Incidence of contralateral occult inguinal hernia found at the time of laparoscopic trans-abdominal pre-peritoneal (TAPP) repair.

BACKGROUND: Trans-abdominal laparoscopic inguinal hernia repair allows rapid assessment and exploration of the contralateral groin and repair of an occult hernia. Although previous studies have shown that the totally extra-peritoneal (TEP) hernia repair can be used to assess the contralateral groin, there is little data pertaining to the trans-abdominal pre-peritoneal (TAPP) approach. The aim of this study was to document the incidence of occult contralateral hernia at the time of TAPP hernia repair. METHODS: Data were collected prospectively from all patients undergoing laparoscopic TAPP hernia repair in a District General Hospital over a three-year period. Two specialist laparoscopic/upper gastrointestinal surgeons undertook all of the operations and telephone follow-up was carried out by a dedicated laparoscopic specialist nurse. RESULTS: A total of 310 patients underwent hernia surgery. Four cases were excluded, leaving 306 patients in the study. The male:female ratio was 10.5:1, with a median age of 59 years. Two hundred and six (67%) patients were booked for a unilateral hernia repair; of these, a contralateral hernia was found and repaired in 45 (22%). In 76 cases where a bilateral repair was planned, 61 (80%) went on to have both groin defects repaired. In the remaining 20%, the clinical suspicion of bilateral hernia was revised at the time of surgery to unilateral only. Twenty (7%) patients were booked to undergo a unilateral repair with the possibility of a contralateral hernia--in this group, the suspected contralateral defect was confirmed in 6 (30%) cases. Four (1%) cases were booked as femoral repairs, one of which was found to be an inguinal hernia. The clinical diagnostic accuracy was 78%. CONCLUSION: Accurate incidence figures of an occult contralateral inguinal hernia will enhance the pre-operative information given to patients and may impact on resource allocation and planning theatre logistics. Finding and repairing an occult contralateral hernia at the time of TAPP has the distinct advantage that it saves the patient from further symptoms and from another operation with its associated potential morbidity.

[Parkinson's disease here and now, a study of 50 cases]. / La maladie de Parkinson ici et maintenant, une étude de cinquante cas.

Over more than ten years, the pathophysiological conceptions as well as the management of the idiopathic Parkinson's disease have considerably improved. We present a study of 50 patients examined during the year 2007 which represent a "cliché" of the pathology current in our area. These patients, whose pathology has been developing since 1 to 16 years (5,5 years average) are still for the time being in an acceptable functional stage. Their motor complications (fluctuations and dyskinesias) do not occur before a period of 5 years; they affect only about 25% of patients and do not engender serious disabilities in most cases. The non-motor symptoms often occur and are developed in our study. These symptoms need to be detected and treated efficiently. Some of these non-motor symptoms (olfactory troubles, rapid eye movement sleep behaviour disorder) are quite specific and can be noticed at the preclinical stage. Depression affects 20 % of the patients whereas dementia is relatively seldom (4 cases). The development of dementia is tied up to the duration of evolution, the age and sex. Levodopa treatment remains the basis for such a disease. After a period of a few years dopamine agonists and catechol-O-methyl transferase inhibitors are combined to Levodopa. A discussion compares our results to the recent literature and provides an overview of the present knowledge concerning this disease.

Generation of an LFA-1 antagonist by the transfer of the ICAM-1 immunoregulatory epitope to a small molecule.

The protein-protein interaction between leukocyte functional antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) is critical to lymphocyte and immune system function. Here, we report on the transfer of the contiguous, nonlinear epitope of ICAM-1, responsible for its association with LFA-1, to a small-molecule framework. These LFA-1 antagonists bound LFA-1, blocked binding of ICAM-1, and inhibited a mixed lymphocyte reaction (MLR) with potency significantly greater than that of cyclosporine A. Furthermore, in comparison to an antibody to LFA-1, they exhibited significant anti-inflammatory effects in vivo. These results demonstrate the utility of small-molecule mimics of nonlinear protein epitopes and the protein epitopes themselves as leads in the identification of novel pharmaceutical agents.

Crystal structure of human insulin-like growth factor-1: detergent binding inhibits binding protein interactions.

