Improved two-dimensional electrophoretic mapping of Japanese human hair proteins; application to curved and straight Japanese human hairs; and protein identification by MALDI MS and MS/MS quadrupole time-of-flight mass spectrometry.

OBJECTIVES: To evaluate improved protein extraction and two-dimensional electrophoresis (2DE) separation methods with Japanese reference human hair (JRH); to determine whether fibre curvature is related to protein composition in curly and straight Japanese women's human hair (JHH) samples; and to identify proteins from JRH 2DE maps and expression differences between curly and straight JHH. METHODS: Hair keratin and keratin-associated proteins (KAPs) were extracted intact with dithiothreitol or tris(2-carboxyethyl) phosphine from JRH or from curved or straight JHH. Extracted proteins were isoelectric-focused on first-dimensional pH gradient gel strips, then separated by molecular weight on laboratory-made, second-dimension, large format gels. The software compared protein abundance between duplicate 2DE gels of curved and straight JHH. Thirty-eight proteins from a JRH 2DE gel were enzyme-cleaved for MALDI-TOF-MS analysis to determine peptide composition, and where possible, de novo sequencing gave peptide sequence data. An in-house human hair protein database incorporating ninety-eight annotated protein sequences assisted MS analysis. RESULTS: 2DE gels of tris(2-carboxyethyl) phosphine-extracted JRH improved keratin and KAP resolution and number compared to those of dithiothreitol-extracted JRH and published commercially made second-dimensional gels. Silver-stained 2DE gels of the straight or curved JHH sets were remarkably similar. Over-staining to reveal basic proteins caused poor resolution of the major acidic protein classes. Software comparisons of fifty-nine resolved proteins revealed two were significantly different in abundance between curved and straight hairs but in insufficient amounts for MS analysis. MS identified twelve proteins from a JRH CBBG-stained 2DE gel: six type II keratins, three type I keratins and three high sulphur proteins. A further eight were potential conformational isoforms and isoelectric variants of the identified proteins bringing the total to twenty identified or partially identified proteins. CONCLUSION: Root-end human hair extraction with tris(2-carboxyethyl) phosphine improves protein resolution and visualizes more proteins on large format 2DE gels. The two minor protein differences between duplicate straight or curved JHH 2DE gels were unlikely to change fibre structure from straight to curved hair. MS results confirmed that multiple isoforms exist of various hair proteins. Low sequence coverage prevented distinction between members in rows of homologous protein spots of similar molecular weight.

OBJECTIFS: évaluer l'amélioration de l'extraction de protéines et les méthodes de séparation bidimensionnelle par électrophorèse (2DE) avec des cheveux humains de référence Japonais (JRH), déterminer si la courbure de la fibre est liée à la composition protéique dans les échantillons de cheveux humains des Japonaises (JHH) bouclés et raides et identifier les protéines issues des cartes JRH 2DE et les différences d'expression entre les JHH bouclés et raides. MÉTHODES: la kératine des cheveux et les protéines associées à la kératine (KAP) ont été extraites intactes avec du dithiothréitol ou du tris (2-carboxyéthyl) phosphine des JRH ou des JHH bouclés ou raides. Les protéines extraites ont subi une focalisation isoélectrique sur des bandes de gel à gradient de pH unidimensionnelles, puis ont été séparées par poids moléculaire sur des gels bidimensionnels de grand format, fabriqués en laboratoire. Le logiciel a comparé l'abondance des protéines entre les deux duplicatas de gels 2DE des JHH bouclés et raides. Trente-huit protéines provenant d'un gel 2DE JRH ont été clivés par enzyme pour l'analyse MALDI-TOF-MS afin de déterminer la composition des peptides, et dans la mesure du possible, un séquençage de novo a donné des données de séquence des peptides. Une base de données interne des protéines capillaires humaines incorporant 98 séquences de protéines annotées a aidé l'analyse MS. RÉSULTATS: les gels 2DE de JRH extraits par le tris (2-carboxyéthyl) ont amélioré la résolution et le nombre de la kératine et du KAP par rapport à ceux du JRH extrait par le dithiothréitol et des gels bidimensionnels fabriqués commercialement. Les gels 2DE à coloration argentée des ensembles de JHH raides ou bouclés étaient remarquablement similaires. La sur-coloration pour révéler les protéines de base a provoqué une mauvaise résolution des principales classes de protéines acides. Les comparaisons logicielles des 59 protéines résolues ont révélé que deux présentaient une différence significative d'abondance entre les cheveux bouclés et raides, mais en quantités insuffisantes pour une analyse MS. La MS a identifié douze protéines provenant d'un gel 2DE coloré CBBG JRH : six kératines de type II, trois kératines de type I et trois protéines à forte teneur en soufre. Huit autres étaient des isoformes conformationnels potentiels et des variantes isoélectriques des protéines identifiées, ramenant le total à 20 protéines identifiées ou partiellement identifiées. CONCLUSION: l'extraction des cheveux humains à la racine avec du tris (2-carboxyéthyl) phosphine améliore la résolution des protéines et permet de visualiser plus de protéines sur les gels 2DE grand format. Les deux différences de protéines mineures entre les duplicatas des gels 2DE JHH raides ou bouclés étaient peu susceptibles de changer la structure des fibres de cheveux raides à bouclés. Les résultats de la MS ont confirmé qu'il existe plusieurs isoformes de diverses protéines capillaires. Une faible couverture de séquence a empêché la distinction entre les protéines homologues de poids moléculaire similaire.

