Search

The functionalization of hydrogels for receptor-mediated cell adhesion is one approach for targeted cell and tissue engineering applications. In this study, polyacrylamide gel surfaces were functionalized with specific cell adhesion ligands via the self-assembly of a peptide-based heterodimer. The system was comprised of a cysteine-terminated monomer, A (MW approximately 5400), grafted to the polyacrylamide gels and a complementary ligand presenting monomer, B(X) (MW approximately 5800) that was designed to heterodimerize with A. Two ligand presenting monomers were synthesized: one presenting the RGDS ligand, B(D), for receptor-mediated cell adhesion, and the other, a control monomer presenting the nonadhesive RGES ligand, B(E). Assembly of the peptide pair A-B(X) by association of the monomers into a coiled coil was verified by circular dichroism in solution. Binding studies were conducted to determine the dissociation constant of the pair A-B(X), which was found to be K(D) approximately 10(-8) m. Polyacrylamide gels functionalized with A-B(X) heterodimers were evaluated for cell adhesion using bovine aortic endothelial cells (BAECs). Endothelial cells cultured on the A-B(D) functionalized surfaces demonstrated typical cell morphologies and expected spreading behavior as a function of the density of RGDS ligand, calculated as the amount of B(D) associated with grafted A on the surface of the gels. In contrast, A-B(E) linked surfaces supported no cell adhesion. The adhesion of the substrate was dynamically altered through the reassembly of A-B(X) dimers as B(D) molecules in the solution replaced B(E) molecules at the substrate. The molecular constructs described here demonstrate the potential to design a broad family of switchable peptides that impart the dynamic control of biofunctionality at an interface, which would be useful for precise manipulation of cell physiology.

Although the analysis and design of turns that connect the strands in antiparallel beta-hairpins has reached an advanced state, much less is known concerning turns between antiparallel helices in helical hairpins. We have conducted an analysis of the structures and sequence preferences of two types of interhelical turns, each of which connects the two helices by a two-residue linker in an alphaL-beta conformation. Based on this analysis, it became apparent that the turn introduced into a designed four-helix bundle protein, DF1, did not occur within an optimal structural context. DF1 is a dimeric model for the diiron class of proteins. A longer loop with a beta-alphaR-beta conformation was inserted between two helices in the protein, and a sequence was chosen to stabilize its conformation. X-ray crystallography and NMR analysis of the protein showed the structure to be in excellent agreement with design.

Here, we describe the folding/unfolding kinetics of alpha3D, a small designed three-helix bundle. Both IR temperature jump and ultrafast fluorescence mixing methods reveal a single-exponential process consistent with a minimal folding time of 3.2 +/- 1.2 micros (at approximately 50 degrees C), indicating that a protein can fold on the 1- to 5-micros time scale. Furthermore, the single-exponential nature of the relaxation indicates that the prefactor for transition state (TS)-folding models is probably >or=1 (micros)-1 for a protein of this size and topology. Molecular dynamics simulations and IR spectroscopy provide a molecular rationale for the rapid, single-exponential folding of this protein. alpha3D shows a significant bias toward local helical structure in the thermally denatured state. The molecular dynamics-simulated TS ensemble is highly heterogeneous and dynamic, allowing access to the TS via multiple pathways.

Site-directed mutagenesis and combinatorial libraries are powerful tools for providing information about the relationship between protein sequence and structure. Here we report two extensions that expand the utility of combinatorial mutagenesis for the quantitative assessment of hypotheses about the determinants of protein structure. First, we show that resin-splitting technology, which allows the construction of arbitrarily complex libraries of degenerate oligonucleotides, can be used to construct more complex protein libraries for hypothesis testing than can be constructed from oligonucleotides limited to degenerate codons. Second, using eglin c as a model protein, we show that regression analysis of activity scores from library data can be used to assess the relative contributions to the specific activity of the amino acids that were varied in the library. The regression parameters derived from the analysis of a 455-member sample from a library wherein four solvent-exposed sites in an alpha-helix can contain any of nine different amino acids are highly correlated (P < 0.0001, R(2) = 0. 97) to the relative helix propensities for those amino acids, as estimated by a variety of biophysical and computational techniques.

