Biomedical subjects

E Polverini

Publications and source records attributed to E Polverini.

8 recordsLinked to original sources

PubMed2004

Small angle x-ray scattering from lipid-bound myelin basic protein in solution.

The structure of myelin basic protein (MBP), purified from the myelin sheath in both lipid-free (LF-MBP) and lipid-bound (LB-MBP) forms, was investigated in solution by small angle x-ray scattering. The water-soluble LF-MBP, extracted at pH < 3.0 from defatted brain, is the classical preparation of MBP, commonly regarded as an intrinsically unfolded protein. LB-MBP is a lipoprotein-detergent complex extracted from myelin with its native lipidic environment at pH > 7.0. Under all conditions, the scattering from the two protein forms was different, indicating different molecular shapes. For the LB-MBP, well-defined scattering curves were obtained, suggesting that the protein had a unique, compact (but not globular) structure. Furthermore, these data were compatible with earlier results from molecular modeling calculations on the MBP structure which have been refined by us. In contrast, the LF-MBP data were in accordance with the expected open-coil conformation. The results represent the first direct structural information from x-ray scattering measurements on MBP in its native lipidic environment in solution.

Computer Simulation↗

PubMed2000

Characterization of a recombinant murine 18.5-kDa myelin basic protein.

A recombinant hexahistidine-tagged 18.5-kDa isoform of murine myelin basic protein has been characterized biochemically and immunogenically, by mass spectrometry, by circular dichroism under various conditions (in aqueous solution, with monosialoganglioside G(M1), and in 89% 2-propanol), and by transmission electron microscopy. The preparations of this protein indicated a high degree of purity and homogeneity, with no significant posttranslational modifications. Circular dichroic spectra showed that this preparation had the same degree of secondary structure as the natural bovine 18.5-kDa isoform of myelin basic protein. Incubation of the recombinant protein with lipid monolayers containing a nickel-chelating lipid resulted in the formation of fibrous assemblies that formed paracrystals of spacings 4.8 nm between fibers and 3-4 nm along them.

Animals↗

PubMed1999

Conformation of bovine myelin basic protein purified with bound lipids.

The basic protein of myelin (called MBP) is an extrinsic protein of the myelin membrane. Its structure and function are still unknown. MBP has been extensively studied in its water-soluble form, but it is also known in a detergent-soluble form, which is purified with endogenous myelin lipids and should correspond to the native form of the protein in the membrane. In order to acquire insight into the structure of MBP, we have carried out circular dichroism (CD) experiments on the protein both in the lipid-free and in the lipid-bound form. Our data clearly show that lipid-free MBP is mainly disordered with only a small amount having alpha-helix and beta-sheet motifs. On the other hand, the lipid-bound form of MBP appears to have a consistent amount of ordered secondary structure. Theoretical predictions, made using different computational methods, substantially confirm the tendency of the protein to assume an ordered secondary structure in accordance with our CD results.

Amino Acid Sequence↗

PubMed1999

The structural basis for the regulation of tissue transglutaminase by calcium ions.

The role of calcium ions in the regulation of tissue transglutaminase is investigated by experimental approaches and computer modeling. A three-dimensional model of the transglutaminase is computed by homology building on crystallized human factor XIII and is used to interpret structural and functional results. The molecule is a prolate ellipsoid (6.2 x 4.2 x 11 nm) and comprises four domains, assembled pairwise into N-terminal and C-terminal regions. The active site is hidden in a cleft between these regions and is inaccessible to macromolecular substrates in the calcium-free form. Protein dynamics simulation indicates that these regions move apart upon addition of calcium ions, revealing the active site for catalysis. The protein dimensions are consistent with results obtained with small-angle neutron and X-ray scattering. The gyration radius of the protein (3 nm) increases in the presence of calcium ions (3.9 nm), but it is virtually unaffected in the presence of GTP, suggesting that only calcium ions can promote major structural changes in the native protein. Proteolysis of an exposed loop connecting the N-terminal and C-terminal regions is linearly correlated with enzyme inactivation and prevents the calcium-induced conformational changes.

Amino Acid Sequence↗

PubMed1998

Purification of bovine P2 myelin protein with bound lipids.

The P2 protein is a neuritogenic, small basic protein present in PNS myelin. It belongs to the family of the cytoplasmic lipid-binding proteins and can be incorporated in lipidic bilayers. P2 has been purified and crystallized only in the lipid-free form. Here we show that the P2 protein can be purified with bound lipids by applying to PNS myelin the same procedure that as used to purify lipid-bound myelin basic protein from CNS myelin. SDS-PAGE showed a single band of 16.5 kDa, and TLC showed the presence of most of the myelin lipids associated with the protein. Lipid-bound P2 revealed different circular dichroism spectra from the corresponding lipid-free form, indicating that lipids influence P2 conformation.

