Protein shimmy & shake

Hemoglobin is a complicated puzzle, not just a carrier of oxygen.

Hemoglobin- the stuff of love and horror, cupid's arrows and Dracula's lust.. what makes it tick? What even makes it red? It certainly isn't the protein. 16,000 daltons of clear protein carry a clear ~700 dalton heme group, whose iron, neatly caged in the middle, only absorbs green to blue light strongly when bound to oxygen. So a very small, if critically important, bit of the protein complex is responsible for all of its color. A bit like the heart being responsible for all of our love.

But the encasing protein does a lot more than just lug the heme ring and its iron around. It is responsible for a few special effects that make our breathing more efficient. These are called allosteric effects, for the way miscellaneous molecules can reshape or regulate a protein, especially its binding of other molecules. One is the Bohr effect, where CO2 (and acidity in general) regulates the oxygen affinity of hemoglobin. In the high CO2 / elevated acid environment of the peripheral tissues, oxygen affinity is reduced, while in the more neutral environment of the lungs, it is raised, appropriately enough.

Another is the cooperativity of the hemoglobin tetramer. Binding one oxygen increases the affinity of the other (and quite distant) three sites for oxygen, again helping speed the process of loading and unloading oxygen in the appropriate places. And a third is the Haldane effect, where, conversely to oxygen, the binding of CO2 is increased in the acidic environment of the periphery. CO2 doesn't bind to the central iron-heme site, but elsewhere on the protein molecule, facilitated by several acid-sensitive histidines.

Fourth is the action of carbon monoxide, which is not really allosteric, but simply competitive, binding 200 times better than oxygen to the central iron binding site, and thereby shutting it down completely, suffocating the victim. The encasing protein of course has other functions as well, and we see one in sickle cell anemia, where the normally cleanly separated tetramers that float around in the red blood cell at very high concentration (35 grams per 100g of packed red blood cells) start to gum up and aggregate due to a single point mutation, (when present in both genetic copies), leading to misshapen red blood cells, and all the other morbidities of this disease.

While most of this molecular intricacy is understood in rough terms, researchers are still nailing down details, and a recent paper used novel statistics and molecular modelling to tease out some more of them. Hemoglobin could be thought of having two stable shapes, called T (tense, with low oxygen affinity, high CO2 affinity), and R (relaxed, with high oxygen affinity, low CO2 affinity). The issue is that in the tetrameric hemoglobin complex of two alpha chains and two beta chains, binding one oxygen in the T state nudges not just its own unit of the tetramer, but all four, toward the R state.

Obviously this requires some complicated transmission mechanism, and that remains the subject of research, including this one. Here is a picture: 

Animation of a hemoglobin tetramer shape changes, focusing on one subunit (reddish) and especially its heme group as it binds O2 (teal).

The researchers tried to break down all the motions of the protein chains into two categories- those that change the relative positions of the four subunits, (quaternary), and those that only affect the internal shape of one subunit, (tertiary), without jutting out to affect the others. This was done with computer simulations based on the many known structures of this protein. Hemoglobin was naturally one of the first protein structures ever solved at the atomic level.

What they found was that they could statistically boil down each of these two classes of shape change into one main value (a principal component). Then the question was how these values and detailed motions relate to each other as the major transition goes along from state to the other. One aspect of the internal (tertiary) motions were clearly correlated to the inter-subunit repositioning, and so could be taken to be part of the allosteric mechanism by which one subunit communicates its binding of oxygen to the others. Much of this analysis is unfortunately cloaked on mathematical abstrusities, ensembles, hyperplanes, etc., so I can neither evaluate it nor fully present it.

They decide that they can differentiate between individual "pushing" and "pulling" contacts between subunits, which significantly channel the interaction. The whole story begins with the binding of oxygen, which pushes away protein arms that reach towards the central heme, and also induces that heme to bend from a bent, to a flat, planar shape:

Edge-on view of hemoglobin heme complex, bent without bound O2, and flat when O2 binds, along with a few other local rearrangements.

Table of dynamic contacts within and between hemoglobin subunits that stretch or switch as oxygen binds and the shape changes from R to T. The amino acids are referred to by single letter codes.

A pushing contact ...

 "A clear example for this is the interaction of lysine 82 of beta 1 and lysine 82 of beta 2 we observed: Close to the T-state both side chains are pointing into the solvent. While moving along cQ [the quaternary-only axis], the two chains approach each other and bring both positively charged side chains unfavourably close. The motion along cTew [the tertiary-only axis] relaxes this repulsive interaction by bending the N-terminal ends of the F helices (the helix notation goes back to Watson, Kendrew and Perutz [16]). Experimental studies introduced cross-links between the two lysines [17], [18]. The derived structure was described to be an intermediate between T- and R-state with characteristics of both states but no cooperativity."

 A pulling contact ...

 "These contacts only stay intact if the system moves along cQ [the quaternary-only axis] and cTew [the tertiary-only axis] together, but break if moving in one or the other direction independently. This is the expected behaviour for contacts which must remain intact for the allosteric mechanism to function. Exemplarily, this was observed for phenylalanine 117 of the alpha subunits and argenine 30 of the beta subunits. The hydrogen bond between the carboxylic oxygen of Phe and the side chain of Arg breaks while moving from the T-state towards the off-diagonal intermediate artificial states (see Figure 3), and forms again when approaching the R-state." 

 Which is to say that the contact stays intact during the actual transition, but was broken here in virtual model terms as the experimenters pursued one or the other of their axes (tertiary or quaternary) alone. It is a sign that this contact helps keep things together in a smooth and concerted fashion as the protein starts to change shape on one side.

A more dramatic type of contact is one that switches during the transition. In the alpha-beta interface, (shown below), contact between apartate 94 on the alpha subunit and tryptophan 37 on the beta subunit (in the T state) is broken and the tryptophan 37 switches to contact asparagine 102 on the beta subunit in the R state. These kinds of distinct shifts help stabilize each of the quasi-stable states, T and R, and the researchers identified four such "switching" contacts.

Cartoon of the subunit border area where tryptophan (W) 37 switches from asparagine (N) 102 to aspartate (D) 94 during the oxygen binding transition, among other changes.

It is remarkable that two atoms- O2- can switch the conformation of an enormous complex with an atomic weight of ~64,000. But it is all in a day's work for proteins, whose structures to start with are relatively floppy in the intense jostling of brownian motion. Putting those flops to work by evolving into structures with two stable states, tickle-able by joined-at-the-hip partners.. that is a bit more challenging, but obviously a life-saver given the absurdly inefficient breathing apparatus we are stuck with.

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