Hirotsugu Tsuruta

Evidence of an associative intermediate on the myoglobin refolding pathway

Time-resolved small-angle x-ray scattering using the stopped-flow method has been applied successfully to investigate the refolding of myoglobin. This is the only method to date that yields direct information on protein physical dimensions during the folding process. It has the potential to detect and probe important processes, such as protein compaction and association, on a millisecond time scale. Initial experiments were performed with horse myoglobin denatured in high concentrations of urea. The denatured protein was diluted rapidly into a buffer containing no urea or low concentrations of urea. The time-course of the forward-scattered intensity shows a decrease in amplitude which is clearly not engendered by the compaction of the protein, but does correspond well to a dimer dissociation process. Initial and final radii of gyration correspond well to a dimer and a monomer, respectively. Kratky plots of the initial and final states also support the transient dimerization model. The apparent dissociation rate constant was obtainable directly from the data. An association rate constant and an equilibrium constant could be estimated. The dimerizing intermediate is speculated to be a globular non-native state with an exposed hydrophobic surface.

Comparing crystallographic and solution structures of nitrogenase complexes

A low-resolution structure from X-ray scattering data of Kp1•(ADP•AlF-_4•Kp2)2 predicted a significant change in the iron protein (Kp2) upon complex formation. This has been subsequently confirmed by the crystallographic structure of the complex in the Av system. New scattering results are provided to demonstrate the similarity of this complex in the two species.

Direct observation of an altered quaternary-structure transition in a mutant aspartate transcarbamoylase

Time‐resolved small‐angle X‐ray scattering (TR‐SAXS) was used to monitor the structural changes that occur upon the binding of the natural substrates to a mutant version of the allosteric enzyme aspartate transcarbamoylase from Escherichia coli, in which the creation of a critical link stabilizing the R state of the enzyme is hindered. Previously, SAXS experiments at equilibrium showed that the structures of the unligated mutant enzyme and the mutant enzyme saturated with a bisubstrate analog are indistinguishable from the T and R state structures, respectively, of the wild‐type enzyme (Tauc et al., Protein Sci. 3:1998–2004, 1994). However, as opposed to the wild‐type enzyme, the combination of one substrate, carbamoyl phosphate, and succinate, an analog of aspartate, did not convert the mutant enzyme into the R state. By using TR‐SAXS we have been able to study the transient steady‐state during catalysis using the natural substrates rather than the nonreactive substrate analogs. The steady‐state in the presence of saturating amount of substrates is a mixture of 60% T and 40% R structures, which is further converted entirely to R in the additional presence of ATP. These results provide a structural explanation for the reduced cooperativity observed with the mutant enzyme as well as for the stimulation by ATP at saturating concentrations of substrates. They also illustrate the crucial role played by domain motions and quaternary‐structure changes for both the homotropic and heterotropic aspects of allostery. Proteins 31:383–390, 1998.

The kinetics of conformational changes of α2-macroglobulin determined by time resolved X-ray solution scattering

The rate of gross conformational change of α2‐macroglobulin (α2M) during its proteinase trapping was directly determined for the first time using time‐resolved X‐ray solution scattering. Decrease of radius of gyration was observed under pseudo‐first‐order conditions with excess proteinases, which exhibited a monophasic timecourse. The rate constants were 0.5 ± 0.1 s−1 and 0.8 ± 0.2 s−1 for the reaction with chymotrypsin and trypsin, respectively. There was no concentration dependence of the observed rate constants. Therefore, the rate‐limiting step of the gross conformational change was not the bimolecular encounter reaction between α2M and proteinases, which requires a new proposal of pre‐trapping of proteinases before the gross conformational change.