Protein Function

Protein Function • Hemoglobin as a model systems for: • Ligand binding • Quaternary structure and symmetry • Cooperative behavior • Allosteric conformational effects • Genetic variation, selection, disease

Myoglobin (a monomeric form of hemoglobin) • Myoglobin and homoglobins were the first protein crystal structures determined, ca. 1960 (John Kendrew and Max Perutz, Nobel Prize, 1962) • Decades long struggle to figure out how to use X-ray diffraction to solve the structure of macromolecules. 50 years later, current methods are mainly modifications of those worked out by Perutz & Kendrew.

An early illustration of the structure of myoglobin emphasizing the heme binding site. Myoglobin binds O2 in the tissues, after is it delivered by hemoglobin. Text, Figure 7-1

The heme cofactor. O2 binds reversibly as the 6th ligand to the Fe. Carbon monoxide binds orders of magnitude more tightly, which is why it is so deadly. Text, Figure 7-2 Figure 7-2

A space-filling model of the region surrounding the heme. The close packing is relevant because (in hemoglobin) itallows binding events to be coupled to conformational changes in the protein Text, Figure 7-3 Figure 7-3

Binding of O2 to myoglobin (which is monomeric) follows simple ‘hyperbolic’ behavior, so name because of the simple asymptotic approach to the saturation point. Y is the fractional saturation Figure 7-4

Binding of O2 to myoglobin (which is monomeric) follows simple ‘hyperbolic’ behavior, so name because of the simple asymptotic approach to the saturation point. work out simple hyperbolic binding curve Figure 7-4

Binding of O2 to hemoglobin (in the blood) is very different. The binding curve is sigmoidal (sort of S-shaped) - The critical advantage of a sigmoidal binding curve (or reaction velocity curve for an enzyme) is that there can be a large change in binding over a narrower change in ligand (i.e. O2) concentration. - This enables optimal delivery of O2 between two regions where the concentration of O2 is different (e.g. lungs vs. muscles). Figure 7-6

Binding of O2 to hemoglobin (in the blood) is very different. The binding curve is sigmoidal (sort of S-shaped) sigmoidal binding (or reaction velocity) behavior results from cooperative binding work out all-or-none (perfectly cooperative) binding curve work out Hill plot and Hill coefficient Figure 7-4

Text, Figure 7-7 Figure 7-7

How does hemoglobin achieve sigmoidal (cooperative) binding behavior? That is, how does binding of O2 at one heme site in the tetramer make it so that the other sites ‘want to’ bind O2 much more strongly than before? • Conformational change in the protein, linked to ligand (O2) binding • Quaternary structure • Coupling of effects between different subunits in the oligomer Figure 7-5

How is O2 binding coupled to protein conformational change? Blue = deoxy Red = oxy O2 binding pulls Fe back into plane of heme, pulling a-helix (F) with it. Thermodynamics and cause-and-effect: Binding of O2 tends to ‘cause’ the protein to shift from one conformation (T) to the other (R). Thermodynamically, this results from the R state having a much higher affinity for the ligand than the T state. Figure 7-8

Role of quaternary structure in hemoglobin cooperative behavior Text, Figure 7-5 Tetrameric assembly (a2b2). Pseudo D2, meaning the four subunits are similar in structure and arranged nearly symmetrically (i.e. equivalent interactions with each other). Figure 7-5

Role of quaternary structure in hemoglobin cooperative behavior • deoxy hemoglobin • protein conformation: T-state • oxy hemoglobin • protein conformation: R-state Text, Figure 7-5 Two different protein conformations, tightly coupled to ligand binding (described earlier). If the conformation of one subunit is tightly coupled to the conformation of other subunits, then the binding of O2 at one site can drive the binding at other sites in the same tetramer. Figure 7-5

The symmetry model or MWC model for cooperativity Recall that having very different affinities for the ligand in the two conformations (i.e. ligand binding is strongly coupled to protein conformation) is a key part of the model (not sufficiently emphasized by the text). So, model can be best understood by considering the limiting case where the T state can’t bind ligand at all. Text, Figure 7-15 Figure 7-15

The symmetry model or MWC model for cooperativity Recall that having very different affinities for the ligand in the two conformations (i.e. ligand binding is strongly coupled to protein conformation) is a key part of the model (not sufficiently emphasized by the text). So, model can be best understood by considering the limiting case where the T state can’t bind ligand at all. Also note that the equilibrium constants should favor T0 over R0, and that binding affinity of the R state for the ligand should be high. Figure 7-15

A diagram of the switch between R and T states Note that a ‘switch’ (rather than a continuous range of possible conformations) is important in transmitting or coupling conformational changes between subunits Text, Figure 7-9 Figure 7-9 part 2

The affinity of hemoglobin for O2 is influenced by several allosteric (meaning ‘other site’) effectors. These have important physiological roles. The Bohr effect: increased O2 affinity at high pH Note that this means uptake of H+ by Hb is coupled to O2 release Text, Figure 7-11 Figure 7-11

The Bohr effect aids in the protonation and deprotonation of CO2 Uptake of H+ by Hb is coupled to O2 release (tissues). Uptake of H helps convert CO2 (gas) to bicarbonate for transport in blood Release of H+ by Hb is coupled to O2+ binding (lungs). Release of H+ helps convert bicarbonate back to CO2 for exhalation. Text, Figure 7-12 Figure 7-12

Examples of other allosteric effectors: CO2, 2,3-phophoglycerate Text, Figure 7-13 Figure 7-13

A single point mutation in hemoglobin (Val6Glu) causes sickle cell anemia by promoting filament formation in the deoxy state Text, Figures (various) Figure 7-17a

Protein Function