Despite efforts spanning considerably more than a decade, a high-resolution view of the family of proteins known as insulin-like growth factors (IGFs) has remained elusive. IGF-1 consists of three helical segments which are connected by a 12-residue linker known as the C-region. NMR studies of members of this family reveal a dynamic structure with a topology resembling insulin but little structural definition in the C-region. We have crystallized IGF-1 in the presence of the detergent deoxy big CHAPS, and determined its structure at 1.8 A resolution by multiwavelength anomalous diffraction, exploiting the anomalous scattering of a single bromide ion and six of the seven sulfur atoms of IGF-1. The structure reveals a well-defined conformation for much of the C-region, which extends away from the core of IGF-1 and has residues known to be involved in receptor binding prominently displayed in a type II beta-turn. In the crystal, these residues form a dimer interface, but analytical ultracentrifugation experiments demonstrate that at physiological concentrations IGF-1 is monomeric. A single detergent molecule contacts residues known to be important for IGF-1 binding protein (IGFBP) interactions. Biophysical and biochemical data show that the detergent binds to IGF-1 specifically and blocks binding of IGFBP-1 and IGFBP-3.

Structure-function analysis of a phage display-derived peptide that binds to insulin-like growth factor binding protein 1.

Highly structured, peptide antagonists of the interaction between insulin-like growth factor 1 (IGF-I) and IGF binding protein 1 (IGFBP-1) have recently been discovered by phage display of naïve peptide libraries [Lowman, H. B., et al. (1998) Biochemistry 37, 8870--8878]. We now report a detailed analysis of the features of this turn-helix peptide motif that are necessary for IGFBP-1 binding and structural integrity. Further rounds of phage randomization indicate the importance of residues contributing to a hydrophobic patch on one face of the helix. Alanine-scanning substitutions confirm that the hydrophobic residues are necessary for binding. However, structural analysis by NMR spectroscopy indicates that some of these analogues are less well folded. Structured, high-affinity analogues that lack the disulfide bond were prepared by introducing a covalent constraint between side chains at positions i and i + 7 or i + 8 within the helix. Analogues based on this scaffold demonstrate that a helical conformation is present in the bound state, and that hydrophobic side chains in this helix, and residues immediately preceding it, interact with IGFBP-1. By comparison of alanine scanning data for IGF-I and the turn-helix peptide, we propose a model for common surface features of these molecules that recognize IGFBP-1.

A minimal peptide scaffold for beta-turn display: optimizing a strand position in disulfide-cyclized beta-hairpins.

Phage display of peptide libraries has become a powerful tool for the evolution of novel ligands that bind virtually any protein target. However, the rules governing conformational preferences in natural peptides are poorly understood, and consequently, structure-activity relationships in these molecules can be difficult to define. In an effort to simplify this process, we have investigated the structural stability of 10-residue, disulfide-constrained beta-hairpins and assessed their suitability as scaffolds for beta-turn display. Using disulfide formation as a probe, relative free energies of folding were measured for 19 peptides that differ at a one strand position. A tryptophan substitution promotes folding to a remarkable degree. NMR analysis confirms that the measured energies correlate well with the degree of beta-hairpin structure in the disulfide-cyclized peptides. Reexamination of a subset of the strand substitutions in peptides with different turn sequences reveals linear free energy relationships, indicating that turns and strand-strand interactions make independent, additive contributions to hairpin stability. Significantly, the tryptophan strand substitution is highly stabilizing with all turns tested, and peptides that display model turns or the less stable C'-C' ' turn of CD4 on this tryptophan "stem" are highly structured beta-hairpins in water. Thus, we have developed a small, structured beta-turn scaffold, containing only natural L-amino acids, that may be used to display peptide libraries of limited conformational diversity on phage.

Tryptophan zippers: stable, monomeric beta -hairpins.

A structural motif, the tryptophan zipper (trpzip), greatly stabilizes the beta-hairpin conformation in short peptides. Peptides (12 or 16 aa in length) with four different turn sequences are monomeric and fold cooperatively in water, as has been observed previously for some hairpin peptides. However, the folding free energies of the trpzips exceed substantially those of all previously reported beta-hairpins and even those of some larger designed proteins. NMR structures of three of the trpzip peptides reveal exceptionally well-defined beta-hairpin conformations stabilized by cross-strand pairs of indole rings. The trpzips are the smallest peptides to adopt an unique tertiary fold without requiring metal binding, unusual amino acids, or disulfide crosslinks.

FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family.

Bronchoalveolar lavage fluid from mice with experimentally induced allergic pulmonary inflammation contains a novel 9.4 kDa cysteine-rich secreted protein, FIZZ1 (found in inflammatory zone). Murine (m) FIZZ1 is the founding member of a new gene family including two other murine genes expressed, respectively, in intestinal crypt epithelium and white adipose tissue, and two related human genes. In control mice, FIZZ1 mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells and in non-neuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the wall of the large and small bowel. During allergic pulmonary inflammation, mFIZZ1 expression markedly increases in hypertrophic, hyperplastic bronchial epithelium and appears in type II alveolar pneumocytes. In vitro, recombinant mFIZZ1 inhibits the nerve growth factor (NGF)-mediated survival of rat embryonic day 14 dorsal root ganglion (DRG) neurons and NGF-induced CGRP gene expression in adult rat DRG neurons. In vivo, FIZZ1 may modulate the function of neurons innervating the bronchial tree, thereby altering the local tissue response to allergic pulmonary inflammation.

Peptide exosite inhibitors of factor VIIa as anticoagulants.

Potent anticoagulants have been derived by targeting the tissue factor-factor VIIa complex with naive peptide libraries displayed on M13 phage. The peptides specifically block the activation of factor X with a median inhibitory concentration of 1 nM and selectively inhibit tissue-factor-dependent clotting. The peptides do not bind to the active site of factor VIIa; rather, they work by binding to an exosite on the factor VIIa protease domain, and non-competitively inhibit activation of factor X and amidolytic activity. One such peptide (E-76) has a well defined structure in solution determined by NMR spectroscopy that is similar to the X-ray crystal structure when complexed with factor VIIa. These structural and functional studies indicate an allosteric 'switch' mechanism of inhibition involving an activation loop of factor VIIa and represent a new framework for developing inhibitors of serine proteases.

Characterization of the binding interface between the E-domain of Staphylococcal protein A and an antibody Fv-fragment.

Staphylococcal protein A (SpA) is a cell-surface component of Staphylococcus aureus. In addition to the well-characterized interaction between SpA and the Fc-region of human IgG, an alternative binding interaction between SpA and the Fab-region of immunoglobulin domains encoded by the V(H)3 gene family has been described. To characterize structurally the interface formed by SpA repeats and type-3 V(H)-domains, we have studied the 32-kDa complex formed between an E-domain mutant (EZ4) and the Fv-fragment of the humanized anti-HER2 antibody (Hu4D5-8) using heteronuclear NMR spectroscopy. Protocols were established for efficient incorporation of (15)N, (13)C, and (2)H into EZ4 and the V(H)- and V(L)-domains of the Fv, allowing backbone resonances to be assigned sequentially for EZ4 and the V(H)-domain in both free and complexed states. Broadening of certain V(H)-resonances in the free and bound Fv-fragment suggests microsecond to millisecond time-scale motion in CDR3. Residues experiencing significant chemical shift changes of backbone (1)H(N), (15)N, and (13)CO resonances upon complex formation delineate contiguous surfaces on EZ4 and the V(H)-domain that define the binding interfaces of the two proteins. The interaction surfaces identified by chemical shift mapping are comprised of predominantly hydrophilic residues. This is in contrast to the SpA-Fc interface which is predominantly hydrophobic in nature. Further analysis of the surface properties suggests a probable binding orientation for SpA- and V(H)3-domains.

Solution structure of the VEGF-binding domain of Flt-1: comparison of its free and bound states.

The extracellular portion of the VEGF and PlGF receptor, Flt-1 (or VEGFR-1), consists of seven immunoglobulin-like domains. The second domain from the N terminus (Flt-1D2) is necessary and sufficient for high affinity VEGF binding. The 1.7 A resolution crystal structure of Flt-1D2 bound to VEGF revealed that this domain is a member of the I-set of the immunoglobulin superfamily, but has several unusual features including a region near the N terminus that bulges away from the domain rather than pairing with the neighboring beta-strand. Some of the residues in this region make contact with VEGF, raising the possibility that this bulge could be a consequence of VEGF binding and might not be present in the absence of ligand. Here we report the three-dimensional structure of Flt-1D2 in its uncomplexed form determined by NMR spectroscopy. A semi-automated method for NOE assignment that takes advantage of the previously solved crystal structure was used to facilitate rapid analysis of the 3D NOESY spectra. The solution structure is very similar to the previously reported VEGF-bound crystal structure; the N-terminal bulge is present, albeit in a different conformation. We also report the 2.7 A crystal structure of Flt-1D2 in complex with VEGF solved in a different crystal form that reveals yet another conformation for the N-terminal bulge region. (1)H-(15)N heteronuclear NOEs indicate this region is flexible in solution; the crystal structures show that this region is able to adopt more than one conformation even when bound to VEGF. Thus, VEGF-binding is not accompanied by significant structural change in Flt-1D2, and the unusual structural features of Flt-1D2 are an intrinsic property of this domain.

