Role of C-terminal region of Staphylococcal nuclease for foldability, stability, and activity.

The role of the C-terminal region of Staphylococcal nuclease (SNase) was examined by deletion mutation. Deletions up to eight residues do not affect the structure and function. The structure and enzymatic activity were partially lost by deleting Ser141-Asn149 (Delta141-149), and deletion of Trp140-Asn149 (Delta140-149) resulted in further loss of structure and activity. A 13-residue deletion showed the same effect as the 10-residue deletion. Both Ser141Gln and Ser141Ala mutations for an eight-residue deletion mutant did not alter properties as well as Ser141A1a for full-length SNase. In contrast, Trp140Ala mutation for Delta141-149 shows the same effect as the deletion of Trp140. Trp140Ala mutation for full-length SNase causes the loss of native structure. These observations indicate the significance of the 140th and the 141st residues. The side-chain of the 140th residue is required to be tryptophan; however, the backbone of the 141st residue is solely critical for foldability, but the side-chain information is not crucial. All of the mutants that take a non-native conformation show enzymatic activity and inhibitor-induced folding, suggesting that foldability is required for the activity.

Structural change of site-directed mutants of PYP: new dynamics during pR state.

The energetics, protein dynamics, and diffusion coefficients of three mutants of photoactive yellow protein, R52Q, P68A, and W119G, were studied by the transient grating and pulsed laser-induced photoacoustic method. We observed a new dynamics with a lifetime of approximately 1 micro s in the transient grating signal, which is silent by the light absorption technique. This fact indicates that, after the structure change around the chromophore is completed (pR(1)), the protein part located far from the chromophore is still moving to finally create another pR (pR(2)) species, which can transform to the next intermediate, pB. Although the kinetics of pR(2)-->pB-->pG are very different depending on the mutants, the enthalpies of the first long-lived (in micro seconds, 100-micro s range) intermediate species (pR(2)) are similar and very high for all mutants. The diffusion coefficients of the parent (pG) and pB species of the mutants are also similar to that of the wild-type photoactive yellow protein. From the temperature dependence of the volume change, the difference in the thermal expansion coefficients taken as indicator of the flexibility of the structure between pG and pR(2) is measured. They are also similar to that of the wild-type photoactive yellow protein. These results suggest that the protein structures of pR(2) and pB in these mutants are globally different from that of pG, and this structural change is not altered so much by the single amino acid residue mutation. This is consistent with the partially unfolded nature of these intermediate species. On the other hand, the volume changes during pR(1)-->pR(2) are sensitive to the mutations, which may suggest that the volume change reflects a rather local character of the structure, such as the chromophore-protein interaction.

Effect of organic anions on the photoreaction of photoactive yellow protein.

In order to clarify changes in the structure and surface properties of photoactive yellow protein (PYP) upon light absorption, the spectroscopic properties and solution structure of its photo-intermediate (PYP(M)) were examined in the presence of various anions. At identical ionic strengths, citrate slowed the decay rate of PYP(M) more than acetate. Although the absorption spectrum in the dark was not affected by organic anions, citrate induced a 5-nm blue shift of the absorption maximum for PYP(M). Solution X-ray scattering experiments indicated that the radius of gyration (Rg) and apparent molecular weight in the dark were constant in all buffer systems. However, the Rg of PYP(M) in citrate buffer at high concentration was 16.2 (+/-0.2) A, while the Rg of PYP(M) in acetate buffer was 15.6 (+/-0.2) A. The apparent molecular weight increased 7% upon PYP(M) formation in citrate buffer at high concentration compared to other conditions. These results suggest that citrate molecules specifically bind to PYP(M). A cluster of basic amino acid residues with a hydrogen bond donor would be exposed upon PYP(M) formation and responsible for the specific binding of citrate.

MR arthrography of shoulders after suture-anchor Bankart repair.

PURPOSE: To determine the ability of magnetic resonance (MR) arthrography to depict the anatomic reestablishment of the capsulolabral complex after suture-anchor Bankart repair. MATERIALS AND METHODS: Thirty patients (25 men, five women; mean age, 28 years) who had undergone suture-anchor Bankart repair of one shoulder underwent MR arthrography before second-look arthroscopy. Ninety-eight anchors were used for the sutures. MR arthrographic diagnosis of anatomic reestablishment of the capsulolabral complex was correlated with arthroscopic findings. Contingency table analysis was performed to determine the relationship between MR arthrographic findings and arthroscopic findings. RESULTS: MR findings of reattachment of the capsulolabral complex were in agreement with arthroscopic findings in 93 anchor points (accuracy, 93 of 98 anchor points; 95%). In 28 shoulders, oblique transverse images obtained with the shoulder in the abduction and external rotation position showed that the anterior band of the inferior glenohumeral ligament (AIGHL) abutted the humeral head and that reattachment of the AIGHL to the glenoid rim was seamless. Arthroscopy revealed satisfactory reestablishment of the capsulolabral complex in these shoulders. In the remaining two shoulders, a pool of contrast material was seen between the AIGHL and humeral head and a "divot" was detected at the point of reattachment of the AIGHL to the glenoid rim. Arthroscopy revealed unsatisfactory reestablishment of the capsulolabral complex. MR arthrographic findings of reattachment of the AIGHL were significantly associated with arthroscopic findings of reestablishment of the capsulolabral complex (P <.01). CONCLUSION: MR arthrography can be reliably used for the postoperative assessment of suture-anchor Bankart repair.