Peptide GVKGDKGNPGWPGAPY (called peptide IV-H1), derived from the protein sequence of human collagen type IV, triple-helix domain residues 1263-1277, represents an RGD-independent, cell-specific, adhesion, spreading, and motility promoting domain in type IV collagen. In this study, peptide IV-H1 has been investigated by 1H NMR (500 MHz) spectroscopy. Cis-trans proline isomerization at each of the three proline residues gives rise to a number of slowly exchanging (500-MHz NMR time scale) conformation states. At least five such states are observed, for example, for the well-resolved A14 beta H3 group, and K3, which is six residues sequentially removed from the nearest proline, i.e., P9, shows two sets. The presence of more than two sets of resonances for residues sequentially proximal to a proline, e.g., A14-cis-P15 and A14-trans-P15, and more than one set for a residue sequentially well-removed from a proline, e.g., K3, indicates long range conformation interactions and the presence of preferred structure in this short linear peptide. Many resonances belonging to these multiple species have been assigned by using mono-proline-substituted analogues. Conformational (isomer) state-specific 2D 1H NMR assignments for the combination of cis and trans proline states have been made via analysis of COSY-type, HOHAHA, and NOESY spectra. Peptide IV-H1 in the all-trans proline state ttt exists in relatively well-defined conformation populations showing numerous short- and long-range NOEs and long-lived backbone amide protons and reduced backbone NH temperature coefficients, suggesting hydrogen-bonding, and structurally informative 3J alpha N coupling constants. The NMR data indicate significant beta-turn populations centered at K3-G4, K5-G6, P9-G10, and P12-G13, and a C-terminal gamma-turn within the A14-P15-Y16 sequence. These NMR data are supported by circular dichroic studies which indicate the presence of 52% beta-turn, 10% helix, and 38% random coil structural populations. Since equally spaced KG and PG residues are found on both sides of peptide IV-H1 in the native collagen type IV sequence, this multiple turn repeat motif may continue through a longer segment of the protein. Synthetic peptide IV-H1 overlapping sequence "walk throughs" indicate that the primary biological activity is localized in the GNPGWPGAP double beta-turn domain, which contains the backbone constraining proline residues. This proline-domain conformation may suggest a collagen type IV receptor-specific, metastatic cell adhesion promoting binding domain.

Specific lengths of the fatty and polyoxyethylene chains of ethoxylated fatty amines are critical to their performance in specific applications, and thus the ability to characterize these surfactants accurately is crucial. Normal-phase high-performance liquid chromatography (HPLC) and matrixassisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry methods were developed to determine with accuracy the molecular weight and degree of ethoxylation of ethoxylated fatty amines. Ethoxylated fatty amines were analyzed using these methods, and comparison was made to molecular weight determinations using proton nuclear magnetic resonance (NMR), neutralization equivalent weight, and hydroxyl value methods. Molecular weight results from normalphase HPLC analyses were in very good agreement with MALDI-TOF results, typically varying less than one ethylene oxide unit. A reversed-phase HPLC method was developed to determine concentrations of polyethylene glycols (PEG) and fatty homologs. PEG interfered with molecular weight determinations by NMR, neutralization equivalent weight, and hydroxyl value methods. PEG caused no interference with molecular weight determinations by normal-phase HPLC and MALDI-TOF methods.

X-band electron spin resonance spectroscopy was used to study the binding of vanadium (IV), or vanadyl, to the brain serine/threonine phosphatase-2B, caicineurin. Spectra were determined on frozen solutions of vanadyl and caicineurin at pH 7.4 in the presence of 20% (v/v) glycerol. The binding of vanadyl to the enzyme was established, and the data suggested the presence of two classes of sites, the higher affinity class of which contained two binding sites for vanadyl. The calcium-binding B subunit of the heterodimeric protein was also shown to bind vanadyl. The holoprotein appeared to be stabilized by vanadyl, and vanadyl enhanced enzymatic activity when assayed with or without calmodulin in the absence of calcium. . It is found in vivo primarily in its V oxidation state, vanadate; in turn, this is reduced intracellulary to the IV oxidation state, vanadyl [12]. ESR spectroscopy has been used to demonstrate that vanadyl binds to a variety of metalloproteins [13,14]. We have shown that vanadyl binds to calmodulin [15,16] and inhibits the stimulation of skeletal muscle myosin light chain kinase activity [17]. In view of the homology of the calcineurin B subunit and calmodulin, particularly in the regions of calcium-binding [1], we performed ESR spectroscopy to ascertain if vanadyl binds to brain calcineurin. Our results show that vanadyl binds to this protein phosphatase and appears to enhance enzymatic activity.