A specific intermolecular association between the regulatory domains of a Tec family kinase.

Interleukin-2 tyrosine kinase (Itk), is a T-cell specific tyrosine kinase of the Tec family. We have examined a novel intermolecular interaction between the SH3 and SH2 domains of Itk. In addition to the interaction between the isolated domains, we have found that the dual SH3/SH2 domain-containing fragment of Itk self-associates in a specific manner in solution. Tec family members contain the SH3, SH2 and catalytic domains common to many kinase families but are distinguished by a unique amino-terminal sequence, which contains a proline-rich stretch. Previous work has identified an intramolecular regulatory association between the proline-rich region and the adjacent SH3 domain of Itk. The intermolecular interaction between the SH3 and SH2 domains of Itk that we describe provides a possible mechanism for displacement of this intramolecular regulatory sequence, a step that may be required for full Tec kinase activation. Additionally, localization of the interacting surfaces on both the SH3 and SH2 domains by chemical shift mapping has provided information about the molecular details of this recognition event. The interaction involves the conserved aromatic binding pocket of the SH3 domain and a newly defined binding surface on the SH2 domain. The interacting residues on the SH2 domain do not conform to the consensus motif for an SH3 proline-rich ligand. Interestingly, we note a striking correlation between the SH2 residues that mediate this interaction and those residues that, when mutated in the Tec family member Btk, cause the hereditary immune disorder, X-linked agamaglobulinemia.

NMR structural studies of human cystatin C dimers and monomers.

Human cystatin C undergoes dimerization before unfolding. Dimerization leads to a complete loss of its activity as a cysteine proteinase inhibitor. A similar process of dimerization has been observed in cells, and may be related to the amyloid formation seen for the L68Q variant of the protein. Dimerization is barrier controlled, and no dimer/monomer interconversion can be observed at physiological conditions. As a consequence, very stable, "trapped" dimers can be easily separated from monomers. A study of the structural aspects of cystatin C dimer formation was undertaken using NMR spectroscopy. The monomer/dimer model was verified by (pulse field gradient NMR) self-diffusion molecular mass measurements. Complete backbone resonance assignments and secondary structure determination were obtained for the monomer using data from triple resonance experiments performed on 13C/15N doubly labeled protein. A marked similarity of the cystatin C secondary structure to that of chicken cystatin was observed. Using uniformly and amino-acid-specific 15N-enriched protein, backbone NH signals were assigned for cystatin C in its dimeric state. Comparison of 1H -15N correlation NMR spectra of the monomer and dimer shows that the three-dimensional structure remains unchanged in the dimer and that only local perturbations occur. These are localized to the amino acid residues comprising the cysteine proteinase binding site. Such a mode of dimerization readily explains the complete loss of the inhibitory activity in the dimer. The NMR results also demonstrate that the dimer is symmetric.

ROESY with water flip back for high-field NMR of biomolecules.

We report a version of the ROESY experiment in which saturation of the water magnetization is avoided without compromising suppression of the water signal during acquisition. Field gradient and selective RF pulses are used to maintain precise control of the water magnetization throughout the experiment and avoid signal losses due to radiation damping and molecular diffusion effects. The pulse sequence includes a delay for intentional radiation damping prior to mixing period. The optimal length of this delay is field and sample dependent, but easily determined from the apparent linewidth of the water signal. NOESY and TOCSY variants of the same experiment are presented which make use of identical manipulations of the water magnetization. The three pulse sequences constitute a suite for which little parameter adjustment is required once one of the experiments has been configured.

Does solvent influence the ground-state tautomeric population of hypericin?

Nuclear magnetic resonance measurements indicate that hypericin exists in the same "normal" tautomeric form irrespective of whether the solvent is dimethyl sulfoxide or tetrahydrofuran. This result is discussed in the context of previous experimental and theoretical work. It is concluded that solvent perturbations cannot induce tautomerization in hypericin.

Action of Azotobacter vinelandii poly-beta-D-mannuronic acid C-5-epimerase on synthetic D-glucuronans.

Eleven different glucans (wheat starch, potato amylopectin, potato amylose, pullulan, alternan, regular comb dextran, alpha-cellulose, microcrystalline cellulose, CM-cellulose, chitin, and chitosan) that had their C-6 primary alcohol groups oxidized to carboxyl groups by reaction with 2,2,6,6-tetramethyl-1-piperidine oxoammonium ion (TEMPO), were reacted with Azotobacter vinelandii poly-beta-(1-->4)-D-mannuronic acid C-5-epimerase. All of the oxidized polysaccharides reacted with the C-5-epimerase, as evidenced by comparing: (1) differences in the relative viscosities; (2) differences in the carbazole reaction; (3) differences in their susceptibility to acid hydrolysis, and (4) differences in their ability to form calcium gels, before and after reaction. We further show the formation of L-iduronic acid from D-glucuronic acid for oxidized and epimerized amylose by 2D NOESY and COSY + 1H NMR.