Ovine keratome: identification, localisation and genomic organisation of keratin and keratin-associated proteins.

Wool is composed primarily of proteins belonging to the keratin family. These include the keratins and keratin-associated proteins (KAPs) that are responsible for the structural and mechanical properties of wool fibre. Although all human keratin and KAP genes have been annotated, many of their ovine counterparts remain unknown and even less is known about their genomic organisation. The aim of this study was to use a combinatory approach including comprehensive cDNA and de novo genomic sequencing to identify ovine keratin and KAP genes and their genomic organisation and to validate the keratins and KAPs involved in wool production using ovine expressed sequence tag (EST) libraries and proteomics. The number of genes and their genomic organisation are generally conserved between sheep, cattle and human, despite some unique features in the sheep. Validation by protein mass spectrometry identified multiple keratins (types I and II), epithelial keratins and KAPs. However, 15 EST-derived genes, including one type II keratin and 14 KAPs, were identified in the sheep genome that were not present in the NCBI gene set, providing a significant increase in the number of keratin genes mapped on the sheep genome.

Application of proteomics for determining protein markers for wool quality traits.

The technique of two-dimensional electrophoresis (2-DE) has been under investigation for its usefulness in identifying protein markers for wool quality traits in sheep. However, before this could be achieved, unique problems relating to the detection and quantitation of wool proteins needed to be overcome so that 2-DE protein maps could be examined using computational programs like Melanie II. Four protein staining regimes were examined. Colloidal Coomassie Blue G-250 was found to be superior to Coomassie Blue R-250 and gave satisfactory staining of all protein classes. Silver staining detects minor strings of keratinous proteins, but unfortunately it negatively stains intermediate filament proteins, the major high sulphur proteins (HSPs) and the high glycine tyrosine proteins and the latter two classes can only be seen by overstaining the background of the gel. In contrast, labeling reduced keratins with [14C]iodoacetamide, followed by autoradiography detection, results in a protein map with low background and all protein spots stained positively. 2-DE has been used to obtain wool protein maps of Lincoln/Merino chimeric sheep to examine wool originating from two genotypes grown with different crimp frequencies within the same fleece. Between fleece, variations have also been examined. Work to date suggests that several major HSPs may be associated with the fibre curvature trait known as crimp frequency. From matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectral mapping, one of these proteins has been identified as being from the B2A family from the HSP class.

Structural features of a peptide corresponding to human kappa-casein residues 84-101 by 1H-nuclear magnetic resonance spectroscopy.

The peptide Val-Arg-Arg-Pro-Asn-Leu-His-Pro-Ser-Phe-Ile-Ala-Ile-Pro-Pro- Lys-Lys-Ile, which corresponds to residues 84-101 of human kappa-casein, has been synthesized and its conformation preferences determined by 1H-nuclear magnetic resonance spectroscopy in dimethyl sulphoxide. The peptide adopted a largely extended chain conformation in solution and there was evidence for the presence of a beta-turn involving residues Pro87-His90 of human kappa-casein. The presence of a turn in this position would make the physiologically significant Arg85 residue of human kappa-casein (which is equivalent to Arg97 in bovine kappa-casein) unavailable for interaction with Asp249 of bovine chymosin, and may partly explain why human kappa-casein is hydrolysed more slowly than its bovine counterpart by bovine chymosin.

Micelle stability: kappa-casein structure and function.