Structure of a CXC chemokine-receptor fragment in complex with interleukin-8.

BACKGROUND: Interactions between CXC chemokines (e.g. interleukin-8, IL-8) and their receptors (e.g. CXCR-1) have a key role in host defense and disease by attracting and upregulating neutrophils to sites of inflammation. The transmembrane nature of the receptor impedes structure-based understanding of ligand interactions. Linear peptides based on the N-terminal, extracellular portion of the receptor CXCR-1 do bind to IL-8, however, and inhibit the binding of IL-8 to the full-length receptor. RESULTS: The NMR solution structure of the complex formed between IL-8 and one such receptor-based peptide indicates that a cleft between a loop and a beta hairpin constitute part of the receptor interaction surface on IL-8. Nine residues from the C terminus of the receptor peptide (corresponding to Pro21-Pro29 of CXCR-1) occupy the cleft in an extended fashion. Intermolecular contacts are mostly hydrophobic and sidechain mediated. CONCLUSIONS: The results offer the first details at an atomic level of the interaction between a chemokine and its receptor. Consideration of other biochemical data allow extrapolation to a model for the interaction of IL-8 with the full-length receptor. In this model, the heparin-binding residues of IL-8 are exposed, thereby allowing presentation of the chemokine from endothelial cell-surface glycosaminoglycans. This first glimpse of how IL-8 binds to its receptor provides a foundation for the structure-based design of chemokine antagonists.

Molecular mimics of insulin-like growth factor 1 (IGF-1) for inhibiting IGF-1: IGF-binding protein interactions.

IGF-1 (insulin-like growth factor 1) is a 70-residue protein hormone which has both metabolic and mitogenic activities mediated through IGF-1 binding to cell surface receptors. However, an unrelated class of proteins, the IGF-binding proteins (IGFBPs) also bind IGF-1 in the serum and tissues and block or modulate its activity in vivo. Therefore, inhibitors of the IGFBPs can alter the distribution between free and bound IGF-1 [Loddick, S. A., Liu, X.-J., Lu, Z.-X., Liu, C., Behan, D. P., Chalmers, D. C., Foster, A. C., Vale, W. W., Ling, N., and De Souza, E. B. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 1894-1898] and potentially affect the distribution of IGF-1 among body tissues. We report here that phage-displayed peptide libraries have yielded a peptide that binds IGFBP-1 and produces IGF-like activity at sub-micromolar concentrations. The 14-residue peptide has an extremely well-defined solution conformation that can aid in the design of smaller, orally active compounds. Interestingly, the peptide structure contains a helix, as does one region of IGF-1 previously implicated in IGFBP binding, yet displays side chains different from those of the IGF-1 helix I. Furthermore, an IGF-1 variant lacking receptor-signaling activity in vitro is shown here to produce IGF-like mitogenic and metabolic activity in vivo. These results suggest that small antagonist mimetics of protein ligands, identified by binding selection to otherwise inhibitory factors, may be useful as indirect agonists for a variety of therapeutic applications.

Transfer of a protein binding epitope to a minimal designed peptide.

Results from protein mutagenesis and x-ray crystallographic studies of the multidomain protein Vascular Cell Adhesion Molecule (VCAM) were used to design cyclic octapeptides that retain the critical structural and binding elements of the epitope of VCAM in the interaction with the integrin alpha 4 beta 1 (VLA-4). Changes in the activities of peptide analogues correlated with the relative activities of protein mutants of VCAM, and predicted the properties of two new mutants that bound alpha 4 beta 1 with improved affinity vs wild type protein. The nmr structures of two peptides revealed a high degree of similarity to the structure of the VCAM binding epitope. These results demonstrate that a compact binding epitope identified via protein structure-function studies may be transferred to a synthetically accessible small peptide with the key structure-activity relationships intact.

