Brandeis BCHM 104A - Tools for studying protein dynamics

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Tools for studying protein dynamicsThe goal of this lecture is to frame protein dynamics in the conceptual framework ofprotein conformational energy landscapes and to discuss the tools we can use todetermine the shape of these landscapes.Charting conformational energy landscapesAs I pointed out during last class our understanding of protein dynamics and its role inprotein function is still in its early infancy. All tools currently available to the study ofprotein dynamics have serious flaws. There is no single technique that allows us to get afull picture of protein motion. In a way we are similar to early cartographers thatgenerated geographic maps by combining direct observations with indirect measurementsand educated guesses. For example, old cartographers used to judge the minimal size of alandmass based on the size of rivers and the number and type of bird species they couldobserve from their boats. If the rivers were big and land birds dominated, then thecartographer would draw a very large land mass even though they had never actually setfoot on the land.The protein dynamics equivalent of a perfect topographic map would be a perfect map ofthe protein energy landscape. We are currently far from such a perfect map and so we arestuck like the early explorers with rough sketches of energy landscapes that represent acombination of experimental observations with a lot of conjecture. However, we knowthat a protein energy landscape exists and that knowing its shape will tell us everythingthere is to know about protein dynamics. So get used to thinking about protein dynamicsin terms of energy landscapes.To get you started, here are three energy landscapes. What are the dynamic properties ofthe molecules that posses these landscapes?ABCThe energy landscape of molecule A is quite flat. There are many different local minimathat all have about the same energy and the barriers between them are rather small. As aresult molecule A would be quite “floppy” or “unstructured”; i.e. molecule A would nothave one dominant structure, but would rapidly exchange between many differentconformations in a diffusion-like manner.The energy landscape of molecule B has one global energy minimum and this minimumis much lower in energy than all other conformations. As a result in a population of type-B molecules the vast majority of molecules will adopt a very similar and well-definedconformation. Most molecular motions of B will be restricted to small structuralfluctuations around the energy minimum. A population of molecule of type C will showthree sub populations of molecules with potentially significant differences in theirstructure as well as their biological properties. Because the energy barriers between themultiple minima are relatively large, the rate with which these populations exchange willbe quite slow.Now that you are hopefully a little more comfortable with the concept of conformationalenergy landscapes lets think about ways to determine the shape of these landscapes.Two approaches to studying protein dynamicsThere are basically two techniques for studying protein dynamics in bulk (i.e. using non-single-molecule techniques). One way is to study equilibrium dynamics. That is we lookat a population of molecules that explore a conformational energy landscape and thisenergy landscape stays the same throughout the entire experiment.The second approach is often referred to as the perturbation-relaxation approach. In thistechnique we introduce a perturbation to our population of molecules that transientlychanges the shape of the conformational energy landscape. As a result we change thedistribution of molecules in our energy landscape. As we remove the perturbation, we cannow follow the process in which the distribution of conformations re-equilibrates to theunperturbed state.Techniques for studying protein dynamicsBelow I will introduce two techniques, which have been used extensively to studyconformational energy landscapes of proteins. I will not have time to cover thesetechniques in detail, so I will just give you a quick overview of these techniques.Two other techniques, X-ray diffraction and NMR spectroscopy will receive their ownlectures.Hole burning spectroscopyThe study of protein conformational energy landscapes has been dominated by oneprotein and one experimental technique. The protein is myoglobin, the oxygen carrier inyour muscles, and the technique is hole burning spectroscopy. Outside this very specificfield, hole burning spectroscopy has not seen much use in the lifesciences, but thetechnique is ideally suited to explain the idea of conformational energy landscapes so Iwill spend some time explaining it to you.The UV-visible absorption bands of light-absorbing ligands bound to protein molecules–generally referred to as chromophores- are usually quite broad. The reason for this isthat the protein’s constant conformational fluctuations continuously alter thechromphore’s molecular environment and thereby alter the energetic difference betweenthe chomophore’s electronic ground state and its electronically excited state. If aparticular conformational state the electronic ground state but disfavors the electronicallyexcited state, then the energy difference between the two electronic states is higher, so thephoton energy that promotes this transition will be greater and the absorption band willbe shifted towards the blue.If this is unclear to you, you may want to go back and have a look at a book on UV-visible spectroscopy. The main thing you have to understand for hole-burningspectroscopy is that conformational changes in the protein alter the molecularenvironment of the chromophore and that these change in the chromophore’s molecularenvironment cause a corresponding shift in its absorption spectrum. In other words theabsorption spectrum we observe for a solution containing many molecules is really thesum of the absorption spectra contributed by a protein’s different conformational states.Each energy minimum in the conformational energy landscape on the right correspondsto a slightly different protein conformation providing a slightly different molecularenvironment for the chromophore and therefore resulting in a slightly different absorptionspectrum. So the overall absorption spectrum reflects the conformational variability ofthe conformational energy landscape.Hole burning spectroscopy exploits this very close relationship between the absorptionspectrum and


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