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S Jayasinghe

Publications and source records attributed to S Jayasinghe.

At least 19 recordsLinked to original sources
PubMed2001

Energetics, stability, and prediction of transmembrane helices.

We show that the peptide backbone of an alpha-helix places a severe thermodynamic constraint on transmembrane (TM) stability. Neglect of this constraint by commonly used hydrophobicity scales underlies the notorious uncertainty of TM helix prediction by sliding-window hydropathy plots of membrane protein (MP) amino acid sequences. We find that an experiment-based whole-residue hydropathy scale (WW scale), which includes the backbone constraint, identifies TM helices of membrane proteins with an accuracy greater than 99 %. Furthermore, it correctly predicts the minimum hydrophobicity required for stable single-helix TM insertion observed in Escherichia coli. In order to improve membrane protein topology prediction further, we introduce the augmented WW (aWW) scale, which accounts for the energetics of salt-bridge formation. An important issue for genomic analysis is the ability of the hydropathy plot method to distinguish membrane from soluble proteins. We find that the method falsely predicts 17 to 43 % of a set of soluble proteins to be MPs, depending upon the hydropathy scale used.

Bacteriorhodopsins↗
PubMed2001

MPtopo: A database of membrane protein topology.

The reliability of the transmembrane (TM) sequence assignments for membrane proteins (MPs) in standard sequence databases is uncertain because the vast majority are based on hydropathy plots. A database of MPs with dependable assignments is necessary for developing new computational tools for the prediction of MP structure. We have therefore created MPtopo, a database of MPs whose topologies have been verified experimentally by means of crystallography, gene fusion, and other methods. Tests using MPtopo strongly validated four existing MP topology-prediction algorithms. MPtopo is freely available over the internet and can be queried by means of an SQL-based search engine.

Algorithms↗
PubMed2000

How to measure and analyze tryptophan fluorescence in membranes properly, and why bother?

Tryptophan fluorescence is a powerful tool for studying protein structure and function, especially membrane-active proteins and peptides. It is arguably the most frequently used tool for examining the interactions of proteins and peptides with vesicular unilamellar model membranes. However, high light scattering associated with vesicular membrane systems presents special challenges. Because of their reduced light scattering compared to large unilamellar vesicles (LUV), small unilamellar vesicles (SUV) produced by sonication are widely used membrane models. Unfortunately, SUV, unlike LUV, are metastable and consequently unsuitable for equilibrium thermodynamic measurements. We present simple and easily implemented experimental procedures for the accurate determination of tryptophan (Trp) fluorescence in either LUV or SUV. Specifically, we show that Trp spectra can be obtained in the presence of up to 6 mM LUV that are virtually identical to spectra obtained in buffer alone, which obviates the use of SUV. We show how the widths and peak positions of such spectra can be used to evaluate the heterogeneity of the membrane conformation and penetration of peptides. Finally, we show how to use a reference fluorophore for the correction of intensity measurements so that the energetics of peptide partitioning into membranes can be accurately determined.

Fluorescence↗
PubMed2000

The secretory carrier membrane protein family: structure and membrane topology.

Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SCAMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain located at the cytoplasmic interface with little exposure of the protein on the ectodomain.

Amino Acid Sequence↗
PubMed1998

Structural features that modulate the transmembrane migration of a hydrophobic peptide in lipid vesicles.

Two approaches employing nuclear magnetic resonance (NMR) were used to investigate the transmembrane migration rate of the C-terminal end of native alamethicin and a more hydrophobic analog called L1. Native alamethicin exhibits a very slow transmembrane migration rate when bound to phosphatidylcholine vesicles, which is no greater than 1 x 10(-4) min(-1). This rate is much slower than expected, based on the hydrophobic partition energies of the amino acid side chains and the backbone of the exposed C-terminal end of alamethicin. The alamethicin analog L1 exhibits crossing rates that are at least 1000 times faster than that of native alamethicin. A comparison of the equilibrium positions of these two peptides shows that L1 sits approximately 3-4 A deeper in the membrane than does native alamethicin (Barranger-Mathys and Cafiso. 1996. Biochemistry. 35:489). The slow rate of alamethicin crossing can be explained if the peptide helix is irregular at its C-terminus and hydrogen bonded to solvent or lipid. We postulate that L1 does not experience as large a barrier to transport because its C-terminus is already buried within the membrane interface. This difference is most easily explained by conformational differences between L1 and alamethicin rather than differences in hydrophobicity. The results obtained here demonstrate that side-chain hydrophobicity alone cannot account for the energy barriers to peptide and protein transport across membranes.

Alamethicin↗
PubMed1994

Structure of micelle-associated alamethicin from 1H NMR. Evidence for conformational heterogeneity in a voltage-gated peptide.

Alamethicin is a 20 amino acid peptide that produces a voltage-dependent conductance in membranes. To understand the mechanism by which this peptide becomes voltage-gated, the structure of alamethicin bound to micelles was examined using high-resolution 1H nuclear magnetic resonance (NMR). Two-dimensional correlation and nuclear Overhauser effect spectroscopy (NOESY) were carried out on alamethicin incorporated into perdeuterated sodium dodecyl sulfate (SDS) micelles, and the 1H NMR spectrum of the peptide in micelles was assigned. The intensities of the HN-HN(i,i+1), H alpha-HN(i,i+1), H alpha-NH(i,i+3), H alpha-H beta (i,i+3), and H alpha-NH(i,i+4) cross peaks in the NOESY spectrum suggest that the N-terminal half of the peptide is predominantly alpha-helical, while the C-terminal half has a less regular or more flexible structure. The exposure of micelle bound alamethicin to the aqueous solution was determined by examining the effect of aqueous paramagnetic reagents on the line widths of the peptide protons. These measurements suggest that alamethicin is buried in the micelle. A set of restraints consisting of 175 distances (derived from NOESY spectra), five dihedral angles, and two hydrogen bond distances were used in a simulated annealing procedure that yielded structures for micelle associated alamethicin. The structures that were generated with simulated annealing were largely helical from residues 4-9 and 12-16. A limited number of structural forms were obtained. The main difference among forms involved the backbone conformations of MeA10, Gly11, and Leu12 and resulted in structures that were straight or had different amounts of bend. The structural forms could be easily interconverted by rotation of the psi and phi angles of residues 10-12. The rotational freedom at or near MeA10 may be a result of Pro14, which would be the normal hydrogen-bonding position for the peptide carbonyl of MeA10. These results suggest that conformation rearrangements at or near MeA10 may play a role in the voltage-gating of alamethicin.

Alamethicin↗