Distinct but overlapping epitopes for the interaction of a CC-chemokine with CCR1, CCR3 and CCR5.

Chemokines play an important role in inflammation. The mechanism via which they bind to more than one receptor and activate them is not well understood. The chemokines are thought to interact with their receptors via two distinct sites, one necessary for binding and the other for activation of signal transduction. In this study we have used alanine scanning mutagenesis to identify residues on RANTES that specifically interact with its receptors CCR1, CCR3, and CCR5 for binding and activation. Residues within a potential receptor binding site known as the N-loop (residues 12-20) and near the N-terminus of RANTES were individually mutated to alanine. The results of this study show that, within the N-loop, the side chain of R17 is necessary for RANTES binding to CCR1, F12 for binding to CCR3, and F12 and I15 for binding to CCR5, thus forming distinct but overlapping binding epitopes. In addition, our finding that P2 is necessary for binding to CCR5 is the first to show that a residue near the N-terminus of a CC-chemokine is involved in binding to a receptor. We have also found that P2, D6, and T7 near the N-terminus are involved in activating signal transduction via CCR1, P2 and Y3 via CCR3, and Y3 and D6 via CCR5. These results indicate that RANTES interacts with each of its receptors in a distinct and specific manner and provide further evidence to support the two-site model of interaction between chemokines and their receptors.

High-resolution NMR structure and backbone dynamics of the Bacillus subtilis response regulator, Spo0F: implications for phosphorylation and molecular recognition.

NMR has been employed for structural and dynamic studies of the bacterial response regulator, Spo0F. This 124-residue protein is an essential component of the sporulation phosphorelay signal transduction pathway in Bacillus subtilis. Three-dimensional 1H, 15N, and 13C experiments have been used to obtain full side chain assignments and the 1511 distance, 121 dihedral angle, and 80 hydrogen bonding restraints required for generating a family of structures (14 restraints per residue). The structures give a well-defined (alpha/beta)5 fold for residues 4-120 with average rms deviations of 0.59 A for backbone heavy atoms and 1.02 A for all heavy atoms. Analyses of backbone 15N relaxation measurements demonstrate relative rigidity in most regions of regular secondary structure with a generalized order parameter (S2) of 0.9 +/- 0.05 and a rotational correlation time (taum) of 7.0 +/- 0.5 ns. Loop regions near the site of phosphorylation have higher than average rms deviation values and T1/T2 ratios suggesting significant internal motion or chemical exchange at these sites. Additionally, multiple conformers are observed for the beta4-alpha4 loop and beta-strand 5 region. These conformers may be related to structural changes associated with phosphorylation and also indicative of the propensity this recognition surface has for differential protein interactions. Comparison of Spo0F structural features to those of other response regulators reveals subtle differences in the orientations of secondary structure in the putative recognition surfaces and the relative charge distribution of residues surrounding the site of phosphorylation. These may be important in providing specificity for protein-protein interactions and for determining the lifetimes of the phosphorylated state.

Model peptide studies demonstrate that amphipathic secondary structures can be recognized by the chaperonin GroEL (cpn60).

The molecular chaperone cpn60 binds many unfolded proteins and facilitates their proper folding. Synthetic peptides have been used to probe the question of how cpn60 might recognize such a diverse set of unfolded proteins. Three hybrid peptides were synthesized encompassing portions of the bee venom peptide, apamin, and the sequence KWLAESVRAGK from an amphipathic helix in the NH2-terminal region of bovine rhodanese. Two disulfides connecting cysteine residues hold the peptides in stable helical conformations with unobstructed faces oriented away from the disulfides. Peptides were designed to present either a hydrophobic or hydrophilic face of the amphipathic helix that is similar to the one near the amino terminus of rhodanese. Aggregation of these peptides was detected by measuring 1,1'-bis(4-anilino)napthalene-5,5'-disulfonic acid (bisANS) fluorescence at increasing peptide concentrations, and aggregation was not apparent below 2 microM. Thus, all experiments with the peptides were performed at a concentration of 1 microM. Reducing agents cause these helical peptides to form random coils. Fluorescence anisotropy measurements of fluorescein-labeled peptide with the exposed hydrophobic face yielded a Kd = approximately 106 microM for binding to cpn60, whereas there was no detectable binding of the reduced form. The peptide with the exposed hydrophilic face did not bind to cpn60 in either the oxidized or reduced states. Fluorescence experiments utilizing bisANS as a probe showed that binding of the helical hydrophobic peptide could induce the exposure of hydrophobic surfaces on cpn60, whereas the same peptide in its random coil form had no effect. Thus, binding to cpn60 is favored by a secondary structure that organizes and exposes a hydrophobic surface, a feature found in amphipathic helices. Further, the binding of a hydrophobic surface to cpn60 can induce further exposure of complementary surfaces on cpn60 complexes, thus amplifying interactions available for target proteins.

