Structure determination of a new protein from backbone-centered NMR data and NMR-assisted structure prediction.

Targeting of proteins for structure determination in structural genomic programs often includes the use of threading and fold recognition methods to exclude proteins belonging to well-populated fold families, but such methods can still fail to recognize preexisting folds. The authors illustrate here a method in which limited amounts of structural data are used to improve an initial homology search and the data are subsequently used to produce a structure by data-constrained refinement of an identified structural template. The data used are primarily NMR-based residual dipolar couplings, but they also include additional chemical shift and backbone-nuclear Overhauser effect data. Using this methodology, a backbone structure was efficiently produced for a 10 kDa protein (PF1455) from Pyrococcus furiosus. Its relationship to existing structures and its probable function are discussed.

Determination of protein backbone structures from residual dipolar couplings.

There are a number of circumstances in which a focus on determination of the backbone structure of a protein, as opposed to a complete all-atom structure, may be appropriate. This is particularly the case for structures determined as a part of a structural genomics initiative in which computational modeling of many sequentially related structures from the backbone of a single family representative is anticipated. It is, however, also the case when the backbone may be a stepping-stone to more targeted studies of ligand interaction or protein-protein interaction. Here an NMR protocol is described that can produce a backbone structure of a protein without the need for extensive experiments directed at side chain resonance assignment or the collection of structural information on side chains. The procedure relies primarily on orientational constraints from residual dipolar couplings as opposed to distance constraints from NOEs. Procedures for sample preparation, data acquisition, and data analysis are described, along with examples from application to small target proteins of a structural genomics project.

Backbone solution structures of proteins using residual dipolar couplings: application to a novel structural genomics target.

Structural genomics (or proteomics) activities are critically dependent on the availability of high-throughput structure determination methodology. Development of such methodology has been a particular challenge for NMR based structure determination because of the demands for isotopic labeling of proteins and the requirements for very long data acquisition times. We present here a methodology that gains efficiency from a focus on determination of backbone structures of proteins as opposed to full structures with all sidechains in place. This focus is appropriate given the presumption that many protein structures in the future will be built using computational methods that start from representative fold family structures and replace as many as 70% of the sidechains in the course of structure determination. The methodology we present is based primarily on residual dipolar couplings (RDCs), readily accessible NMR observables that constrain the orientation of backbone fragments irrespective of separation in space. A new software tool is described for the assembly of backbone fragments under RDC constraints and an application to a structural genomics target is presented. The target is an 8.7 kDa protein from Pyrococcus furiosus, PF1061, that was previously not well annotated, and had a nearest structurally characterized neighbor with only 33% sequence identity. The structure produced shows structural similarity to this sequence homologue, but also shows similarity to other proteins, which suggests a functional role in sulfur transfer. Given the backbone structure and a possible functional link this should be an ideal target for development of modeling methods.

NMR solution structure and backbone dynamics of the CC chemokine eotaxin-3.

Eotaxin-3 is one of three related chemokines that specifically activate chemokine receptor CCR3. We report the 3D structure and backbone dynamics of eotaxin-3 determined by NMR spectroscopy. Eotaxin-3 is monomeric under the conditions in this study and consists of an unstructured N-terminus before the first two conserved cysteine residues, an irregularly structured N-loop following the second conserved cysteine, a single turn of 3(10)-helix, a three-stranded antiparallel beta-sheet, an alpha-helix, and an unstructured C-terminal tail. As in other chemokines, the alpha-helix packs against one face of the beta-sheet. The average backbone and heavy atom rmsd values of the 20 structures (residues 9-65) are 0.44 and 1.01 A, respectively. A comparison between the structures of eotaxin-3 and related chemokines suggests that the electrostatic potential in the vicinity of a surface groove and the structure of the beta2-beta3 turn may be important for maintaining receptor specificity. The backbone dynamics of eotaxin-3 were determined from 15N NMR relaxation data using the extended model free dynamics formalism. Large amplitude motions on the picosecond to nanosecond time scale were observed in both termini and in some residues in the N-loop, the beta1-beta2 turn, and the beta3 strand; the location of these residues suggests a possible role for dynamics in receptor binding and activation. In contrast to eotaxin, eotaxin-3 exhibits no substantial mobility on the microsecond to millisecond time scale.

Factors affecting choice of surgical residency training program.