This report investigates the concept that severe constipation requiring major abdominal surgery may result from one of three common causes: 1) colonic inertia, 2) pelvic hiatal hernia, or 3) both colonic inertia and pelvic hernia. This study evaluates the symptoms, anatomy and outcome in 201 patients with severe surgical constipation treated by a single surgeon. In 2042 patients with constipation referred to one colon and rectal surgeon, 211 major abdominal surgical procedures were performed on 201 patients for severe constipation between 1989 and 1999. There were 187 women and 14 men. Mean age was 49 years (range, 9-84). Five high-risk patients had ileostomy; 196 had major colonic surgery for anatomic or physiologic causes of constipation, excluding malignancy, diverticular disease, and inflammatory bowel disease. Pelvic hiatal hernia was defined as the herniation of bowel through the hiatus of the pelvic diaphragm seen on pelvic videofluoroscopy or physical examination. Of these 196 patients, 44 per cent had pelvic hiatal hernia repair (PHHR), 27 per cent had total abdominal colectomy and ileorectal anastomosis for colonic inertia, and 29 per cent had surgery for both colonic inertia and pelvic hiatal hernia. Of the 144 patients undergoing PHHR, 95 had Gore-Tex patch (W. L. Gore and Associates, Inc., Phoenix, AZ) sacral colpopexy. PHHR for pelvic hiatal hernia without colonic inertia included sigmoid resection, rectopexy, and Gore-Tex patch sacral colpopexy. Mean duration of follow-up was 20 months. Symptoms noted preoperatively included abdominal pain (84%), straining at stool (90%), incomplete rectal emptying (85%), painful bowel movements (74%), pelvic pain (69%), vaginal bulge (55%), digital assistance with evacuation (35%), and incontinence of stool (38%). Outcome assessed by symptom relief was successful in 89.1 per cent of patients. 8.6 per cent of patient conditions were unchanged, and 2.3 per cent were unsatisfied with the outcome. There were no postoperative deaths. The complication rate was 6.1 per cent (small bowel obstruction, 7; anastomotic leak, 2; ureteral stenosis, 2; and patch erosion, 1). In our experience, severe surgical constipation can be due to colonic inertia, pelvic hiatal hernia, or both. Careful preoperative evaluation identifies these disorders, and surgical therapy aimed at correction of anatomic and physiologic defects results in high patient satisfaction and improvement in bowel function.

We studied the thermal denaturation of eglin c by using CD spectropolarimetry and differential scanning calorimetry (DSC). At low protein concentrations, denaturation is consistent with the classical two-state model. At concentrations greater than several hundred microM, however, the calorimetric enthalpy and the midpoint transition temperature increase with increasing protein concentration. These observations suggested the presence of intermediates and/or native state aggregation. However, the transitions are symmetric, suggesting that intermediates are absent, the DSC data do not fit models that include aggregation, and analytical ultracentrifugation (AUC) data show that native eglin c is monomeric. Instead, the AUC data show that eglin c solutions are nonideal. Analysis of the AUC data gives a second virial coefficient that is close to values calculated from theory and the DSC data are consistent with the behavior expected for nonideal solutions. We conclude that the concentration dependence is caused by differential nonideality of the native and denatured states. The nondeality arises from the high charge of the protein at acid pH and is exacerbated by low buffer concentrations. Our conclusion may explain differences between van't Hoff and calorimetric denaturation enthalpies observed for other proteins whose behavior is otherwise consistent with the classical two-state model.

IgE antibodies bind to specific high-affinity receptors on mast cells, leading to mast cell degranulation and release of mediators, such as histamine, which produce symptoms associated with allergy. Hence, anti-IgE antibodies that block binding of IgE to its high-affinity receptor are of potential therapeutic value in the treatment of allergy. These antibodies must also not bind to IgE once it is bound to the receptor because this would trigger histamine release. This study describes the humanization of a murine antibody, MaE11, with these characteristics. Variants of the humanized antibody were evaluated to probe the importance of framework residues on antibody binding and to determine which charged residues in the CDR interacted with IgE. We found that only five changes in human framework residues were required to provide for binding comparable to that of the original murine antibody.