Solution structure of the E-domain of staphylococcal protein A.

The E-domain of staphylococcal protein A is one of five homologous IgG-binding domains designated E, D, A, B, and C that comprise the extracellular portion of protein A. The E-domain binds tightly to Fc fragments of IgG and binds certain Fv fragments with micromolar affinity. To explore further the structural features of Fc binding by protein A, and as a first step in developing a structural understanding of E-domain/Fv complex formation, we have determined the solution structure of the uncomplexed E-domain using 2D homonuclear and heteronuclear NMR spectroscopy. Complete 1H and 15N resonance assignments were obtained, and the structure was determined from 383 NOE-derived distance restrains, 34 phi and 19 chi 1 dihedral angle restraints, and 54 restraints for 27 H-bonds. 3JH alpha-H beta coupling constants and long-range NOEs involving Phe11 indicate the side chain exists in more than one conformation with differing chi 1 values. NOE restraints that were incompatible with chi 1 = -60 degrees were removed from one set of structure calculations, and those incompatible with chi 1 = 180 degrees were removed from a second set to allow Phe11 to explore both rotamer wells. Thus, two sets of 20 final structures, having no distance or dihedral angle restraint violations greater than 0.12 A or 1.6 degrees, respectively, represent the solution structure of the E-domain. Backbone atomic rms differences with respect to the mean coordinates for each set of 20 structures for residues 8-53 averaged 0.41 +/- 0.06 and 0.35 +/- 0.06 A. No significant differences in the overall structure result from the different orientations of Phe11. The solution structure of the E-domain consists of three alpha-helices that pack together to form a compact helical bundle. A detailed comparison between the E-domain ensembles and the previously determined structure for the B-domain in complex with Fc indicates that only the 180 degrees chi 1 rotamer of Phe11 is competent for binding; the -60 degrees chi 1 rotamer must reorient to 180 degrees to create a cavity that is filled by Ile253 from the CH2 domain of Fc in the Fc-bound complex.

RhNGF slow unfolding is not due to proline isomerization: possibility of a cystine knot loop-threading mechanism.

The unfolding of recombinant human beta-NGF (NGF) in guanidine hydrochloride (GdnHCl) was found to be time dependent with the denaturation midpoint moving to lower GdnHCl concentration over time. Dissociation and extensive unfolding of the NGF dimer occurred rapidly in 5 M GdnHCl, but further unfolding of the molecule occurred over many days at 25 degrees C. Fluorescence spectroscopy, size-exclusion and reversed-phase HPLC, ultra-centrifugation, and proton NMR spectroscopy were used to ascertain that the slow unfolding step was between two denatured monomeric states of NGF (M1 and M2). Proton NMR showed the monomer formed at early times in GdnHCl (M1) had little beta-sheet structure, but retained residual structure in the tryptophan indole and high-field methyl regions of the spectrum. This residual structure was lost after prolonged incubation in GdnHCl giving a more fully unfolded monomer, M2. From kinetic unfolding experiments in 5 M GdnHCl it was determined that the conversion of M1 to M2 had an activation energy of 26.5 kcal/mol, a half-life of 23 h at 25 degrees C, and the rate of formation of M2 was dependent on the GdnHCl concentration between 5 and 7.1 M GdnHCl. These properties of the slow unfolding step are inconsistent with a proline isomerization mechanism. The rate of formation of the slow folding monomer M2 increases with truncation of five and nine amino acids from the NGF N-terminus. A model for the slow unfolding reaction is proposed where the N-terminus threads through the cystine knot to form M2, a loop-threading reaction, increasing the conformational freedom of the denatured state.