium exch. are
consistent with residue exposure.
- - Chemical reactivity of residues are consistent with residue
exposure.
- - Optical probes of overall shape (e.g. light and x-ray scattering)
are consistent.
- - Hydrodynamic studies of size and shape (e.g. sedimentation,
gel filtration) are consistent.
- - Optical probes of regularity/helicity (e.g. Circular dichroism
and ORD) are consistent.
- - Probes of local environment (e.g. NMR, CD & ORD, Fluorescence,
UV) are consistent.
- Note that any "non-rigid" region of the protein
will not show up on X-ray diffraction image, or will be "fuzzy."
- Thus quite confident of structures.
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3-D Structure of Proteins IV
Protein Folding
- Primary structure specifies tertiary (& therefore quaternary)
structure. This is known from in vitro denaturation/renaturation
studies of small proteins. (Denaturation means to unfold to non-functional
state, often achieve a "random coil" in solution; renaturation
means to return to the properly folded, natural, and functional
state.)
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- The classic study involved Ribonuclease: Reduce (break) -S-S-
bonds, denature with urea to random coil. Now can renature by
gently removing denaturant (urea) and oxidize -S-S- bonds. [overhead
5.41, P] Enzyme activity fully recovered. X-ray diffraction image
same! Note - no gremlins, no magic, done in "test tube."
Other small proteins, such as Myoglobin and proinsulin, fold
up spontaneously in the same manner as Ribonuclease. Hoever, insulin
fails to fold correctly, since a peptide essential to folding
has been cleaved off.
But, note, this experiment has not been repeated with large
proteins. Now known that many proteins are aided in folding process
by Chaperones: appear to stabilize unfolded conformation, allowing
time to find correct folding pattern. Some chaperons are known
to have a barrel-shape into which new or partially denatured protein
is inserted, native protein is released. Chaperones require ATP
energy to function. The so-called Heat-shock proteins are a family
of chaperons.
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- Last modified 21 September 2001
Pathway Diagrams
