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.

 

Stages of Protein Folding

Protein folding follows a sequence of stages (Figure 4.31, p 109) [overhead, V&V 8.5]:

• Burst phase driven by hydrophobic collapse: hydrophobic groups come together expelling most of hydrating water within a few milliseconds.
  • Radius of gyration is reduced dramatically, to about 50% for a 100 residue protein.
  • Most of the secondary structure is formed in this period.
  • This initial collapsed phase known as a molten globule.
    • The molten globule has most of the native secondary structure and native fold.
    • The molten globule has a radius of gyration that is only 5-15% larger than the native protein.
• Intermediate folding phase:
  • Native-like tertiary structure appears over 5ms to 1 s.
    • Secondary structure stabilized and tertiary structure starts to form, probably as sub-domains.
  • Side-chains of amino acids are most likely still quite mobil.
• Final folding phase:
  • Native structure is achieved over a period of several seconds for small single-domain proteins (longer for more complex proteins).
  • Relatively rigid core packing of native protein is achieved with expulsion of the last water molecules from hydrophobic core.

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.

• Protein folding pathways seem to be specified by primary structure.
• Protein structure is determined by specification of folding pathways as well as folding possibilities.

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.

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:

• Atomic fluctuations (10^-15- 10^-11 sec; 0.001 - 0.1 nanometer) Myoglobin example [overhead v&v 8.9, 8.10]
• Collective motions of covalently linked atoms, from aa R-groups to domains (10^-12 - 10^-3 sec; 0.001 - >0.5 nanometer)
• Triggered conformational changes: in response to ligand binding, covalent modification etc.

How do we know about the mobility of protein structures?

• X-ray diffraction studies of proteins with and without ligand bound
• NMR (phe, his ring protons/carbons show up on edges of signal envelope)
• H-exchange
• Antibody binding: make antibodies to normally interior aa residues, over time protein ppt forms as interior groups momentarily exposed.

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]:

Pathway Diagrams

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Last modified 12 February 2007