Effect of pH on molecular constitution and distribution of hemoglobin in living erythrocyte

The molecular constitution of in situ hemoglobin (Hb) and their distribution in living erythrocyte were investigated versus pH using the technique of confocal Raman microscopy. Both Raman point spectra and line mapping measurements were performed on living erythrocytes in suspensions with pH values from 4.82 to 9.70. It was found that the Hb inside a living erythrocyte would dissociate into monomer/dimer when the cells are in low and high pH environments. In contrast to the homogeneous distribution of the Hbs in the cells in neutral suspension, there are more Hbs distributing around the cell membrane or binding to the membrane as pH increases. While in low pH, as the cell become spherical, most of the Hbs distribute to the central part of the cell. In summary, our investigation suggests that the variation of the external pH not only brings changes in the morphology and membrane structure of an erythrocyte, but also affects the constitution and distribution of its intracellular Hbs, thereby the flexibility of the cell membrane and the oxygenation ability of the Hb. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 348–354, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Conformational studies of the alpha-helical 28-43 fragment of the B3 domain of the immunoglobulin binding protein G from Streptococcus

To determine whether the alpha-helix in the B3 immunoglobulin binding domain of protein G from group G Streptococcus has conformational stability as an isolated fragment, we carried out a CD and NMR study of the 16-residue peptide in solution corresponding to this alpha-helix. Based on two-dimensional H-NMR spectra recorded at three different temperatures (283, 305, and 313 K), it was found that this peptide is mostly unstructured in water at these temperatures. Weak signals corresponding to i,i+3 or i,i+4 interactions, which are characteristic of formation of turn-like structures, were observed in the ROE spectra at all temperatures. The absence of a stable three-dimensional structure of the investigated peptide supports an earlier study (Blanco and Serrano, Eur J Biochem 1995, 230, 634-649) of a possible mechanism for folding of other (B1 and B2) immunoglobulin binding domains of Protein G. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 1032-1044, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Investigations of thermotropic phase behavior of newly developed synthetic PEGylated lipids using Raman spectro-microscopy

In this article, a temperature-controlled Raman spectro-microscopic technique has been utilized to detect and analyze the phase behaviors of two newly developed synthetic PEGylated lipids trademarked as QuSomestrade mark, which spontaneously form liposomes upon hydration in contrast to conventional lipids. The amphiphiles considered in this study differ in their hydrophobic hydrocarbon chain length and contain different units of polyethylene glycol (PEG) hydrophilic headgroups. Raman spectra of these new artificial lipids have been recorded in the spectral range of 500-3100 cm(-1) by using a Raman microscope system in conjunction with a temperature-controlled sample holder. The gel to liquid phase transitions of the sample lipids composed of pure 1,2-dimyristoyl-rac-glycerol-3-dodecaethylene glycol (GDM-12) and 1,2-distearoyl-rac-glycerol-3-triicosaethylene glycol (GDS-23) have been revealed by plotting peak intensity ratios in the C--H stretching region as a function of temperature. From this study, we have found that the main phase transitions occur at a temperature of approximately 5.2 and 21.2 degrees C for pure GDM-12 and GDS-23, respectively. Furthermore, the lipid GDS-23 also shows a postphase transition temperature at 33.6 degrees C. To verify our results, differential scanning calorimetry (DSC) experiments have been conducted and the results are found to be in an excellent agreement with Raman scattering data. This important information may find application in various studies including the development of lipid-based novel substances and drug delivery systems. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 1012-1020, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Binding of lectins to "young" and "old" human erythrocytes