BACKGROUND: A significant problem facing American surgery today is the lack of participation from women and minorities. In 1995 and 1996, 15.1 and 15.8% of United States general surgical residency graduates were women. Of our 71 graduates in the last 12 years, 38% were women. The aim of this study was to identify the factors influencing our residents' choice of training program and the reasons why our program has a high percentage of female graduates. METHODS: Between 1989 and 2000, 27 women and 44 men completed general surgical training at our university and 44/71 (59%) responded to our survey. The age at residency completion was 34 +/- 2.2 years for men and 33.9 +/- 2.8 years for women. Fifty-five percent of men and 30% of women went on to fellowship training; and 36% of men and 20% of women are in academia. RESULTS: Factors influencing our graduates' selection of training program are: Only 23% of men had a female faculty as their mentor, whereas 90% of women had a male faculty as their mentor during training. Only 59% of men but 80% of women (P < 0.05) agreed that female medical students need role models of successful female faculty members. Fifty-five percent of men and 45% of women would encourage a female medical student to choose surgery as a career, but 82% of men and 50% of women would encourage a male medical student to do so. Ninety-one percent of men and 85% of women would choose surgery as a career again. CONCLUSIONS: A surgical residency training program with strong leadership, good clinical experience, and high resident morale will equally attract both genders. Women may pay more attention to the program's gender mix and geographic location.

Childbearing and child care in surgery.

HYPOTHESIS: The responsibility for childbearing and child care has a major effect on general surgical residency and subsequent surgical practice. METHODS: A survey of all graduates from a university general surgical training program between 1989 and 2000. RESULTS: Twenty-seven women and 44 men completed general surgical training at our university during the period, and 42 (59%) responded to our survey. The age at completion of the residency was 34.0 +/- 2.2 years for men and 33.9 +/- 2.8 years for women. During residency, 64% (14/22) of the men and 15% (3/20) of the women had children. At the time of the survey, 21 (95%) of the men and 8 (40%) of the women had children. Most residents (24 [57%] of 42) relied on their spouse for child care. During surgical practice, 18 (43%) indicated that they rely on their spouse; 19 (45%) use day care, home care, or both; and (8%) of 26 are unsatisfied with their current child care arrangement. During training, 38% (5/13) of men and 67% (2/3) of women took time off for maternity leave, paternity leave, or child care. Two of 3 surgeons would like to have had more time off during residency; most men (70%, or 7 of 10) recommended a leave of 1 to 3 months, and all women preferred a 3-month maternity or child care leave of absence. During surgical practice, only 12% (2/17) of men but 64% (7/11) of women have taken time off for either childbearing or child care. Half of the respondents (21/42) have a formal leave of absence policy at work, 52% (11/21) of which are paid leave programs. Although the workweek of our practicing graduates is 69 +/- 16 hours for men and 64 +/- 12 hours for women, 62% (26/42) spend more than 20 hours per week parenting. More than 80% (27/32) would consider a part-time surgical practice for more parenting involvement; one third of the responders suggested that 30 hours a week constitutes a reasonable part-time practice, one third preferred fewer than 30 hours, and one third favored more than 30 hours per week. Data are presented as mean +/- SD. CONCLUSIONS: Childbearing and child care may have an enormous impact on one's decision to pursue a career in surgery. To attract and retain the best candidates for future surgeons, formal policies on the availability of child care services in the residency program and the workplace should be studied and implemented. Furthermore, national studies are needed to define appropriate, acceptable workweeks for part-time or flexible practices and the duration of leaves of absence for childbearing or child care.

Laparoscopic suturing evaluation among surgical residents.

BACKGROUND: Laparoscopic suturing is an integral part of advanced laparoscopic surgery training. The objective of this study was to evaluate the performance and preference of surgical residents performing intracorporeal and extracorporeal knot-tying techniques using conventional and Endo Stitch instruments. The residents were also evaluated on their suturing techniques using conventional instruments, the Endo Stitch, and the Suture Assistant. METHODS: Using an inanimate laparoscopic trainer model, 39 residents were evaluated as they performed laparoscopic knot tying exercises. Endpoints of the study were execution time and subjective preference of surgical residents with respect to the type of instrument used for knot tying. Forty-three residents were evaluated as they performed laparoscopic suturing exercises with three different types of suturing instruments using the same endpoints. RESULTS: The intracorporeal technique was the preferred (89%) method of knot tying among surgical residents. The time for completion of laparoscopic suturing was significantly (P < 0.05) shorter with the Endo Stitch (114 +/- 64 s) than with the conventional instrument (206 +/- 107 s) or the Suture Assistant (151 +/- 70 s). Residents preferred the use of the Endo Stitch in all three categories for suturing, knot tying, and handling. CONCLUSION: The Endo Stitch enhanced laparoscopic skills and was the preferred instrument for laparoscopic knot tying and suturing among surgical residents.

