Kazumoto Kimura

Unfolding of dimeric creatine kinase in urea and guanidine hydrochloride as measured using small angle X-ray scattering with synchrotron radiation

The denaturation of dimeric creatine kinase (CK) induced by urea and guanidine hydrochloride (GuHCl) has been studied by small angle X‐ray scattering (SAXS), which is a direct way to measure the changes in the overall dimensions of a protein molecule. The radii of gyration (R g) of CK are 29±0.4 Å in the native state and 46±1.5 Å in the unfolded state in either 8 M urea or 3 M GuHCl. The transition curves of urea denaturation derived from the R g values and the zero angle intensity (I(0)) are similar to that from intrinsic fluorescence, indicating that the changes in the molecular shape, the tertiary structure and the dissociation of the subunits proceed simultaneously. In the case of GuHCl‐induced denaturation, the dramatic increases both in R g and in I(0) in 0.3–0.5 M GuHCl suggest clearly that soluble aggregates form at low GuHCl concentrations. The aggregates dissociate and the molecule unfolds at higher GuHCl concentrations. The results suggest that the mechanisms of CK denaturation in urea and in GuHCl are somewhat different and the intermediate in GuHCl denaturation can much more easily form soluble aggregates.

Stopped‐flow apparatus for x‐ray scattering at subzero temperature

A stopped‐flow apparatus was constructed for x‐ray scattering study at subzero temperature. It can be operated over a wide temperature range down to −20 °C with highly viscous solution (20 cP) successfully. We have applied the stopped‐flow x‐ray scattering method to many biological reactions. In particular, the association of E. coli ribosomal subunits was detected at −10 °C which was too fast to be detected at room temperature. Dissociation of E. foetida hemoglobin was measured by the stopped‐flow x‐ray scattering method combined with a time‐resolved imaging plate as a detector.

GroES co‐chaperonin small‐angle X‐ray scattering study shows ring orifice increase in solution

GroES consists of seven identical 10 kDa subunits and is involved in assisting protein folding as the partner of another oligomeric protein, the GroEL chaperonin. Here we studied the GroES structure in solution using small‐angle X‐ray scattering (SAXS). The SAXS pattern, calculated for the GroES crystal structure, was found to be different from the experimental one measured in solution. The synchronic shift in the radial direction and some turning of the protein subunits eliminate the difference and result in the increase of the hole diameter in the GroES ring‐like structure from 8 Å in the crystal to 21 Å in solution.

Structural kinetics of the allosteric transition of aspartate transcarbamylase produced by physiological substrates

We have studied the kinetics of the quaternary structure change associated with the allosteric transition of aspartate transcarbamylase (ATCase) (E. coli), inducing this change by exposure to the natural substrates (carbarnyl phosphate and L‐aspartate). The presence of 30% ethylene glycol slowed the quaternary structure change sufficiently for it to be followed by stopped‐flow X‐ray scattering at − 5°C. After adding substrates to the enzyme, the change occurred, with a half‐life of a few seconds, yielding a mixture of the two standard quaternary structures (or, conceivably, a state intermediate between them). This mixture persisted until the enzyme reduced the substrate concentration below a threshold value.

The compactness of ribonuclease A and reduced ribonuclease A

The compactness of ribonuclease A with intact disulfide bonds and reduced ribonuclease A was investigated by synchrotron small‐angle X‐ray scattering. The R g values and the Kratky plots showed that non‐reduced ribonuclease A maintain a compact shape with a R g value of about 17.3 Å in 8 M urea. The reduced ribonuclease A is more expanded, its R g value is about 20 Å in 50 mM Tris‐HCl buffer at pH 8.1 containing 20 mM DTT. Further expansions of reduced ribonuclease A were observed in the presence of high concentrations of denaturants, indicating that reduced ribonuclease A is more expanded and is in neither a random coil [A. Noppert et al., FEBS Lett. 380 (1996) 179–182] nor a compact denatured state [T.R. Sosnick and J. Trewhella, Biochemistry 31 (1992) 8329–8335]. The four disulfide bonds keep ribonuclease A in a compact state in the presence of high concentrations of urea.