"Old" human erythrocytes showed a 21.2% decrease in cell surface area and a 2% decrease in the number of WGA receptor sites, but a 27% increase in the distribution density of the WGA (lectin) receptor site, when compared with "young" human erythrocytes. For a list of lectin abbreviations, see Materials and methods). Both "young" and "old" erythrocytes exhibited very weak binding activity for 125I-labeled PNA, but there was no difference in binding activity for PNA between "young" erythrocytes and "old" ones. Compared with "young" erythrocytes, decreases in the number and distribution density of receptor sites for five lectins including LPA, Con A, RCA-II, SBA and BPA on the cell surface were observed in aged erythrocytes. "Old" erythrocytes also showed a decrease in the number of PHA-E receptor sites, while the distribution density of the same receptor site remained unchanged. In view of these and other observations, it is thought that human erythrocyte aging is accompanied by elimination of some glycoconjugates which have affinity for six lectins, LPA, Con A, RCA-II, PHA-E, SBA and BPA, whereas no WGA receptor-containing glycoconjugates are released from erythrocyte membranes. Elimination of the glycoconjugates results in shrinkage of erythrocytes to reduce their cell surface areas.     

Influence of chondroitin sulfate on collagen gel structure and mechanical properties at physiologically relevant levels

The ability to alter collagen organization could lead to more physiologically relevant scaffolds for tissue engineering. This study examined collagen organization in the presence of polysaccharide and the resulting effects on viscoelastic properties. Fibrillogenesis in the presence of chondroitin sulfate (CS) resulted in changes in the collagen network organization with an increase in void space present. The increased void space caused by CS addition correlated with a decreased stiffness of the collagen gel. These changes occurred with physiologically relevant ratios of collagen to CS, at physiological pH and ionic strength, and without a decrease in the amount of collagen incorporated into fibrils. The addition of dextran, an uncharged polysaccharide, yielded no change in network void space or mechanical properties. Changes in fibril diameter caused by CS or dextran were not correlated with mechanical properties. The results of this study demonstrate that collagen organization can be modified by the addition of GAG, leading to altered matrix mechanical properties. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 841-851, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

The role of weakly polar and H-bonding interactions in the stabilization of the conformers of FGG, WGG, and YGG: an aqueous phase computational study

The energetics of intramolecular interactions on the conformational potential energy surface of the terminally protected N-Ac-Phe-Gly-Gly-NHMe (FGG), N-Ac-Trp-Gly-Gly-NHMe (WGG), and N-Ac-Tyr-Gly-Gly-NHMe (YGG) tripeptides was investigated. To identify the representative conformations, simulated annealing molecular dynamics (MD) and density functional theory (DFT) methods were used. The interaction energies were calculated at the BHandHLYP/aug-cc-pVTZ level of theory. In the global minima, 10%, 31%, and 10% of the stabilization energy come from weakly polar interactions, respectively, in FGG, WGG, and YGG. In the prominent cases 46%, 62%, and 46% of the stabilization energy is from the weakly polar interactions, respectively, in FGG, WGG, and YGG. On average, weakly polar interactions account for 15%, 34%, and 9% of the stabilization energies of the FGG, WGG, and YGG conformers, respectively. Thus, weakly polar interactions can make an important energetic contribution to protein structure and function. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 1002-1011, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

The molecular structure and physical properties of elastin fibers as revealed by Raman microspectroscopy

Raman microspectroscopy has been used to investigate the structure of alpha-elastin and fibrous elastin from ligament and aorta, and to explore changes associated with mechanical strain and temperature. Although no vibrational modes associated with cross-linking of the fibers could be identified, the secondary structure of dehydrated fibrous elastin was significantly different from alpha-elastin. The former differed from previous experimental measurements, but was close to the theoretical predictions with 36% beta-structures, 46% unordered, and 18% alpha-helix. alpha-Elastin contained 29% beta-structures, 53% unordered, and 18% alpha-helix. In nuchal fibers the amide I mode was polarized, consistent with the peptide bond. Strains of up to 60% in ligament fiber bundles resulted in no significant shifts in peak position or in secondary structure. Polarization measurements revealed that the peptide bonds and several side chains re-orientated closer to the fiber axis. Heating nuchal fibers to 60 degrees C to increase the energetic component of the elasticity was associated with a 30% increase in the proportion of beta-structures in the amide I band, a 50% increase in the amide III band, and a 50% reduction in the signal from bound water. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 931-940, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Self-crosslinked and reducible fusogenic peptides for intracellular delivery of siRNA