Laparoscopic umbilical herniorrhaphy.

BACKGROUND: Laparoscopic technique is an alternative approach to ventral hernia repair. This study evaluated the feasibility of performing umbilical hernia repair using a single 5-mm trocar technique. PATIENTS AND METHODS: During February 1999 to November 1999, we performed laparoscopic umbilical hernia repair in 16 consecutive patients. All operations were performed under general anesthesia. One 5-mm port was used to visualize the defect. A second 5-mm port was inserted only if there was incarcerated omentum requiring reduction. The Endo Close was inserted through a 2-mm incision made directly over the hernia to perform transabdominal closure of the defect using nonabsorbable suture. RESULTS: The mean size of the umbilical hernia defects was 1.2 cm +/- 0.4 (range 1.0-2.0 cm). All operations were completed laparoscopically with no intraoperative or postoperative complications. The mean operative time was 35 +/- 15 minutes (range 21-75 min). All cases were performed in an outpatient setting. There have been no recurrences at a mean follow-up of 5.9 months. CONCLUSIONS: Laparoscopic umbilical herniorrhaphy is safe and technically feasible. Its potential advantages, such as a lower rate of recurrence, will need to be validated with longer follow-up.

NMR solution structure and receptor peptide binding of the CC chemokine eotaxin-2.

The human CC chemokine eotaxin-2 is a specific agonist for the chemokine receptor CCR3 and may play a role in the recruitment of eosinophils in allergic diseases and parasitic infections. We report the solution structure of eotaxin-2 determined using heteronuclear and triple resonance NMR methods. A family of 20 structures was calculated by hybrid distance geometry-simulated annealing from 854 NOE distance restraints, 48 dihedral angle restraints, and 12 hydrogen bond restraints. The structure of eotaxin-2 (73 amino acid residues) consists of a helical turn (residues 17-20) followed by a 3-stranded antiparallel beta-sheet (residues 22-26, 37-41, and 44-49) and an alpha-helix (residues 54-66). The N-loop (residues 9-16) is packed against both the sheet and the helix with the two conserved disulfide bonds tethering the N-terminal/N-loop region to the beta-sheet. The average backbone and heavy atom rmsd values of the 20 structures (residues 7-66) are 0.52 and 1.13 A, respectively. A linear peptide corresponding to the N-terminal region of CCR3 binds to eotaxin-2, inducing concentration-dependent chemical shift changes or line broadening of many residues. The distribution of these residues suggests that the peptide binds into an extended groove located at the interface between the N-loop and the beta2-beta3 hairpin. The receptor peptide may also interact with the N-terminus of the chemokine and part of the alpha-helix. Comparison of the eotaxin-2 structure with those of related chemokines indicates several structural features that may contribute to receptor specificity.

Backbone dynamics of the human CC-chemokine eotaxin.

Eotaxin is a CC chemokine with potent chemoattractant activity towards eosinophils. 15N NMR relaxation data have been used to characterize the backbone dynamics of recombinant human cotaxin. 15N longitudinal (R1) and transverse (R2) auto relaxation rates, heteronuclear ¿1H¿-15N steady-state NOEs, and transverse cross-relaxation rates (eta xy) were obtained at 30 degrees C for all resolved backbone secondary amide groups using 1H-detected two-dimensional NMR experiments. Ratios of transverse auto and cross relaxation rates were used to identify NH groups influenced by slow conformational rearrangement. Relaxation data were fit to the extended model free dynamics formalism, yielding parameters describing axially symmetric molecular rotational diffusion and the internal dynamics of each NH group. The molecular rotational correlation time (tau m) is 5.09 +/- 0.02 ns, indicating that eotaxin exists predominantly as a monomer under the conditions of the NMR study. The ratio of diffusion rates about unique and perpendicular axes (D parallel/D perpendicular) is 0.81 +/- 0.02. Residues with large amplitudes of subnanosecond motion are clustered in the N-terminal region (residues 1-19), the C-terminus (residues 68-73) and the loop connecting the first two beta-strands (residues 30-37). N-terminal flexibility appears to be conserved throughout the chemokine family and may have implications for the mechanism of chemokine receptor activation. Residues exhibiting significant dynamics on the microsecond-millisecond time scale are located close to the two conserved disulfide bonds, suggesting that these motions may be coupled to disulfide bond isomerization.

