Gautam Sanyal

The structure of human acidic fibroblast growth factor and its interaction with heparin

The secondary and tertiary structure of recombinant human acidic fibroblast growth factor (aFGF) has been characterized by a variety of spectroscopic methods. Native aFGF consists of ca. 55% beta-sheet, 20% turn, 10% alpha-helix, and 15% disordered polypeptide as determined by laser Raman, circular dichroism, and Fourier transform infrared spectroscopy; the experimentally determined secondary structure content is in agreement with that calculated by the semi-empirical methods of Chou and Fasman (Chou, P. Y., and Fasman, G. C., 1974, Biochemistry 13, 222-244) and Garnier et al. (Garnier, J. O., et al., 1978, J. Mol. Biol. 120, 97-120). Using the Garnier et al. algorithm, the major secondary structure components of aFGF have been assigned to specific regions of the polypeptide chain. The fluorescence spectrum of native aFGF is unusual in that it is dominated by tyrosine fluorescence despite the presence of a tryptophan residue in the protein. However, tryptophan fluorescence is resolved upon excitation above 295 nm. The degree of tyrosine and tryptophan solvent exposure has been assessed by a combination of ultraviolet absorption, laser Raman, and fluorescence spectroscopy; the results suggest that seven of the eight tyrosine residues are solvent exposed while the single tryptophan is partially inaccessible to solvent in native aFGF, consistent with recent crystallographic data. Denaturation of aFGF by extremes of temperature or pH leads to spectroscopically distinct conformational states in which contributions of tyrosine and tryptophan to the fluorescence spectrum of the protein vary. The protein is unstable at physiological temperatures. Addition of heparin or other sulfated polysaccharides does not affect the spectroscopic characteristics of native aFGF. These polymers do, however, dramatically stabilize the native protein against thermal and acid denaturation as determined by differential scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The interaction of aFGF with such polyanions may play a role in controlling the activity of this growth factor in vivo.

Static quenching of tryptophan fluorescence by oxidized dithiothreitol

The quenching of fluorescence of 5-methoxyindole, N-acetyl-L-tryptophanamide and two single tryptophan containing peptides, melittin and mastoparan X, by oxidized dithiothreitol was studied. The slopes of the Stern-Volmer plots for steady-state fluorescence quenching were 133 M-1, 71.2 M-1, 75.5 M-1 and 35.0 M-1 at 21 degrees C and pH 7.0 for 5-methyoxyindole, N-acetyl-L-tryptophanamide, melittin and mastoparan X respectively. Fluorescence lifetimes of indole or tryptophan in these compounds, as determined by multifrequency phase fluorometry, were decreased by 15% or less at concentrations that produced 50% or more quenching of steady-state fluorescence. Thus, quenching of fluorescence by oxidized dithiothreitol for these derivatives of indole appears to be largely static in nature, suggesting a ground-state interaction.

Stabilization of the FK506 binding protein by ligand binding

Although the rotamase activity of the FK506 binding protein is inhibited by ligand binding, it is hypothesized that the ligand/protein complex itself may be responsible for the immunosuppressive effects of FK506. We have therefore examined the structure of the FK506 binding protein in the presence of an analog of FK506 (FK520) by a combination of fluorescence, CD, FTIR and calorimetry. While only small changes in the overall structure of the protein may be induced by ligand, a large change in thermal stability of the binding protein is observed.

Biophysical Characterization and Formulation of TP40: A Chimeric Protein that Requires a pH-Dependent Conformational Change for Its Biological Activity

is a genetically engineered chimeric protein composed of transforming growth a 5-kDa peptide (Massague, 1990), fused to 40-kDa segment (PE40) of Pseudomonas exotoxin (PE). In its unaltered form PE contains three distinct domains: (a) a cell binding portion that presumably binds to an macroglobulin/low density lipoprotein receptor which is present on most eukaryotic cell surfaces (Kounnas et al., 1992), (b) a translocation domain which is responsible

Tryptophan Fluorescence as a Structural Probe of Interaction of Amphiphilic Peptides with Calmodulin

Several peptides exhibit calcium-dependent high affinity binding (dissociation constant ≃10-9M) to calmodulin (CaM), the ubiquitous calcium binding protein (Malencik and Anderson, 1983; Malencik & Anderson, 1984; Cox et al., 1985). These peptides, such as the three mastoparans, competitively displace enzymes (e. g. myosin light chain kinase) off the 1:1 CaM-enzyme complex to form the CaM-peptide complex (Malencik and Anderson, 1983). Consequently they are considered to be viable structural models for the CaM-interacting surfaces on the CaM-dependent enzymes. It was proposed independently by us and by others, that the ability to form a basic amphiphilic helix is probably the common driving force for these peptides to bind to CaM (Cox et al., 1985; McDowell et al., 1985). The purpose of this study was to probe the environment of the CaM-interacting region of some amphiphilic peptides by using the fluorescence signal from their single tryptophan (Trp) residues (CaM does not contain Trp). The single letter amino acid sequences of these peptides are shown in Table 1. With the exception of melittin the Trp in each peptide forms a part of the hydro-phobic face of the putative helical wheel (not shown, see McDowell et al., 1985). They all have large helical hydrophobic moments (Eisenberg et al., 1982) and difference circular dichroic (CD) spectra suggest that a high degree of helicity is induced in the peptides upon CaM binding (Maulet and Cox, 1983; McDowell et al., 1985).