A novel self-crosslinked and reducible peptide was synthesized for stable formation of nanoscale complexes with an siRNA-PEG conjugate to enhance transfection efficiency in serum containing condition without compromising cytotoxicity. A fusogenic peptide, KALA, with two cysteine residues at both terminal ends was crosslinked via disulfide linkages under mild DMSO oxidation condition. The reducible crosslinked KALA (cl-KALA) was used to form nano-complexes with green fluorescent protein (GFP) siRNA. Size and morphology of various polyelectrolyte complexes formulated with KALA and cl-KALA were comparatively analyzed. cl-KALA exhibited more reduced cell cytotoxicity and formed more stable and compact polyelectrolyte complexes with siRNA, compared with naked KALA and polyethylenimine (PEI), probably because of its increased charge density. The extent of gene silencing was quantitatively evaluated using MDA-MB-435 cells. cl-KALA/siRNA complexes showed comparable gene silencing efficiency with those of cytotoxic PEI. In a serum containing medium, cl-KALA/siRNA-PEG conjugate complexes exhibited superior gene inhibition because of the shielding effect of PEG on the surface. The formulation based on the self-crosslinked fusogenic peptide could be used as a biocompatible and efficient nonviral carrier for siRNA delivery. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 881-888, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Hot-spot residues at the E9/Im9 interface help binding via different mechanisms

Protein-protein association involves many interface interactions, but they do not contribute equally. Ala scanning experiments reveal that only a few mutations significantly lower binding affinity. These key residues, which appear to drive protein-protein association, are called hot-spot residues. Molecular dynamics simulations of the Colicin E9/Im9 complex show Im9 Glu41 and Im9 Ser50, both hot-spots, bind via different mechanisms. The results suggest that Im9 Ser50 restricts Glu41 in a conformation auspicious for salt-bridge formation across the interface. This type of model may be helpful in engineering hot-spot clusters at protein-protein interfaces and, consequently, the design of specificity. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 916-920, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Structural properties of bombesin-like peptides revealed by surface-enhanced Raman scattering on roughened silver electrodes

This work presents a Fourier-transform absorption infrared, Fourier-transform Raman, and surface-enhanced Raman scattering (SERS) study of the following peptides belonging to the bombesin-like family: phyllolitorin, [Leu(8)]phyllolitorin, NMB, NMC, and PG-L. The SERS study was undertaken to understand the adsorption mechanism of bombesin-like peptides on an electrochemically roughened silver electrode surface and to show changes in the adsorption mechanism with alterations in amino acids and small tertiary structures. The SERS spectra presented here shows bands mainly associated with the Trp(8) residue vibrations. The presence of mainly pyrrole coring vibrations for phyllolitorin and [Leu(8)]phyllolitorin and mainly benzene coring modes for NMB and NMC indicated that these groups interact with the roughened silver electrode surface. Furthermore, N(1)--C(8) and C(3)--C(9) bonds of the PG-L indole ring seemed to have nearly a vertical orientation on the electrode surface. In addition, distinct vibrations of the C--S fragment were observed in the SERS spectra of [Leu(8)]phyllolitorin and PG-L. The strong enhancement of the nu(C==O) vibration in the [Leu(8)]phyllolitorin SERS spectrum yielded evidence that the intact C==O bond(s) bind strongly to the silver electrode surface, whereas NMC, phyllolitorin, and NMB were located near the silver surface. This finding was supported by the presence of the nu(C--C(==O)) mode. The amide I band observed at 1642 and 1634 cm(-1) for NMB and NMC, respectively, and the Raman amide III band seen in the 1282-1249 cm(-1) range for all peptides except PG-L, indicate that the strongly hydrogen-bonded alpha-helical conformation and random-coil structure are favored for binding to the surface. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 980-992, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Complexation of beta-lactam antibiotic drug carbenicillin to bovine serum albumin: energetics and conformational studies