Laparoscopic Roux-en-Y gastric bypass for morbid obesity.

Surgery is currently the only effective treatment for morbid obesity. The two most commonly accepted operations are the Roux-en-Y gastric bypass and vertical banded gastroplasty. Although multiple authors have reported on a laparoscopic approach to gastric banding, the Roux-en-Y gastric bypass is a complex operation to be replicated using laparoscopic techniques. In this article, we describe our technique of the Roux-en-Y gastric bypass using a laparoscopic approach in four cases.

The solution structure of photosystem I accessory protein E from the cyanobacterium Nostoc sp. strain PCC 8009.

PsaE is a small basic subunit located on the stromal (cytoplasmic) side of photosystem I. In cyanobacteria, this subunit participates in cyclic electron transport and modulates the interactions of the complex with soluble ferredoxin. The PsaE protein isolated from the cyanobacterium Synechococcus sp. strain PCC 7002 adopts the beta topology of an SH3 domain, with five beta strands (betaA through betaE) and a turn of 3(10) helix between strands betaD and betaE [Falzone, C. J., Kao, Y.-H., Zhao, J., Bryant, D. A., and Lecomte, J. T. J. (1994) Biochemistry 33, 6052-6062]. The primary structure of the PsaE protein is strongly conserved across all oxygen-evolving photosynthetic organisms. However, variability in loop lengths, as well as N- or C-terminal extensions, suggests that the structure of a second representative PsaE subunit would be useful to characterize the interactions among photosystem I polypeptides. In this work, the solution structure of PsaE from the cyanobacterium Nostoc sp. strain PCC 8009 was determined by NMR methods. Compared to PsaE from Synechococcus sp. strain PCC 7002, this PsaE has a seven-residue deletion in the loop connecting strands betaC and betaD, and an eight-residue C-terminal extension. Angular and distance restraints derived from homonuclear and heteronuclear NMR experiments were used to calculate structures by a distance-geometry/simulated-annealing protocol. A family of 20 structures (rmsd of 0.24 A in the regular secondary structure) is presented. Differences between the two cyanobacterial proteins are mostly confined to the CD loop region; the C-terminal extension is disordered. The thermodynamic stability of Nostoc sp. strain PCC 8009 PsaE toward urea denaturation was measured by circular dichroism and fluorescence spectroscopy, and thermal denaturation was monitored by UV absorption spectroscopy. Chemical and thermal denaturation curves are modeled satisfactorily with two-state processes. The DeltaG degrees of unfolding at room temperature is 12.4 +/- 0.3 kJ mol(-1) (pH 5), and the thermal transition midpoint is 59 +/- 1 degrees C (pH 7). Interactions with other proteins in the photosystem I complex may aid in maintaining PsaE in its native state under physiological conditions.

Cardiopulmonary responses to experimental venous carbon dioxide embolism.

BACKGROUND: Although the low-flow CO2 insufflation rate used to initiate pneumoperitoneum may reduce the severity of potential venous embolism, its safety is not established. METHODS: Anesthetized pigs were ventilated with room air at a fixed minute ventilation. After 1 h of baseline, they were intravenously infused with CO2 at the rate of 0.3, 0.75, or 1.2 ml/kg/min for 2 h (n = 5 for each group), followed by 1 h of recovery. RESULTS: All animals experienced pulmonary hypertension, depressed stroke volume, hypoxemia, hypercarbia, and acidemia during intravenous CO2 infusion. They had systemic hypertension at the low rate of hypotension at the highest rate of infusion. End-tidal CO2 levels briefly decreased, then increased in all cases. In the highest rate group, three of the five animals (60%) died at 50, 65, and 100 min of infusion. These three animals had severe hypotension and hypoxemia, with visible coronary gas embolism. There was no patent foramen ovale at necropsy in any animals. CONCLUSIONS: The low-flow insufflation rate exceeds the fatal rate of continuous intravenous CO2 infusion. End-tidal CO2 levels were increased in venous CO2 embolism, not decreased as seen in venous air embolism. Severe hypoxemia and hypotension are predictors of potentially fatal cases.