Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2007

Lecture Notes: 12 February

© R. Paselk 2006
 
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Protein Folding, cont.

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.

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 (Figure 4.31, p 109) [overhead, V&V 8.5]:

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.

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.
 

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 different 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 accommodate a protein with >600 residues
    • Hsp90
    • Nucleoplasmins (necessary for assembly of nucleosomes in eukaryotic chromosomes).
  • Protein disulfide isomerases.
  • Pepidyl prolyl cis-trans isomerases.

 

MYOGLOBIN AND HEMOGLOBIN AS EXAMPLE PROTEINS

Myoglobin: Myoglobin is a 153 residue globular protein in the globin family. Eight alpha helices form its single domain (myoglobin fold) tertiary structure; about 80% alpha helix (high for globular proteins). (Figure 4.43) [overhead 7-41, V&V] Interior almost exclusively hydrophobic residues, with water excluded from interior. Surface has mix of hydrophobic and hydrophilic residues, with ionizable groups on surface.

Myoglobin functions to store and facilitate the diffusion of oxygen in muscle. Oxygen binds to a heme {Fe (II)-protoporphyrin IX} prosthetic grp. Four of irons six ligands are to heme nitrogens, with a fifth to a histidine nitrogen. The final ligand bond goes to oxygen. (Figure 4.44, p 116) [overhead, S, Figure 7-5, 7-8] Breathing motions (see below) are necessary to allow the exchange of oxygen, since the heme is in a closed pocket. [overhead 8-9, 8-10, V&V]

PROTEIN DYNAMICS

"Breathing" motions:

How do we know about the mobility of protein structures?

Hemoglobin: Hemoglobin is an alpha-alpha-beta-beta oligomeric protein: its quaternary structure consists of a tetramer of myoglobin like subunits. (Figure 4.49, p 120) [overhead 9-13a, V&V] The two types of chain are slightly shorter than myoglobin chains (alpha= 141 aa residues, beta= 146 aa residues). 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. (Figure 4.47, p 119) [overheads 9-13a vs. 9-13b, V&V] The change of conformation affects the binding of oxygen [overhead Fig 9-16, V&V] {oxygen binding is reduced in the "blue" form due to steric hindrance between the oxygen and the heme}.

OXYGEN BINDING

Let's look at binding in terms of saturation, Y, where if Y = 1 every site of every Myoglobin is occupied by an oxygen molecule (thus if Y = 0.5, then 50% of the myoglobin are binding oxygen and 50% are "empty"). Mb/Hb binding curve [overhead 33 V&V]:

 


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