Contryphans from Conus textile venom ducts.

Contryphans are unusual Conus peptides which contain a distinctive post-translational modification, D-tryptophan or D-leucine. cDNA clones encoding new contryphans from the mollusc-hunting cone snail Conus textile were identified and the inferred mature peptides were synthesized: contryphan-Tx (Gly-Cys-Hyp-D-Trp-Gln-Pro-Tyr-Cys-NH(2)), Leu-contryphan-Tx (Cys-Val-D-Leu-Tyr-Pro-Trp-Cys-NH(2)) and contryphan R/Tx which is identical to contryphan-R [Jimenez et al., 1996. Contryphan is a D-tryptophan containing Conus peptide. J. Biol. Chem. 281, 28002-28005]. Leu-contryphan-Tx exhibits a single peak, but contryphan-Tx shows two peaks under reverse-phase high-performance liquid chromatography conditions. Ultraviolet resonance Raman spectroscopy demonstrates a difference in the D-tryptophan dihedral angle for the two contryphan-Tx equilibrium conformers. Both the sequences and in vivo effects of all contryphans isolated suggest that there are two major branches of the contryphan family.

A critical review of current nursing faculty practice.

PURPOSE: To critically examine the current literature on nursing faculty practice, using the National Organization of Nurse Practitioner Faculties (NONPF) Guidelines for Evaluation of Faculty Practice, and to examine faculty practice models' strengths, weaknesses, and barriers. DATA SOURCES: Thirty-five articles describing models of faculty practice were identified through an exhaustive search on CINAHL and Medline. Two NONPF monographs on nursing faculty practice were used as guidelines for the critical review. CONCLUSIONS: Faculty practice has become an integral component of faculty-role expectations at many schools of nursing. Workload, especially without adequate compensation, remains a hindrance to practice. The value of faculty practice time and expertise has not been sufficiently demonstrated. Integration of practitioner, educator and researcher roles remains extremely difficult and sometimes elusive. IMPLICATIONS FOR PRACTICE: Faculty practice offers many advantages to schools of nursing, including educational and research opportunities for faculty and students, as well as practice sites and affordable community healthcare. Providing health care in the community presents an opportunity for independent and collaborative practice. To fully utilize the great research opportunities provided by faculty practice, more emphasis must be placed on gathering and analyzing descriptive data.

UV resonance raman spectra of ligand binding intermediates of sol-gel encapsulated hemoglobin.

We report for the first time specific conformational changes for a homogeneous population of ligand-bound adult deoxy human hemoglobin A (HbA) generated by introducing CO into a sample of deoxy-HbA with the effector, inositol hexaphosphate, encapsulated in a porous sol-gel. The preparation of ligand-bound deoxy-HbA results from the speed of ligand diffusion relative to globin conformational dynamics within the sol-gel (1). The ultraviolet resonance Raman (UVRR) difference spectra obtained reveal that E helix motion is initiated upon ligand binding, as signaled by the appearance of an alpha14beta15 Trp W3 band difference at 1559 cm(-1). The subsequent appearance of Tyr (Y8a and Y9a) and W3 (1549 cm(-1)) UVRR difference bands suggest conformational shifts for the penultimate Tyralpha140 on the F helix, the "switch" region Tyralpha42, and the "hinge" region Trpbeta37. The UVRR results expose a sequence of conformational steps leading up to the ligation-induced T to R quaternary structure transition as opposed to a single, concerted switch. More generally, this report demonstrates that sol-gel encapsulation of proteins can be used to study a sequence of specific conformational events triggered by substrate binding because the traditional limitation of substrate diffusion times is overcome.

Solution-active structural alterations in liganded hemoglobins C (beta6 Glu --> Lys) and S (beta6 Glu --> Val).

