Ultrafast Biophysical Studies of Proteins: Time-resolved WAXS Studies

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Abstract

Time-resolved Laue crystallography, as implied by its name, can only be performed on crystalline samples. The intermolecular forces that maintain crystalline order constrain large amplitude conformational motion, and this loss of flexibility may perturb or even inhibit the function of a protein. Nonetheless, Laue crystallography stands alone in its ability to acquire near-atomic structural information on ultrafast time scales. On the other hand, X-rays can also extract structural information from molecules in solution where the full range of conformational motion is permitted. Because there is no long-range order in protein solutions, Wide-Angle-X-ray-Scattering (WAXS) from the protein is diffuse. However, diffraction rings in the WAXS pattern are influenced by the size and shape of the protein. Though this structural information is not at atomic resolution, it does provide a fingerprint that can be correlated via models with the protein structure. Time-dependent changes of the WAXS fingerprint can therefore be used to assess which models best describe the reaction pathway. [unreadable] [unreadable] When we first set out to study the quaternary structure transition of hemoglobin with this technique, we observed a substantial change of the WAXS spectrum at the earliest time we could measure, which suggested that the WAXS spectrum is sensitive to tertiary structure changes as well as quaternary structure changes. To prove this point, we recorded the WAXS spectrum of photolyzed carbon monoxymyoglobin (MbCO) and observed a sizable change in the WAXS pattern. According to X-ray structures of MbCO and deoxy Mb, the rms difference in their atomic positions is less than 0.5 Angstrom. This unexpected ability to sense such small but systematic structure changes is quite encouraging, and has spurred us to continue our efforts to develop this new experimental methodology.[unreadable] [unreadable] Our efforts to characterize the quaternary structure transition by time-resolved WAXS has proven more challenging than envisaged. One of the problems faced in these studies is the substantial geminate ligand rebinding that follows laser photolysis. Even though we can photolyze the majority of the bound ligands with a 2-ns pump pulse, more than 30% of them rebind after the pump pulse is over, and a heterogeneous mixture of ligation states are formed. Some of these states are capable of undergoing the R→ T quaternary structure transition, but at different rates. For example, it is known that Hb(CO)2 can undergo a quaternary structure transition, provided the two ligands are not both bound to alpha chains or both bound to beta chains. One would expect HbCO to undergo the transition at a faster rate, and Hb (no bound ligands) would be fastest. To produce a more homogeneous distribution of ligation states, we sacrificed our time resolution by photolyzing the Hb(CO)4 with a 100-150 ns duration laser pulse. Because the pump pulse was long compared to the 33 ns geminate rebinding time, the laser pulse had multiple opportunities to drive CO away from the primary docking site and thereby generated a high population of Hb with no CO bound. Samples for these studies were prepared at a relatively high concentration (2 mM) to maximize the protein scattering signal. At this concentration, complete photolysis produces a CO concentration in solution of 9 mM, which is 9-fold higher that the equilibrium concentration of CO in the solution (when equilibrated with 1 atm CO). The excess CO leads to rapid bimolecular rebinding to the hemes, which ultimately limits the fractional population that can attain the T state. We aim to continue these studies at a lower protein concentration; however, our desire to further this project has been thwarted by a lack of beamtime at the ESRF. Thankfully, preliminary studies at the APS have demonstrated that we will be able to pursue these experiments in the USA in FY2009. [unreadable] [unreadable] The ability to extract structural information from proteins in solution will help pave the way to study proteins involved in signaling, in which global conformational changes are required. Because the shape of the WAXS spectrum can be calculated from a structural model, the temporally evolving fingerprint will provide incisive information that can be used to validate putative models for the reaction pathway. Through our collaboration with the group of Dr. Gerhard Hummer, we aim to further explore this possibility. Preliminary analysis suggests that their computational methods are capable of reproducing some of the fine details observed in time-resolved WAXS studies of photolyzed MbCO.