Distal interphalangeal joint arthrodesis: an analysis of complications.

One hundred thirty-nine patients underwent 181 arthrodeses of finger distal interphalangeal joints (144) and/or thumb interphalangeal joints (37). Techniques included (1) crossed Kirschner pins (111 joints), (2) interfragmentary wire and longitudinal Kirschner pin (43 joints), and (3) Herbert screw (27 joints). Each technique had a similar nonunion rate. There were 21 nonunions: 13 were pain free, 6 were successfully fused on the second attempt, 1 was painful (but the patient refused further surgery), and 1 was amputated. Inadequate bone stock, inadequate bone resection, premature pin removal, and infection appear to complicate the attainment of bony union. Twenty percent of the fusions had major complications (nonunion, malunion, deep infection, and osteomyelitis). Minor complications (dorsal skin necrosis, cold intolerance, proximal interphalangeal joint stiffness, paresthesias, superficial wound infection, and prominent hardware) occurred in 16% of the joints fused.

Trapeziometacarpal arthrodesis in primary osteoarthritis: a minimum two-year follow-up study.

We retrospectively reviewed 49 patients with primary osteoarthritis who underwent 59 trapeziometacarpal (TM) arthrodeses to assess the efficacy of this procedure with respect to patient satisfaction and radiographic evidence of peritrapezial arthritis. The average patient age was 54 years (range, 41-73 years) and the average follow-up period was 7 years (range, 2-20 years). All TM arthrodeses used K-wires; 61 had a supplemental distal radius bone graft. There were 10 bilateral arthrodeses. There were 4 (7%) nonunions. Three nonunions were painless and 1 was painful. There were 2 patients who required a second procedure; 1 had a painful nonunion that was successfully treated with a repeat arthrodesis and the other had a solid but painful thumb that was treated with trapezial excision and soft tissue interposition. The average pain score at the time of the follow-up examination was 1.5 of 10. Thirty-three patients had postoperative radiographs; 7 had radiographic evidence of peritrapezial arthritis. Based on our findings we believe that isolated, symptomatic TM osteoarthritis in patients older than 40 years old is an indication of TM arthrodesis.

Gradient-enhanced TOCSY experiments with improved sensitivity and solvent suppression.

Gradient-enhanced versions of the homonuclear TOCSY experiment are described, with solvent suppression and sensitivity superior to that of a conventional TOCSY experiment. The pulse sequences are constructed by appending a WATERGATE module to a z-filtered TOCSY experiment. Pulsed-field gradients and appropriately phased selective rf pulses are used to maintain precise control of the water magnetization vector. Problems associated with radiation damping and spin-locking of the water magnetization are thus alleviated. The water magnetization is returned to equilibrium prior to each acquisition, which improves water suppression and minimizes signal losses due to saturation transfer.

The role of cation-pi interactions in biomolecular association. Design of peptides favoring interactions between cationic and aromatic amino acid side chains.

