Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2007

Lecture Notes: 8 February

© R. Paselk 2006
 
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3-D Structure of Proteins 4

Domains, cont.

Note that domains will have their own tertiary structures, made up of secondary and frequently supersecondary elements. Domains can be categorized into four main groups:

  1. All alpha
  2. All beta
  3. alpha/beta (have alternating alpha and beta structures, such as in the beta-alpha-beta motif)
  4. alpha + beta (local clusters of alpha and beta in same chain with each cluster consisting of contiguous primary structure).

Groups of motifs forming the core of the tertiary structures of domains are referred to as Folds. (p 99) Over 600 folds have been discovered, with an expectation that about 1,000 exist. (a bunch, but well below the infinite number possible!) Common examples include (Fig 4.24) [overhead]:

Folds/Motifs are often more highly conserved than sequences, and so are used along with sequences to trace relatedness among molecules and thus organisms. An example of conservation for a domain is seen in Cytochrome c as shown in your text in Figure 4.21.

Quaternary Protein Structure

ternary (4°) structures (Fig. 4.25; overheads: MvH 6.26, Fig 25): Geometrically specific associations of protein subunits; the spatial arrangement of protein subunits.

Folding Hierarchy Overview

 

Rationale for quaternary: There are a variety of advantages to large structures:

Quaternary structures allows the assembly of large to extremely large structures. 

 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. [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. However, insulin fails to fold correctly, since a peptide essential to folding has been cleaved off.

Accesory 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.

Chaperones - See next week's discussion.

  • 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. (Figures 4.32 & 4.33) Some chaperones require ATP energy to function. A number of differnt type:
    • The so-called Heat-shock proteins (Hsp70) are a family of chaperons.
    • The Chaperonins (Hsp60 or GroEL and Hsp10 or GroES) -barrel-like proteins, provide internal folding environment, require ATP energy for optimum function. GroEL is large enough to accomodate a protein with >600 residues
    • Hsp90
    • Nucleoplasmins (necessary for assembly of nucleosomes in eukaryoptic chromosomes).
  • Protein disulfide isomerases.
  • Pepidyl prolyl cis-trans isomerases.

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!


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Last modified 8 February 2007