Binding of the antibiotic drug carbenicillin to bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC) in combination with fluorescence and circular dichroism (CD) spectroscopies. The thermodynamic parameters of binding have been evaluated as a function of temperature, ionic strength, and in the presence of anionic, cationic and nonionic surfactants, tetrabutylammonium bromide, and sucrose. The values of van't Hoff enthalpy do not agree with the calorimetric enthalpy indicating conformational changes in the protein upon drug binding. These observations are supported by the intrinsic fluorescence and CD spectroscopic measurements. A reduction in the binding affinity of carbenicillin to BSA is observed with increase in ionic strength of the solution, thereby suggesting, prevailing of electrostatic interactions in the binding process. The involvement of hydrophobic interactions in the binding of the drug to the protein is also indicated by a slight reduction in binding constant in the presence of tetrabutylammonium bromide. The experiments in the presence of sucrose suggest that hydrogen bonding is perhaps not dominant in the binding. The anionic surfactant sodium dodecyl sulphate (SDS) is observed to completely interfere in the ionic interactions in addition to its partial denaturing capacity. However, the presence of cationic surfactant hexadecyl trimethylammonium bromide (HTAB) and nonionic surfactant Triton-X 100 induce a slight reduction in the values of binding affinity. These calorimetric and spectroscopic results, provide quantitative information on the binding of carbenicillin to BSA and suggests that the binding is dominated by electrostatic interactions with contribution from hydrophobic interactions. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 831-840, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Bombesin-modified 6-14 C-terminal fragments adsorption on silver surfaces: influence of a surface substrate

Surface-enhanced Raman scattering (SERS) spectroscopy has been applied to investigate the interaction with a silver colloidal surface of following seven 6-14 fragments of bombesin (BN) C-terminus: cyclo[D-Phe(6),His(7),Leu(14)]BN(6-14), [D-Phe(6),Leu-NHEt(13),des-Met(14)]BN(6-14), [D-Phe(6),Leu(13)-(R)-p-chloro-Phe(14)]BN(6-14), [D-Phe(6),beta-Ala(11),Phe(13),Nle(14)]BN(6-14), [D-Tyr(6),beta-Ala(11),Phe(13),Nle(14)]BN(6-14), [D-Tyr(6),beta-Phe(11),Phe(13),Nle(14)OH]BN(6-14), and [D-Cys(6),Asn(7),D-Ala(11),Cys(14)]BN(6-14), potent r-GRP-R receptor antagonists used in chemotherapy and potential effective drugs in cancer treatment. The adsorption active sites and molecular orientations on the colloidal silver surface have been determined on the basis of SERS "surface selection rules" subsequent to a detailed SERS analysis. In addition, the similarities and differences of these spectra with the SERS spectra of the peptides immobilized on a roughened silver electrode surface have been examined. From the data, suggestion has been made about structural properties of these peptides on the colloidal surface. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 941-950, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Stable intermediates determine proteins' primary unfolding sites in the presence of surfactants

Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS) and cationic (DTAC) surfactant concentrations corresponding to specific conformational transitions, using the surfactant‐robust broad‐specificity proteases Savinase and Alcalase. Cleavage sites are identified by SDS‐PAGE and N‐terminal sequencing. We observe well‐defined cleavage fragments, which suggest that flexibility is limited to certain regions of the protein. Cleavage sites for α‐lactalbumin and myoglobin correspond to regions identified in other studies as partially unfolded at low pH or in the presence of organic solvents. For Tnfn3, which does not form partially folded structures under other conditions, cleavage sites can be rationalized from the structure of the protein's folding transition state and the position of loops in the native state. Nevertheless, they are more sensitive to choice of surfactant and protease, probably reflecting a heterogeneous and fluctuating ensemble of partially unfolded structures. Thus, for proteins accumulating stable intermediates on the folding pathway, surfactants encourage the formation of these states, while the situation is more complex for proteins that do not form these intermediates. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 221–231, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Thermal stability and conformational structure of salmon calcitonin in the solid and liquid states