The stability of the casein micelle is dependent on the presence of kappa-casein (CN) on the surface of the micelle where it functions as an interface between the hydrophobic caseins of the micelle interior and the aqueous environment. kappa-Casein is also involved in thiol-catalyzed disulfide interchange reactions with the whey proteins during heat treatments and, after rennet cleavage, in the facilitation of micelle coagulation. These functions of kappa-CN are regulated by the three-dimensional structure of the protein on the micelle surface. The usual means of determining structure are not available for kappa-CN because this protein is strongly self-associating and has never been crystallized. Instead, algorithms were used to predict selected secondary structures and circular dichroism spectroscopy on kappa-CN and the macropeptide released by chymosin. Three peptides were synthesized to cover the chymosin-sensitive site (His98-Lys111), the region in the macropeptide that could be helical (Pro130-Ile153), and the region between. Nuclear magnetic resonance spectroscopy showed that the peptide His98-Lys111 was probably a beta-strand with tight turns at each end. This hypothesis was confirmed by a study of the molecular dynamics showing that the C variant of kappa-CN interacted less strongly with chymosin; consequently, the slow renneting time of milk that contains this protein was explainable. Both circular dichroism and nuclear magnetic resonance indicated that the peptide Pro130-Ile153 was probably helical under normal physiological conditions. A preliminary study using nuclear magnetic resonance showed that the intervening peptide had no discernible secondary structure. Consequently, most of the beta-sheet structure of kappa-CN is likely in the para-kappa-CN region.

Solution conformation of a peptide corresponding to bovine kappa-casein B residues 130-153 by circular dichroism spectroscopy and 1H-nuclear magnetic resonance spectroscopy.

The peptide Pro130-Thr-Ser-Thr-Pro-Thr-Ile-Glu-Ala-Val-Glu140- Ser-Thr-Val-Ala-Thr-Leu-GLu-Ala-Ser-Pro150-Glu-Val-Ile, which corresponds to residues 130-150 of kappa-casein B, was synthesized and the conformation of the peptide in solution investigated by circular dichroism (CD) spectroscopy, structure prediction algorithms and 1H-nuclear magnetic resonance spectroscopy. In a solution containing the structure-enhancing solvent trifluoroethanol the CD spectrum was typical of a peptide in the alpha-helical conformation and nuclear magnetic resonance showed that the amino acids between Ile136 and Ser149 (kappa-casein numbering) were predominantly in the alpha-helical conformation but that Pro130 to Thr135 and Pro150 to Ile153 were not. In addition, Thr133-Pro134 and Ser-149-Pro150 were primarily in the trans conformation, the residues from Thr131 to Thr135 were in unordered structures and the residues from Glu151 to Ile153 were in an extended conformation. Residues Glu137 to Glu140 and Thr145 to Ala148 also displayed some 3(10)-helix character. When the peptide was dissolved in 10 mM-cetyltrimethylammonium chloride solution at pH 6, the CD spectra indicated that the proportion of helical structure was comparable to that of the peptide in trifluoroethanol solution (400 ml/l), whereas when the peptide was dissolved in buffer alone in 10 mM-SDS solution, the CD spectra were consistent with a low helical content. Acidification of these solutions to pH 2.85 resulted in a slight increase in the helical content of the peptide in buffer and more markedly in buffer containing SDS. When the peptide was in 5 mM-CaCl2 solution at neutral pH, the CD spectrum indicated that some ordered structure was present. Taken together these results indicate that the ionizable residues Glu137, Glu140, Glu147 and Glu151 could be important in determining the stability of the putative helix. The structure predictions found that the sequence from Glu137 to Pro150 would be more likely to be in a helical than any other conformation in the intact bovine protein, but that pig, sheep and goat kappa-caseins did not give a prediction of a strongly helical region in this part of the molecule.

Restrained molecular dynamics study of the interaction between bovine kappa-casein peptide 98-111 and bovine chymosin and porcine pepsin.

The cleavage of bovine kappa-casein at the Phe105-Met106 bond by chymosin or pepsin is the first stage in casein micelle coagulation and casein digestion. The nature of the interaction of the peptide His98-Pro-His-Pro-His-Leu-Ser-Phe105-Met-Ala-Ile-Pro-Pro- Lys111 with chymosin and porcine pepsin was investigated using molecular modelling and energy minimization techniques. This study verified and extended a proposed model that electrostatic binding (involving His98, His100, His102 and Lys111 or Lys112) at either end of the active site cleft of chymosin is important for the positioning of residues 103-108 in the cleft. The peptide conformation remained unchanged in going from solution to binding into the active site cleft, with the exception that optimum binding of substrate to chymosin required the isomerization of the His98-Pro99 peptide bond from the trans to the cis conformation. The study also identified an acidic region in porcine pepsin that is in a position to form strong electrostatic interactions with the histidines at the N-terminus of the peptide.

