Biomedical subjects

Shiamalee Perumal

Publications and source records attributed to Shiamalee Perumal.

3 recordsLinked to original sources

PubMed2005

Surface-induced unfolding of human lactoferrin.

We have determined the structural conformations of human lactoferrin adsorbed at the air/water interface by neutron reflectivity (NR) and its solution structure by small angle neutron scattering (SANS). The neutron reflectivity measurements revealed a strong structural unfolding of the molecule when adsorbed at the interface from a pH 7 phosphate buffer solution (PBS with a total ionic strength at 4.5 mM) over a wide concentration range. Two distinct regions, a top dense layer of 15-20 angstroms on the air side and a bottom diffuse layer of some 50 angstroms into the aqueous subphase, characterized the unfolded interfacial layer. At a concentration around 1 g dm(-3), close to the physiological concentration of lactoferrin in biological fluids, the adsorbed amount was 5.5 x 10(-8) mol m(-2) in the absence of NaCl, but the addition of 0.3 M NaCl reduced protein adsorption to 3.5 x 10(-8) mol m(-2). Although the polypeptide distributions at the interface remained similar, quantitative analysis showed that the addition of NaCl reduced the layer thickness. Parallel measurements of lactoferrin adsorption in D2O instead of null reflecting water confirmed the unfolded structure at the interface. Furthermore, the D2O data indicated that the polypeptide in the top layer was predominantly protruded out of water, consistent with it being hydrophobic. In contrast, the scattering intensity profiles from SANS were well described by a cylindrical model with a diameter of 47 angstroms and a length of 105 angstroms in the presence of 0.3 M NaCl, indicating a retention of the globular framework in the bulk solution. In the absence of NaCl but with the same amount of phosphate buffer, the length of the cylinder increased to some 190 angstroms and the diameter remained constant. The length increase is indicative of changes in distance and orientation between the bilobal monomers due to the change in charge interactions. The results thus demonstrate that the surface structural unfolding was caused by the exposure of the protein molecule to the unsymmetrical energetic balance following surface adsorption.

Adsorption↗

PubMed2004

Interfacial nano-structuring of designed peptides regulated by solution pH.

The in-situ conformations of peptide layers formed from the adsorption of two different synthetic 15-mer peptides at the hydrophilic silicon oxide/aqueous solution interface have been determined using neutron reflectivity (NR). The first peptide is based on the native sequence of a protein-binding domain within a heteromeric transcriptional activator, HAP2, identified from yeast Saccharomyces cerevisiae, with tyrosine (Y) present at the 1st, 8th and 15th amino acid positions, hence we denote this YYY15. Substitution of tryptophan (W) at the same locations gives WWW15. Both peptides have alpha-helical structure in phosphate buffer, as determined by circular dichroism (CD) spectra. D(2)O was used as solvent in the NR experiments to highlight structural heterogeneity across the hydrogenated peptide layers. At pH 7, YYY15 was found to form a weakly adsorbed interfacial monolayer. However, the mutant WWW15 showed strong interfacial adsorption, with the interfacial layer characterized by a middle hydrophobic sublayer of 7-8 A with lower scattering length density and two almost symmetrical hydrophilic outer sublayers of 6-8 A with higher scattering length density, suggesting the formation of a "sideways-on" helical conformation. An increase in pH to 9 resulted in the improved packing within the interfacial layer with similar structure. However, decrease in pH to 5 reduced the interfacial adsorption, mainly due to the enhanced solubility of the peptides associated with the protonation of arginine (R) and lysine (K) groups and the decreasing concentration of divalent HPO(4)(2-) in the phosphate buffer. Subsequent assessment of the reversibility of adsorption showed that once the peptide layers were formed they did not desorb. These interfacial structures may provide feasible routes to interfacial nano-templating.

Amino Acid Sequence↗

PubMed2003

Adsorption of beta-hairpin peptides on the surface of water: a neutron reflection study.

Neutron reflectivity has been used to determine the thickness and surface coverage of monolayers of two 14-residue beta-hairpin peptides adsorbed at the air/water interface. The peptides differed only in that one was labeled with a fluorophore, while the other was not. The neutron reflection measurements were mainly made in null reflecting water, NRW, containing 8.1% D(2)O. Under this isotopic contrast the water is invisible to neutrons and the specular signal was then only from the peptide layer. At the highest concentration of ca. 4 microg/mL studied, the area per peptide molecule (A) was found to be 230 +/- 10 and 210 +/- 10 A(2) for the peptides with and without a BODIPY-based fluorophore, respectively. The thickness of the peptide layers was about 10 A for a Gaussian distribution. With decreasing bulk peptide concentration, both surface excess and layer thickness showed a steady trend of decrease. While the neutron results clearly indicate structural changes within the peptide monolayers with increasing bulk concentration, the outstanding structural feature is the formation of rather uniform peptide layers, consistent with the structural characteristics typical of beta-strand peptide conformations. These structural features are well supported by the parallel measurements of the adsorbed layers in D(2)O. With this isotopic contrast the neutron reflectivity provides an estimate about the extent of immersion of the peptide layers into water. The results strongly suggest that the 14-mer peptide monolayers were fully afloat on the surface of water, with only the carboxy groups on Glu residues hydrated.

Adsorption↗