Solution X-ray scattering study on the chaperonin GroEL from Escherichia coli

The molecular architecture of native GroEL has been studied by solution X-ray scattering. The radius of gyration for the native molecule was estimated to be 66.0 A in 50 mM Tris-HCl, 100 mM KCl at pH 7.5 and 25 degrees C. The maximum dimension was estimated to be 170 A, based on the pair distance distribution function. A cylindrical structure or two heptameric rings was found to be the best for native GroEL among structures examined by using a multi-sphere model analysis in which the radius of constituent sphere was 6 A. The results of the model analysis show that the radius of GroEL is 68.0 A and the height is 150.7 A. Unexpectedly, the central penetrating hole through GroEL was not confirmed in the best-fit structure.

Dynamics of oligomer formation by denatured carbonic anhydrase II

Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. Many proteins unrelated to amyloidoses also fibrillate at the appropriate conditions. These proteins serve as a model for studying the processes of protein misfolding, oligomerization and fibril formation. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation. The urea-induced unfolding of bovine carbonic anhydrase II, BCA II, is characterized by a combination of high-resolution NMR, circular dichroism spectroscopy and small angle X-ray scattering. It is shown that the formation of associates of protein molecules in complex with solvent (water and urea), APS, takes place in the presence of 4-6 M urea. The subsequent increase in urea concentration to 8 M is accompanied by a disruption of APS and leads to a complete unfolding of a protein molecule. Analysis of BCA II self-association in the presence of 4.2 M urea revealed that APS are relatively large mostly beta-structural blocks with the averaged molecular mass of 190-220 kDa. This work also demonstrates some novel NMR-based methodological approaches that provide useful information on protein self-association.

Kinetic studies of unfolding process of aspergillopepsin II by pH-jump methods

pH-induced unfolding process of the acid proteinase aspergillopepsin II was studied kinetically by stopped-flow methods using circular dichroism and small angle X-ray scattering. Native aspergillopepsin II consists of two polypeptide chains, a light chain and a heavy chain, which are bound noncovalently to each other at acidic pH. Above neutral pH the two chains are known to dissociate and swell due to electrostatic repulsion inside the molecule. The present studies suggested that the polypeptide chains dissociate first and that the dissociated heavy chain expands subsequently. This two-phased pathway is consistent with the accumulation of an intermediate species in the pH-induced unfolding of the enzyme.

Dissociation of Limulus polyphemus (horseshoe crab) hemocyanin. I. Solution X-ray scattering study in equilibrium.

A solution X-ray scattering study has been performed on Limulus polyphemus (horseshoe crab) hemocyanin and its dissociated fragments at various pH values in the presence and absence of Ca2+. The scattering patterns of native hemocyanin (48-mer), the half molecule (24-mer), quarter molecule (12-mer) and monomer fraction were measured. The radii of gyration for the four molecular species were calculated from the Guinier plots to be 110.7, 91.3, 77.3, and 36.5 A, respectively. Models which yield good fits to the experimental data are presented. The models were constructed using eight, four and two spheres with a radius of 58 A, assuming the sphere to be the submultiple composed of six subunits. The radii of gyration were calculated on the basis of the model and the values found to be 106, 94 and 73 A, respectively, in good agreement with the experimental results.

Calcium Dependent Allosteric Modulation and Assembly of the Giant Hemoglobin from the Earthworm, Eisenia Foetida

Extracellular hemoglobins(Hb) from annelids have been known to show no heterotropic interaction with the organic phosphate, such as 2,3-diphosphoglycerate. In an attempt to investigate allosteric effectors of annelid Hb, we estimated the oxygen equilibrium of Eisenia Hb and found that Ca plays an important role on the modulation of the oxygen affinity and cooperativity, as a result of the binding to the oxy-form of the Hb, under a physiological range of Ca concentration(1).