Techniques

Spectroscopy

Spectroscopy is chiefly the study of how light interacts with matter; because the electromagnetic spectrum contains such a wide variety of light (visible, ultraviolet, infrared, microwave, etc.) we are able to gain a large amount of information by studying how various types of light interact with our heme proteins. By using ultraviolet and visible light (UV-Vis), we can gain general information about the electronic structure of our protein and heme cofactor by monitoring at which wavelength(s) each absorbs. If we concentrate a laser using visible light with a wavelength near where the heme absorbs, we can gain information about the oxidation state and ligation motif of the heme by monitoring inelastically scattered light in a technique known as resonance Raman (rR) spectroscopy. By examining the change in the light that is emitted following absorption (fluorescence), we can gain information about the changing environment of aromatic residues or selectively placed fluorophores in the protein. Monitoring the differential absorption of circularly-polarized light (CD) can give us information about the secondary structure of the protein; if we add a superconducting magnetic (up to 7 Tesla!) with a field parallel to the propagating CD light (MCD) we can further resolve the electronic absorption spectrum and discern the spin state and ligation motif of the iron in the heme cofactor. To probe the unpaired spin density on the heme, we can apply microwave radiation in the presence of a magnet in a technique known as electron paramagnetic resonance (EPR) spectroscopy. In the Burstyn group, we use all of these techniques to gain an insight into the nature of our heme proteins.

 

Thermodynamics

Not only is the Burstyn group concerned with the mechanisms of allosteric control for heme-containing proteins, but it is also interested in the thermodynamics which govern these processes.  A modern perspective of protein behavior views proteins as dynamic systems which achieve a broad population of conformational states in a relatively short time frame.  One of the models of allostery suggests that the population of conformational states changes depending on whether the protein is effector-bound or free; the effector-bound form of the protein shifts the dynamic equilibrium towards a set of global conformations which promote the “effector activated” function of the protein.  The Burstyn group is interested in studying the thermodynamics of both the free and effector-bound protein in order to get a better picture of how effector binding causes global changes to the protein, how cooperativity between protein domains plays a role in this process, and how effector binding subsequently “turns on” or “turns off” protein function through a redistribution of conformational states.  Protein unfolding studies have already been done for the Fe (III) form of CooA transcription factor in guanidinium hydrochloride, using heme absorption, tryptophan fluorescence, and α-helical ultraviolet circular dichroism to monitor the progress of unfolding.  The results are summarized in the below figure.

 Thermodynamic studies