Based upon existing crystallographic evidence, HbS, HbC, and HbA have essentially the same molecular structure. However, important areas of the molecule are not well defined crystallographically (e.g. the N-terminal nonhelical portion of the alpha and beta chains), and conformational constraints differ in solution and in the crystalline state. Over the years, our laboratory and others have provided evidence of conformational changes in HbS and, more recently, in HbC. We now present data based upon allosteric perturbation monitored by front-face fluorescence, ultraviolet resonance Raman spectroscopy, circular dichroism, and oxygen equilibrium studies that confirm and significantly expand previous findings suggesting solution-active structural differences in liganded forms of HbS and HbC distal to the site of mutation and involving the 2,3-diphosphoglycerate binding pocket. The liganded forms of these hemoglobins are of significant interest because HbC crystallizes in the erythrocyte in the oxy form, and oxy HbS exhibits increased mechanical precipitability and a high propensity to oxidize. Specific findings are as follows: 1) differences in the intrinsic fluorescence indicate that the Trp microenvironments are more hydrophobic for HbS > HbC > HbA, 2) ultraviolet resonance Raman spectroscopy detects alterations in Tyr hydrogen bonding, in Trp hydrophobicity at the alpha1beta2 interface (beta37), and in the A-helix (alpha14/beta15) of both chains, 3) displacement by inositol hexaphosphate of the Hb-bound 8-hydroxy-1,3,6-pyrenetrisulfonate (the fluorescent 2,3-diphosphoglycerate analog) follows the order HbA > HbS > HbC, and 4) oxygen equilibria measurements indicate a differential allosteric effect by inositol hexaphosphate for HbC approximately HbS > HbA.

The conformational and dynamic basis for ligand binding reactivity in hemoglobin Ypsilanti (beta 99 asp-->Tyr): origin of the quaternary enhancement effect.

Hemoglobin Ypsilanti (HbY) is a stable tetrameric hemoglobin that binds oxygen with little or no cooperativity and with high affinity [Doyle, M. L., et al. (1992) Proteins: Struct., Funct., Genet. 14, 351-362]. It displays an especially large quaternary enhancement effect. An X-ray crystallographic study [Smith, F. R., et al. (1991) Proteins: Struct., Funct., Genet. 10, 81-91] of the carboxy derivative of this hemoglobin (COHbY) revealed a new quaternary structure that partially resembles the recently described R2 structure [Silva, M. M., et al. (1992) J. Biol. Chem. 267, 17248-17256]. Very little is known about either the solution phase conformations of the liganded and deoxy forms of HbY or the molecular basis for the large quaternary enhancement effect (Doyle et al., 1992). In this study, near-IR absorption, Soret-enhanced Raman, and UV (229 nm) resonance Raman spectroscopies are used to probe the liganded and deoxy derivatives of HbY in solution. Nanosecond time-resolved near-IR absorption measurements are used to expose the relaxation properties of the photoproduct of COHbY. Time-resolved (Soret band) absorption is used to generate the geminate and solvent phase ligand rebinding curves for photodissociated COHbY. The spectroscopic results indicate that COHbY has an R-like conformation with respect to both the proximal heme pocket and the hinge region of the alpha 1 beta 2 interface. The deoxy derivative of HbY has spectroscopic features that are very similar to those observed for species assigned to the deoxy R or half-liganded R conformations of human adult hemoglobin (HbA). The 10 ns to 100 micros relaxation properties of the photoproduct of COHbY are distinctly different from those of HbA in that for HbY, little if any tertiary or quaternary relaxation is observed. The near-absence of relaxation in the HbY photoproduct explains the differences in the geminate and solvent phase CO recombination between HbA and HbY. The impact of the conformational and relaxation properties of HbY on the geminate rebinding process forms the basis of a model that accounts for the large quaternary enhancement effect reported for HbY (Doyle et al., 1992). In addition, the spectroscopic data and the X-ray crystallographic results explain the slow relaxation for HbY and the near-absence of cooperative ligand binding for this protein based on the behavior of the penultimate tyrosines.

Rapid loop dynamics of Yersinia protein tyrosine phosphatases.

