Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2010

Lecture Notes: 15 February

© R. Paselk 2006


3-D Structure of Proteins 4

 FYI - Review/Enhancement: X-ray Diffraction determination of protein structure.

  • Review x-ray diffraction learned in Chem 109
  • Note the each crystal point now becomes many points, each with its own "reflecting" plane.
  • Rotation to determine relative locations in three space results in thousands of reflections
  • Need to add heavy metals to act as "beacons" to locate positions in absolute space, and need to do a couple of times isomorphically (without altering the proteins structure - isomorphic replacements).

Finally use Fourier transforms to convert angles and intensities of reflections to 3-D map of protein.

Aside: The reality of X-ray diffraction structures. Trouble is that most of our detailed knowledge of protein 3-D structure is due to X-ray diffraction. Problem: Non-solution, look at very concentrated, crystal structures for proteins.

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 exchange 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.

Protein Folding

Primary structure specifies tertiary (& therefore quaternary) structure. This is known from in vitro denaturation/renaturation studies of small proteins.
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. [Fig 4.27-4.29] 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. However, insulin fails to fold correctly, since a peptide essential to folding has been cleaved off.

Accessory Folding Proteins

The ribonuclease renaturation-type experiment has not been repeated with large proteins, which seem to require the participation of "folding catalysts," the chaperones, to aid their folding.

Let's look at folding in another way: You might guess a protein would fold to lowest free energy conformation. Problem: is there time? ("Levinthal's Paradox", formulated by Cyrus Levinthal in 1968) Stryer calculation (very conservative): Assume 100 aa residue protein with 3 possible conformations/residue; then get 3100or 5 x 1047 possible conformations. If search at a rate of one structure/10-13sec then get (5 x 1047)(10-13)= 5 x 1034 sec or 1.6 x 1027 years to search (and thus to fold protein). This is greater than the age of our Universe (13.7 x 109 yrs). Rawn calculation (perhaps more realistic): same but assume 10 conformations, then get 1087sec or 3 x 1080 yrs!

Obviously from these calculations not searching all possible conformations (or we have the process wrong!), so cannot say protein achieves the lowest global free energy, but rather a local free energy minima. (Like a valley in mountain range: a local energy minima, but not lowest [Marianas trench].) [overhead - note represents 'local' minimum, not global minimum)] Which valley the protein reaches will depend on folding "paths." That is folding appears to depend on kinetics as well as thermodynamics. [Fig 4.30]

Some structures fold faster than others: alpha-helix seems fastest folding, due to cooperativity. That is, once a couple of H-bonds of helix are formed subsequent residues are aligned to form H-bonds, thus a cooperative process. Beta-structures are second most rapidly formed. These kinetic factors probably contribute to the commonness of these two structure types. Another factor making them common, is that helices and sheets are the most compact forms polymers can take - the proportion of a flexible chain in helix and sheet conformation increases as the chain is forced to become more compact. H-bonding and phi and psi angles then determine they will be alpha and beta structures. Thus we expect rapid formation of alpha and beta structures. However, unless they are stabilized by interacting with each other and by compaction, they may unfold and try other combinations until stable associations result.

[See D. Baker. (4 May 2000) A surprising simplicity to protein folding. Nature, 405. p 39-42.]

Stages of Protein Folding

Protein folding follows a sequence of stages [Fig 4.31]:

Protein folding also appears to be hierarchical, that is it begins with folding of low stability local structures, which interact locally etc. Note that this leads naturally to a sub-domain type structure and hierarchical structures as seen in proteins.

Note that some aa residues favor one or another secondary structure. Unfortunately, such tendencies by themselves have not proved effective in predicting protein structures. However, using this kind of information in a "local" (nucleation) and "hierarchical" (extensions/higher levels) way can predict some small protein structures reasonably well.

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Last modified 15 February 2010