Animals↗

PubMed1998

Conformational changes of neuromedin B and delta sleep-inducing peptide induced by their interaction with lipid membranes as revealed by spectroscopic techniques and molecular dynamics simulation.

Static and dynamic spectroscopic properties of the tryptophanil emission in conjunction with circular dichroism (CD) spectroscopy and molecular dynamics are used to investigate the interactions of the neuropeptide neuromedin B (NMB) and the membrane-permeable delta sleep-inducing peptide (DSIP) with the membrane lipid phase. Our data indicate that in solution both peptides exist in energetically equivalent conformations, whereas in the presence of the membrane specific conformational states are stabilized. By changing from the aqueous to the lipid phase, the static and the dynamic fluorescence properties of the NMB's tryptophan residue are clearly affected: the fluorescence steady-state spectrum as well as the resolved fluorescence decay-associated spectra (DAS) are shifted to the blue with a significant increase of the fluorescence intensity of the second lifetime component (tau 2-DAS). On the other hand, in the lipid environment the same parameters of DSIP are negligibly affected as compared to the aqueous buffer. The CD and molecular dynamics analyses are consistent with these results and indicate that, while NMB assumes a helix-like conformation with the tryptophan residue in the apolar surface, DSIP adopts a globule-like structure with the indole ring that is surface-exposed. As previously found for neuromedin C (Polverini, E., Neyroz, P., Fariselli, P., Casadio, R., and Masotti, L., Biochem. Biophys. Res. Commun. 214, 663-668, 1995), for NMB the stabilized "lipophilic" structure also may favor the correct peptide-receptor contact and recognition. For DSIP, the lipid-stabilized conformation does not support an amphiphilic structure-driven peptide-membrane interaction and suggests a hydrophobicity-driven diffusion across the bilayer.

Circular Dichroism↗

PubMed1996

Intrinsic fluorescence properties and structural analysis of p13(suc1) from Schizosaccharomyces pombe.

p13(suc1) acts in the fission yeast cell division cycle as a component of p34(cdc2). In the present work, structural information contained in the intrinsic fluorescence of p13(suc1) has been extracted by steady-state and time-resolved fluorescence techniques. In its native form, the steady-state emission spectrum of p13(suc1) is centered at 336 nm. Upon denaturation by guanidine HCl (4.0 M), the emission spectrum is shifted to 355-360 nm and the fluorescence intensity decreases 70%. The same changes are not obtained with p13(suc1) at 56 degrees C or after incubation at 100 degrees C, and the protein appears to be substantially temperature-stable. The fluorescence decay of p13(suc1) is best described by three discrete lifetimes of 0.6 ns (tau1), 2.9 ns (tau2), and 6.1 ns (tau3), with amplitudes that are dependent on the native or unfolded state of the protein. Under native conditions, the two predominant decay-associated spectra, DAS-tau2 (lambdamax = 332 nm) and DAS-tau3 (lambdamax = 340 nm), derive from two different excitation DAS. Moreover distinct quenching mechanisms and collisional accessibilities (kq(tau2)>>kq(tau3)) are resolved for each lifetime. An interpretation in terms of specific tryptophan residue (or protein conformer)-lifetime assignments is presented. The decay of the fluorescence anisotropy of native p13(suc1) is best described by a double exponential decay. The longer correlation time recovered (9 ns </= phi2 </= 15ns) can be associated with the rotational motion of the protein as a whole and a Stokes radius of 21.2 A has been calculated for p13(suc1). Anisotropy measurements obtained as a function of temperature indicate that, in solution, the protein exists exclusively as a prolate monomer. In 1 mM zinc, changes of the anisotropy decay parameters are compatible with subunits oligomerization.

Cell Cycle Proteins↗

PubMed1995

The effect of membranes on the conformation of neuromedin C.

The combination of static and dynamic spectroscopy data with the information obtained by computational methods has been used to investigate the conformation of neuromedin C in the absence and in the presence of lipid membranes. Upon addition of lipids, the peptide steady-state emission spectrum is blue shifted (355 nm vs 345 nm) and all the recovered fluorescence decay constants are significantly longer. CD measurements show that fluorescence changes are consistent with alpha-helix inducing structural transitions. Molecular dynamics studies show that, in solution, the peptide conformation rapidly exchanges between energetically equivalent disordered and ordered states, whereas, in the presence of membrane, the peptide conformation is stabilized in an ordered state.

Animals↗