The Yersinia protein tyrosine phosphatases (PTPase) contain a single and invariant tryptophan (W354) located at one of the hinge positions of the flexible loop (WpD loop), which is essential for catalysis. The wild-type Yersinia PTPase and an active site mutant in which the esential Cys 403 has been replaced by serine (C403S) have been examined using both time-resolved fluorescence anisotropy and steady-state UV resonance Raman (UVRR) spectroscopies. Both enzymes were examined with and without the bound inhibitor arsenate. The UVRR spectra indicate that in solution the ligand-free, wild-type PTPase exists as an equilibrium mixture of two tryptophan rotamer structures with chi2,1 dihedral angles of -4 degrees and -90 degrees. The two rotamers have been attributed to the presence of both "closed" and "open" WpD loop conformers of the ligand-free enzyme. Conversely, the UVRR spectra of the arsenate-ligated, wild-type PTPase and of ligand-free and arsenate-ligated C403S PTPase contain a single W3 band which is correlated to the -4 degrees rotamer of W354, indicating a predominance of the closed WpD loop conformer. The tryptophan fluorescence anisotropy decay measurements of the ligand-bound, wild-type Yersinia PTPase and of both ligation states of the C403S PTPase reveal a single correlation time of 30-48 ns due to the rotational motion of the protein, while the ligand-free, wild-type PTPase is found to have two correlation times of 31 and 3.8 ns. The 3.8 ns correlation time of the ligand-free enzyme is attributed to the hinged movement of the WpD loop which contains W354. These results indicate that under physiological conditions, the nonligated, wild-type Yersinia PTPase alternates between an open WpD loop and a closed loop form with a rate constant of approximately 2.6 x 10(8) s(-1). We conclude that the rate of WpD loop closure of the wild-type Yersinia PTPase is thus independent of the presence of ligand, whereas in the presence of ligand the rate of opening is dramatically reduced resulting in a closed conformation on ligand binding. In contrast, the ligand-free and ligated C403S PTPase remain in the loop closed configuration over the time course of our dynamic measurements. The lack of WpD loop motion in the C403S PTPase is believed to be due to either a loss of repulsive potential between the anionic thiolate and Asp 356 of the WpD loop and/or the formation of a hydrogen bond or water bridged hydrogen bond between Ser 403 and Asp 356.

Probing the hemoglobin central cavity by direct quantification of effector binding using fluorescence lifetime methods.

Time-resolved fluorescence methods have been used to show that 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT), a fluorescent analog of 2,3-diphosphoglycerate, binds to the central cavity of carboxyhemoglobin A (HbACO) at pH 6.35. A direct quantitative approach, based on the distinctive free and bound HPT fluorescent lifetimes of 5.6 ns and approximately 27 ps, respectively, was developed to measure the binding affinity of this probe. HPT binds to a single site and is displaced by inositol hexaphosphate at a 1:1 mol ratio, indicating that binding occurs at the 2,3-diphosphoglycerate site in the central cavity. Furthermore, the results imply that low pH HbACO exists as an altered R state and not an equilibrium mixture of R and T states. The probe was also used to monitor competitive effector binding and to compare the affinity of the binding site in several cross-bridged HbA derivatives.

Orientation and linear dichroism of Mastigocladus laminosus phycocyanin trimer and Nostoc sp. phycocyanin dodecamer in stretched poly(vinyl alcohol) films.

The linear dichroism (LD) spectra of the C-phycocyanin (C-PC) trimer disks oriented in poly(vinyl alcohol) films (PVA) at room temperature and at 95 K were determined. Utilizing the known atomic coordinates of the chromophores (Schirmer, T., Bode, W. and Huber, R. (1987) J. Mol. Biol. 196, 677-695) and theoretical estimates of the orientations of the transition dipole moments relative to the molecular framework, the LD spectra were simulated using the pairwise exciton interaction model of Sauer and Scheer (Biochim. Biophys. Acta 936 (1988) 157-170); in this model, the alpha 84 and beta 84 transition moments are coupled by an exciton mechanism, while the beta 155 chromophore remains uncoupled. Linear dichroism spectra calculated using this exciton model, as well as an uncoupled chromophore (molecular) model, were compared with experimental LD spectra. Satisfactory qualitative agreement can be obtained in both the exciton and molecular models using somewhat different relative values of the theoretically estimated magnitudes of the beta 155 oscillator strength. Because the relative contributions of each of the chromophores (and thus exciton components) to the overall absorption of the C-PC trimer are not known exactly, it is difficult to differentiate successfully between the molecular and exciton models at this time. The linear dichroism spectra of PC dodecamers derived from phycobilisomes of Nostoc sp. oriented in stretched PVA films closely resemble those of the C-PC trimers from Mastigocladus laminosus, suggesting that the phycocyanin chromophores are oriented in a similar manner in both cases, and that neither linker polypeptides nor the state of aggregation have a significant influence on these orientations and linear dichroism spectra. The LD spectra of oriented phycocyanins in stretched PVA films at low temperatures (95 K) appear to be of similar quality and magnitude as the LD spectra of single C-PC crystals (Schirmer, T. and Vincent, M.G. (1987) Biochim. Biophys. Acta 893, 379-385).