Spectrochemical studies on the blue copper protein azurin from Alcaligenes denitrificans.

Spectroscopic and electrochemical studies, incorporating electronic spectra, electron paramagnetic resonance (EPR) spectra, resonance Raman (RR) spectra, and measurements of the redox potential, have been carried out on the blue copper protein azurin, from Alcaligenes denitrificans. These data are correlated with the refined crystal structure of this azurin and with corresponding data for other blue copper proteins. The electronic spectrum, characterized by an intense (epsilon = 5100 M-1 cm-1) charge-transfer band at 619 nm, the EPR spectral parameters (g perpendicular = 2.059, g parallel of = 2.255, A parallel of = 60 X 10(-4) cm-1), and the resonance Raman spectrum are similar to those obtained from other azurins and from plastocyanins. Both the electronic spectrum and the EPR spectrum are unchanged over the pH range 4-10.5, but major changes occur above pH 12 and below pH 3.5. A small reversible change occurs at pH approximately 11.4. In the RR spectrum the Cu-S stretching mode is shown to contribute to all of the five principal RR peaks. Deuterium substitution produces shifts in at least seven of the peaks; these shifts may be attributable, at least in part, to the NH...S hydrogen bond to the copper-ligated Cys-112. Measurements of the redox potential, using spectroelectrochemical methods, over the temperature range 4.8-40.0 degrees C, give values for delta H0' and delta S0' of -55.6 kJ mol-1 and -97.0 J K-1 mol-1, respectively. The redox potential of A. denitrificans azurin at pH 7.0, Eo', is 276 mV. These data are interpreted in terms of a copper site, in azurin, comprising three strong bonds, in an approximately trigonal plane, from Cys-112, His-46, and His-117 and much longer axial approaches from Met-121 and the peptide carbonyl oxygen of Gly-45. Spectral differences within the azurin family and between azurin and plastocyanin are attributed to differences in the strengths of these axial interactions. Likewise, the distinctly lower Eo values for azurins, as compared with plastocyanins, are related to the more copper(II)-like site in azurin [with a weaker Cu-S(Met) interaction and a Cu-O interaction not found in plastocyanin]. On the other hand, the relative constancy of the EPR parameters between azurin and plastocyanin suggests they are not strongly influenced by weakly interacting axial groups.

Studies on human lactoferrin by electron paramagnetic resonance, fluorescence, and resonance Raman spectroscopy.

Investigations of metal-substituted human lactoferrins by fluorescence, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy confirm the close similarity between lactoferrin and serum transferrin. As in the case of Fe(III)- and Cu(II)-transferrin, a significant quenching of apolactoferrin's intrinsic fluorescence is caused by the interaction of Fe(III), Cu(II), Cr(III), Mn(III), and Co(III) with specific metal binding sites. Laser excitation of these same metal-lactoferrins produces resonance Raman spectral features at ca. 1605, 1505, 1275, and 1175 cm-1. These bands are characteristic of tyrosinate coordination to the metal ions as has been observed previously for serum transferins and permit the principal absorption band (lambda max between 400 and 465 nm) in each of the metal-lactoferrins to be assigned to charge transfer between the metal ion and tyrosinate ligands. Furthermore, as in serum transferrin the two metal binding sites in lactoferrin can be distinguished by EPR spectroscopy, particularly with the Cr(III)-substituted protein. Only one of the two sites in lactoferrin allows displacement of Cr(III) by Fe(III). Lactoferrin is known to differ from serum transferrin in its enhanced affinity for iron. This is supported by kinetic studies which show that the rate of uptake of Fe(III) from Fe(III)--citrate is 10 times faster for apolactoferrin than for apotransferrin. Furthermore, the more pronounced conformational change which occurs upon metal binding to lactoferrin is corroborated by the production of additional EPR-detectable Cu(II) binding sites in Mn(III)-lactoferrin. The lower pH required for iron removal from lactoferrin causes some permanent change in the protein as judged by altered rates of Fe(III) uptake and altered EPR spectra in the presence of Cu(II). Thus, the common method of producing apolactoferrin by extensive dialysis against citric acid (pH 2) appears to have an adverse effect on the protein.