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Protein Dynamics IntroFrom rigid structures to motions on energy landscapesDo you all remember Anfinsen? What concept now associated with his name madeAnfinsen famous? Right, it is the concept that the structure of a functionally foldedprotein represents the global conformational energy minimum of that protein. Oneminimum -> one structure.Throughout the previous section of the course we always assumed that there was onesingle structure for the protein; i.e. a protein folds once and then stays in that folded stateuntil it is degraded by the cell.In the following few classes I hope I will convince you that proteins are by no meansrigid structures and that motion is, in fact, just as important for protein function asadopting a stable structure. The picture that should emerge at the end of this section isthat proteins achieve a fine balance between adopting a defined structure and maintaininga high degree of flexibility. A protein’s structure has to be stable enough to organize andorient side chains into active sites or to form specific protein-protein interactioninterfaces. At the same time the protein has to be flexible enough to let substrates intoactive sites or to under go conformational changes to transmit signals etc.The Folding-Unfolding Reaction is a dynamic Equilibrium.Lets think about an extreme case of protein dynamics first. We know that the stability ofa protein is often on the order of 10 kcal/mol. What fraction of the molecules then iscompletely unfolded, if we have our protein sitting around at room temperature?† DG = -RT lnKeqsoKeq= e-DGRTforDG = 10kcal / molKeq=117 ⋅106The calculation shows us that on average one in every 17 million molecules is completelyunfolded. Given that the folding/unfolding process of many proteins takes place on theorder of milliseconds, each of these protein molecules will visit the completely unfoldedstate within a few hours. Put a different way, while the energies that hold together aprotein will lead the vast majority of molecules to adopt a conformation very close to theoverall conformational energy minimum, thermal energy alone is sufficient to let proteinsexplore areas of conformational space that is very far from the global conformationalenergy minimum.We can measure this unfolding/refolding process using hydrogen deuterium exchangeand NMR. Many hydrogen atoms, like those bound to backbone-amide nitrogens, existin a rapidly exchanging equilibrium in which the protons dissociate and re-associate withthe protein. If the amid group is exposed to water the proton that dissociates is notnecessarily the same proton that re-associates and a proton originally bound to the proteinis rapidly exchanged with a proton from solvent.If we take a protein and place it in deuterated water, then those hydrogen atoms that arechemically predisposed to exchange and are exposed to the water will be replaced bydeuterium ions. NMR spectroscopy allows us to distinguish between normal hydrogenatoms and deuterium atoms. If we set our NMR spectrometer to record hydrogen dataonly, then we can follow the rate with which individual hydrogen atoms are replaced bydeuteriums atoms.The experimental observations we get from those hydrogen-deuterium exchangeexperiments agree quite well with the predictions from our unfolding/refolding picture.Small, fast-folding proteins exchange the majority of their amide hydrogens, even thosethat are completely buried in the protein’s interior in the fully folded state, within a fewhours. The only possible explanation is that each protein has sampled a completelyunfolded conformation at least once during this time period.Protein Motion a nuisance or a necessityI hope this little example above convinces you that protein motions, up to and includingthe complete unfolding, are very real and commonplace. The question now is whetherprotein motion is just a nuisance, something the protein has to live with, or is proteinmotion actually essential for protein function? There are two lines if evidence that thelatter is true, that protein motions are essential for protein function!The first line of evidence comes from protein structure. The active sites of many enzymesare completely buried in the interior of the protein and are not accessible to solvent, letalone a substrate molecule. Myoglobin and Acetylcholine esterase are two classicexamples. In both cases, the structure of the free protein gives no indication of how thesubstrate might possibly be able to get into the active site.The second line of evidence is the temperature dependence of protein motions and ofprotein function. Most proteins undergo a glass transition around 200 deg. Kelvin. Abovethis temperature proteins display relatively large and concerted molecular motions, belowthis temperature molecular motions seem to be restricted to simple, small-scale, harmonicvibrations. It is possible to see this transition in protein dynamics by a variety ofexperimental methods including dynamic neutron scattering, Moesbauer spectroscopy,and hole burning spectroscopy as well as X-ray crystallography. Associated with thecessation of these larger-scale, concerted and non-harmonic motions is a dramatic dropoff in protein function. Enzyme catalysis for example does not just slow down, it stopscompletely below this temperature. Greg Petsko and Dagmar Ringe have written a verynice review article on this topic. (Ringe D, Petsko GA. Biophys Chem. Vol. 105, pp. 667-80 (2003)).Two physical pictures of molecular motionNow that I have hopefully convinced you about the importance of protein motion, letstake a step back and think about the motions of individual atoms and see if this can teachus something about the way proteins move.One way we can think about the motion of atoms in a protein is as a Newtonian particle,i.e. a particle that has a certain mass and a certain amount of kinetic energy and that thisparticle just keeps going in a straight line, until it experiences a force that alters itscourse. An alternative picture is that of a diffusing particle propelled by Brownian motionon a stochastic trajectory. Lets do some model calculations for both models.Atoms as Newtonian ParticlesKinetic gas theory tells us that thermal and kinetic energy of a molecule is one and thesame thing. Specifically, this theory predicts that the translational energy of a moleculeis 3/2 kT. Lets use this information to calculate the speed with which a carbon atommoves at room temperature. How long does it take


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Brandeis BCHM 104A - Protein Dynamics Intro

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