Humboldt State University ® Department of Chemistry

Richard A. Paselk

Chem 438

Introductory Biochemistry

Spring 2010

Lecture Notes: 12 February

© R. Paselk 2006
 
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Protein Folding, cont.

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 4.40) 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. (Figure 4.39) 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) Breathing motions (see below) are necessary to allow the exchange of oxygen, since the heme is in a closed pocket. (Figure 4.45 - note motion possibilities)

Hemoglobin

Hemoglobin is an alpha-alpha-beta-beta oligomeric protein: its quaternary structure consists of a tetramer of myoglobin like subunits. (Figure 4.43, 4.42) 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) 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 (Figure 4.46):

 

Myogloboin and hemoglobin binding curves showing saturation (Y) vs. oxygen pressure

Reviewing the curve in terms of saturation, Y, if Y = 1 then 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").

Can describe binding as dissociation equilibrium,then:

MbO2 Mb + O2 ; equilibrium expression for the dissociation of oxymyoglobin & saturation expression for myoglobin, Y = concentration of myoglobin over concentration of myoglobin + concentration of myoglobin

for saturation. Substituting, myoglobin saturation curve in terms of oxygen concentration, Y = concentration of oxygen over concentration of oxygen + K, the equation of a hyperbola. If expressed as pressures, then myoglobin saturation curve in terms of oxygen pressure, Y = oxygen pressure over oxygen pressure + oxygen pressure at 50% saturation where P50 = pO2 @ 50% saturation. Note that the binding curve for Mb is indeed hyperbolic in shape.

What about Hb? Obviously more complex. The sigmoid shape (s-shape) of the curve indicates cooperativity. That is, if one site binds, another is more likely to as well (it cooperates with the first site). If the subunits of Hb are fully cooperative (if one subunit binds oxygen they all must bind oxygen, if one releases oxygen they must all release oxygen) then

chemical equation for the dissociation of oxy hemoglobin to hemoglobin + four oxygens; equilibrium expression for oxyhemoglobin dissociation

and for saturation, substituting as above with Mb:

hemooglobin saturation curve in terms of oxygen pressure, Y = oxygen pressure to the fourth power over oxygen pressure to the fourth power + oxygen pressure at 50% saturation to the fourth power

But this assumption of total cooperativity doesn't work, the curve is too steep.


FYI

Hill equation: We can rearrange our equation to find the degree of cooperativity. If we generalize:Hill equation, Y = oxygen pressure to the nth power over oxygen pressure to the nth power + oxygen pressure at 50% saturation to the nth power. Rearranging:

Hill equation rearranged for Hill plot, Y over 1 - Y = oxygen pressure to the nth power over oxygen pressure at 50% saturation both to the nth power

 

In this equation n is the cooperativity, that is the apparent number of fully cooperative sites.

Hill plot: If we take logs of both sides of the Hill equation and plot the results, the exponent, n, shows up as the slope. Thus we can readily find the apparent cooperativity by plotting saturation vs. oxygen pressure (or concentration). For Hb the slope turns out to be n = 2.8. That is, Hb is partially cooperative - its 4 cooperating subunits only partially cooperate to behave like 2.8 completely cooperative subunits.

Plot showing cooperativity in center and at extreme concentrations

Note limiting situations at extremes with n = 1. At high concentrations this results because the effective equilibrium is:

Hb(O2)4 equilibrium arrows Hb(O2)3 + O2

In other words it acts like it has a single site (only one site is available at any moment) like myoglobin! At low concentrations of oxygen the opposite effect gives the same result:

HbO2equilibrium arrows Hb + O2

Again, only one site is effectively operating (not enough O2 to fill more than subunit one site at any given time), so again mimics myoglobin.


Pathway Diagrams

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Last modified 17 February 2010