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 (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.

Text, Figure 7-1 An early illustration of the structure of myoglobin emphasizing the heme binding site. Myoglobin binds O 2 in the tissues, after is it delivered by hemoglobin.

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

Figure 7-3 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 7-4 Binding of O 2 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 O 2 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 7-6 Binding of O 2 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. O 2 ) concentration. - This enables optimal delivery of O 2 between two regions where the concentration of O 2 is different (e.g. lungs vs. muscles).

Figure 7-4 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 Binding of O 2 to hemoglobin (in the blood) is very different. The binding curve is sigmoidal (sort of S-shaped)

Figure 7-7 Text, Figure 7-7

Figure 7-5 How does hemoglobin achieve sigmoidal (cooperative) binding behavior? That is, how does binding of O 2 at one heme site in the tetramer make it so that the other sites ‘want to’ bind O 2 much more strongly than before? Conformational change in the protein, linked to ligand (O 2 ) binding Quaternary structure Coupling of effects between different subunits in the oligomer

Figure 7-8 How is O 2 binding coupled to protein conformational change? Blue = deoxy Red = oxy O2 binding pulls Fe back into plane of heme, pulling  -helix (F) with it. Thermodynamics and cause-and-effect: Binding of O 2 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 7-5 Tetrameric assembly (  2  2 ). Pseudo D 2, meaning the four subunits are similar in structure and arranged nearly symmetrically (i.e. equivalent interactions with each other). Role of quaternary structure in hemoglobin cooperative behavior Text, Figure 7-5

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 O 2 at one site can drive the binding at other sites in the same tetramer. deoxy hemoglobin protein conformation: T-state oxy hemoglobin protein conformation: R-state Role of quaternary structure in hemoglobin cooperative behavior Text, Figure 7-5

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. 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 7-9 part 2 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 7-11 The affinity of hemoglobin for O 2 is influenced by several allosteric (meaning ‘other site’) effectors. These have important physiological roles. The Bohr effect: increased O 2 affinity at high pH Note that this means uptake of H+ by Hb is coupled to O 2 release Text, Figure 7-11

Figure 7-12 The Bohr effect aids in the protonation and deprotonation of CO 2 Uptake of H + by Hb is coupled to O 2 release (tissues). Uptake of H helps convert CO 2 (gas) to bicarbonate for transport in blood Release of H + by Hb is coupled to O 2 + binding (lungs). Release of H + helps convert bicarbonate back to CO 2 for exhalation. Text, Figure 7-12

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

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