Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 431	Biochemistry	Fall 2007
Lecture Notes: 21 September	© R. Paselk 2007
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Domains

Large proteins (>200 aa's) usually fold up in smaller pieces of 100-200 aa's called domains. Recall that we define a Domain as an independent folding region in a protein. Often defined by clefts in 3-D structure giving globular elements connected by "hinges" (single strand segments connecting the domains). Domains have the advantages of speeding up the folding process (fold domains independently, then assemble resultant folded domains - effectively processing folding of domains in parallel). Another advantage of domain structure is that nature can take bits of DNA specifying particular domains with particular functions and assemble them in new combinations to get new activities (e.g. combine an ATP binding site and a sugar binding site to give a sugar phosphorylating protein).

Example: IgG , domains, exons and evolution. [overheads: IgG/proteins; 7.23 MvH] (text Figure 5-23)

• IgG made up of four independently synthesized proteins, 2 heavy chains with 4 domains each, and 2 light chains with 2 domains each.
  • Domain types: -meander [anti-parallel -sheet], "collapsed -barrel domain"(immunoglobulin fold). (Note that Motifs and Domains often use the same nomenclature, and indeed often overlap. Can in fact have Motif = Domain = Tertiary structure!)
• Domains correspond to exons of DNA (frequently, but not always the case)
  • The domains are all apparently related through gene duplication in the remote past.
• The active site of IgG (2/IgG) is made up between two domains, one from a heavy chain and one from a light chain.
• When immune system is developing individual cells express single IgG molecules made from randomly expressed heavy and light chains.

In a similar manner we see that many enzymes have active sites created between two domains, often one domain binds one substrate while the second binds a second substrate.

Its as if these proteins were designed by taking "off-the-shelf" components, assembling them, and then over time (and generations) tuning the combination up.

Groups of motifs forming the core of the tertiary structures of domains are referred to as Folds. Over 600 folds have been discovered, with an expectation that about 1,000 exist, as we saw in our earlier discussion. (Note that 1,000 is a bunch, but well below the infinite number possible!)

Note also that protein classes often apply to domains of multidomain proteins, with no overall class applicable to the entire protein. Thus for IgG we see multiple domains of the same class (all beta) to make up an all beta protein, but many enzymes are made up of domains with entirely distinct folds and evolutionary backgrounds.

Quaternary Protein Structure

Quaternary (4°) structures : Geometrically specific associations of protein subunits; the spatial arrangement of protein subunits.

• Hemoglobin as an example: Hemoglobin is an alpha-beta-alpha-beta oligomeric protein: its quaternary structure consists of a tetramer of myoglobin like (all-alpha) subunits. (text Figure 4-23) The two types of chain are slightly shorter than myoglobin chains (alpha= 141 aa residues, beta= 146 aa residues vs. 153 aa residues in Mb). There are extensive contacts between an alpha and a beta subunit to give a dimer. The dimers have additional contacts to give the tetramer. Oxygen binding results in a change of conformation in Hb which we will look at later.

Folding Hierarchy Overview

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

• Increasing the size of a protein allows better "fits" for catalysis and binding - many weak bonds are needed to maintain specific structures.
• Can bring sequential active sites of metabolic pathways into close proximity.
• However, large peptides have some problems:
  • The process of folding slows tremendously with increasing size, thus folding individual subunits, and assembling these subunits can greatly enhance folding efficiency.
  • Get about 1 error / 10³ aa residues due to the precision of the translation of messenger RNA to protein. Thus need to keep residue number down.
• Interacting subunits provide mechanisms for regulation.

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

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." NMR has now been successfully applied to a variety of proteins, again confirming the structures determined via X-ray. 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.

• 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.)

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. (text Figure 4-27) with the enzyme activity fully recovered. X-ray diffraction image is also the 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.

Pathway Diagrams

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Last modified 21 September 2007