Salmon calcitonin (sCT) was selected as a model protein drug for investigating its intrinsic thermal stability and conformational structure in the solid and liquid states by using a Fourier transform infrared (FT‐IR) microspectroscopy with or without utilizing thermal analyzer. The spectral correlation coefficient (r) analysis between two second‐derivative IR spectra was applied to quantitatively estimate the structural similarity of sCT in the solid state before and after different treatments. The thermal FT‐IR microspectroscopic data clearly evidenced that sCT in the solid state was not effected by temperature and had a thermal reversible property during heating–cooling process. Moreover, the high r value of 0.973 or 0.988 also evidenced the structural similarity of solid‐state sCT samples before and after treatments. However, sCT in H₂O exhibited protein instability and thermal irreversibility after incubation at 40°C. The temperature‐induced conformational changes of sCT in H₂O was occurred to transform the α‐helix/random coil structures to β‐sheet structure and also resulted in the formation of intramolecular and intermolecular β‐sheet structures. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 200–207, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Determination of the secondary structure of proteins in different environments by FTIR-ATR spectroscopy and PLS regression

The secondary structures of proteins (alpha-helical, beta-sheet, beta-turn, and random coil) in the solid state and when bound to polymer beads, containing immobilized phenyl and butyl ligands such as those as commonly employed in hydrophobic interaction chromatography, have been investigated using FTIR-ATR spectroscopy and partial least squares (PLS) methods. Proteins with known structural features were used as models, including 12 proteins in the solid state and 7 proteins adsorbed onto the hydrophobic surfaces. A strong PLS correlation was achieved between predictions derived from the experimental data for 4 proteins adsorbed onto the phenyl-modified beads and reference data obtained from the X-ray crystallographic structures with r(2) values of 0.9974, 0.9864, 0.9924, and 0.9743 for alpha-helical, beta-sheet, beta-turn, and random coiled structures, respectively. On the other hand, proteins adsorbed onto the butyl sorbent underwent greater secondary structural changes compared to the phenyl sorbent as evidenced from the poorer PLS r(2) values (r(2) are 0.9658, 0.9106, 0.9571, and 0.9340). The results thus indicate that the secondary structures for these proteins were more affected by the butyl sorbent, whereas the secondary structure remains relatively unchanged for the proteins adsorbed onto the phenyl sorbent. This study has important ramifications for understanding the nature of protein secondary structural changes following adsorption onto hydrophobic sorbent surfaces. This knowledge could also enable the development of useful protocols for enhancing the chromatographic purification of proteins in their native bioactive states. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 895-905, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Vibrational spectroscopic studies of newly developed synthetic biopolymers

Vibrational spectroscopic techniques such as near‐infrared (NIR), Fourier transform infrared (FTIR), and Raman spectroscopy are valuable diagnostic tools that can be used to elucidate comprehensive structural information of numerous biological samples. In this review article, we have highlighted the advantages of nanotechnology and biophotonics in conjunction with vibrational spectroscopic techniques in order to understand the various aspects of new kind of synthetic biopolymers termed as polyethylene glycol (PEG)ylated lipids. In contrast to conventional phospholipids, these novel lipids spontaneously form liposomes or nanovesicles upon hydration, without the supply of external activation energy. The amphiphiles considered in this study differ in their hydrophobic acyl chain length and contain different units of PEG hydrophilic headgroups. We have further explored the thermotropic phase behaviors and associated changes in the conformational order/disorder of such lipids by using variable‐temperature FTIR and Raman spectroscopy. Phase transition temperature profiles and correlation between various spectral indicators have been identified by either monitoring the shifts in the vibrational peak positions or plotting vibrational peak intensity ratios in the C? H stretching region as a function of temperature. To supplement our observations of phase transformations, a thermodynamic approach known as differential scanning calorimetry (DSC) has been applied and revealed a good agreement with the infrared and Raman spectroscopic data. Finally, the investigation of thermal properties of lipids is extremely crucial for numerous purposes, thus the results obtained in this work may find application in a wide variety of studies including the development of PEGylated lipid based drug and substances delivery vehicles. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 403–417, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Defining resonance Raman spectral responses to substrate binding by cytochrome P450 from Pseudomonas putida