Cation-pi interactions between amino acid side chains are increasingly being recognized as important structural and functional features of proteins and other biomolecules. Although these interactions have been found in static protein structures, they have not yet been detected in dynamic biomolecular systems. We determined, by (1)H NMR spectroscopic titrations, the energies of cation-pi interactions of the amino acid derivative AcLysOMe (1) with AcPheOEt (2) and with AcTyrOEt (3) in aqueous and three organic solvents. The interaction energy is substantial; it ranges from -2.1 to -3.4 kcal/mol and depends only slightly on the dielectric constant of the solvent. To assess the effects of auxiliary interactions and structural preorganization on formation of cation-pi interactions, we studied these interactions in the association of pentapeptides. Upon binding of the positively-charged peptide AcLysLysLysLysLysNH(2) (5) to the negatively-charged partner AcAspAspXAspAspNH(2) (6), in which X is Leu (6a), Tyr (6b), and Phe (6c), multiple interactions occur. Association of the two pentapeptides is dynamic. Free peptides and their complex are in fast exchange on the NMR time-scale, and 2D (1)H ROESY spectra of the complex of the two pentapeptides do not show intermolecular ROESY peaks. Perturbations of the chemical shifts indicated that the aromatic groups in peptides 6b and 6c were affected by the association with 5. The association constants K(A) for 5 with 6a and with 6b are nearly equal, (4.0 +/- 0.7) x 10(3) and (5.0 +/- 1.0) x 10(3) M(-)(1), respectively, while K(A) for 5 with 6c is larger, (8.3 +/- 1.3) x 10(3) M(-)(1). Molecular-dynamics (MD) simulations of the pentapeptide pairs confirmed that their association is dynamic and showed that cation-pi contacts between the two peptides are stereochemically possible. A transient complex between 5 and 6 with a prominent cation-pi interaction, obtained from MD simulations, was used as a template to design cyclic peptides C(X) featuring persistent cation-pi interactions. The cyclic peptide C(X) had a sequence in which X is Tyr, Phe, and Leu. The first two peptides do, but the third does not, contain the aromatic residue capable of interacting with a cationic Lys residue. This covalent construct offered conformational stability over the noncovalent complexes and allowed thorough studies by 2D NMR spectroscopy. Multiple conformations of the cyclic peptides C(Tyr) and C(Phe) are in slow exchange on the NMR time-scale. In one of these conformations, cation-pi interaction between Lys3 and Tyr9/Phe9 is clearly evident. Multiple NOEs between the side chains of residues 3 and 9 are observed; chemical-shift changes are consistent with the placement of the side chain of Lys3 over the aromatic ring. In contrast, the cyclic peptide C(Leu) showed no evidence for close approach of the side chains of Lys3 and Leu9. The cation-pi interaction persists in both DMSO and aqueous solvents. When the disulfide bond in the cyclic peptide C(Phe) was removed, the cation-pi interaction in the acyclic peptide AC(Phe) remained. To test the reliability of the pK(a) criterion for the existence of cation-pi interactions, we determined residue-specific pK(a) values of all four Lys side chains in all three cyclic peptides C(X). While NOE cross-peaks and perturbations of the chemical shifts clearly show the existence of the cation-pi interaction, pK(a) values of Lys3 in C(Tyr) and in C(Phe) differ only marginally from those values of other lysines in these dynamic peptides. Our experimental results with dynamic peptide systems highlight the role of cation-pi interactions in both intermolecular recognition at the protein-protein interface and intramolecular processes such as protein folding.

Spectroscopic, kinetic, and mechanistic study of a new mode of coordination of indole derivatives to platinum(II) and palladium(II) ions in complexes.

Binding of tryptophan residue to intrinsic metal ions in proteins is unknown, and very little is known about the coordinating abilities of indole. Indole-3-acetamide displaces the solvent ligands from cis-[Pt(en)(sol)2]2+, in which sol is acetone or H2O, in acetone solution and forms the complex cis-[Pt(en)(indole-3-acetamide)]2+ (3) of spiro structure, in which the new bidentate ligand coordinates to the Pt(II) atom via the C(3) atom of the indolyl group and the amide oxygen atom. This structure is supported by 1H, 13C, 15N, and 195Pt NMR spectra and by UV, IR, and mass spectra. Molecular mechanical simulations by Hyperchem and CHARMM methods give consistent structural models; the latter is optimized by density-functional quantum chemical calculations. Dipeptide-like molecules N-(3-indolylacetyl)-L-amino acid in which amino acid is alanine, leucine, isoleucine, valine, aspartic acid, or phenylalanine also displace the solvent ligands in acetone solution and form complexes cis-[Pt(en) N-(3-indolylacetyl)-L-amino acid)]2+ (6), which structurally resemble 3 but exist as two diastereomers, detected by 1H NMR spectroscopy. The bulkier the amino acid moiety, the slower the coordination of these dipeptide-like ligands to the Pt(II) atom. The indolyl group does not coordinate as a unidentate ligand; a second donor atom is necessary for bidentate coordination of this atom and the indolyl C(3) atom. The solvent-displacement reaction is of first and zeroth orders with respect to indole-3-acetamide and cis-[Pt(en)(sol)2]2+, respectively. A mechanism consisting of initial unidentate coordination of the ligand via the amide oxygen atom followed by closing of the spiro ring is supported by 1H NMR data, the kinetic effects of acid and water, and the activation parameters for the displacement reaction. In the case of N-(3-indolylacetyl)-L-phenylalanine, the bulkiest of the entering ligands, the reaction is of first order with respect to both reactants. The bidentate indole-3-acetamide ligand in 3 is readily displaced by (CH3)2SO and 2-methylimidazole, but not by CNO-, CH3COO-, and CH3CN. Complexes cis-[Pd(en)(sol)2]2+ and cis-[Pd(dtco)(sol)2]2+ react with indole-3-acetamide more rapidly than their Pt(II) analogues do and yield complexes similar to 3. This study augments our recent discovery of selective, hydrolytic cleavage of tryptophan-containing peptides by Pd(II) and Pt(II) complexes.