Resonance Raman spectra are reported for substrate-free and camphor-bound cytochrome P450cam and its isotopically labeled analogues that have been reconstituted with protoheme derivatives that bear -CD(3) groups at the 1, 3, 5, and 8-positions (d12-protoheme) or deuterated methine carbons (d4-protoheme). In agreement with previous studies of this and similar enzymes, substrate binding induces changes in the high frequency and low frequency spectral regions, with the most dramatic effect in the low frequency region being activation of a new mode near 367 cm(-1). This substrate-activated mode had been previously assigned as a second "propionate bending" mode (Chen et al., Biochemistry, 2004, 43, 1798-1808), arising in addition to the single propionate bending mode observed for the substrate-free form at 380 cm(-1). In this work, this newly activated mode is observed to shift by 8 cm(-1) to lower frequency in the d12-protoheme reconstituted enzyme (i.e., the same shift as that observed for the higher frequency "propionate bending" mode) and is therefore consistent with the suggested assignment. However, the newly acquired data for the d4-protoheme substituted analogue also support an earlier alternate suggestion (Deng et al., Biochemistry, 1999, 38, 13699-13706) that substrate binding activates several heme out-of-plane modes, one of which (gamma(6)) is accidentally degenerate with the 367 cm(-1) propionate bending mode. Finally, the study of the enzyme reconstituted with the protoheme-d4, which shifts the macrocycle nu(10) mode, has now allowed a definitive identification of the vinyl C==C stretching modes. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 1045-1053, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Self-assembling peptides: sequence, secondary structure in solution and film formation

Peptides of alternating charge and hydrophobic amino acids have a tendency to adopt unusually stable beta-sheet structures that can form insoluble macroscopic aggregates under physiological conditions. In this study, analogues of a well-known self-assembling peptide, characterized by the same polar/nonpolar periodicity but with different residues, were designed to study the relationship between sequence, conformation in solution and film-forming capacity in saline solution. Peptide conformation, evaluated by circular dichroism, correlated with film forming capacity observed by inverted optical microscopy after addition of saline solution and subsequent drying. We found that polar/nonpolar periodicity of several analogues is not criterion enough to induce beta-sheet and thus film formation and that conformations different from beta-sheet also allow self-assemblage. Furthermore, addition of the short adhesive sequence RGD to a known self-assembling sequence was shown to not prevent the self-assembling process. This finding might prove useful for the design of biomimetic scaffolds. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 906-915, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Micro-Raman characterisation of the R to T state transition of haemoglobin within a single living erythrocyte

We present the first recorded Raman spectra of haemoglobin in both the R and T states from within a single living erythrocyte using 632.8 nm excitation. Bands characteristic of low spin haems are observed in oxygenated and carboxylated erythrocytes at approx. 1636 (nu(10)), 1562-1565 (nu(2)), 1250-1245 cm(-1) (nu(13)) and 1226-1224 cm(-1) (nu(5)+nu(8)). The spectra of deoxygenated and methaemoglobin erythrocytes have characteristic high spin bands at approx. 1610-1606 cm(-1) (nu(10)), 1582-1580 (nu(37)), 1547-1544 (nu(11)), 1230-1220 cm(-1) (nu(13)) and 1215-1210 cm(-1) (nu(5)+nu(8)). Bands at 1172 (nu(30)), 976 (nu(45)) and 672 (nu(7)) cm(-1) appear to be enhanced at 632.8 nm in low spin haems. The oxidation state marker band (nu(4)) at 1364-1366 cm(-1) appeared invariant within this domain in all single cells and conditions investigated contrary to other resonance Raman studies on haem isolates. The information gained by in vivo single erythrocyte molecular analysis has important ramifications to the understanding of fundamental physiological processes and may have applications in the diagnosis and treatment of red blood cell disorders.