Cement precursor proteins of the reef-building polychaete Phragmatopoma californica (Fewkes).

Two distinctive 3,4-dihydroxyphenyl-L-alanine-(DOPA-) containing proteins (Pc-1 and Pc-2) have been isolated and partially characterized from the thorax of the reef-building sabellariid Phragmatopoma californica. They are the first such reported from the phylum Annelida. The proteins are presumed to be soluble precursors of the quinone-tanned cement used to bind particulate materials in the construction of the tubes that serve as habitats for the worms. The proteins have apparent molecular weights ranging from 18,000 to 20,000 and isoelectric point greater than or equal to 8.0. Both proteins consist of repeated sequence motifs in their primary structure. Pc-1 has repeats of (XGGY*GY*GAK) where X = V, L, I, AA, or KV, and Y* is DOPA or tyrosine. Pc-2, in contrast, appears to have repeats of (X1-[GGY*]n-[GA]m-X2-[HP(A)V]p-HK) where X1 can be AL, A, or F; X2 can be WG or absent; n and m can be 1 or 2, and p = 0-2. Both protein families appear to share the same C-terminal sequence ALGGY*GAGA. Of the DOPA-containing proteins characterized from other phyla, Phragmatopoma cement precursors most resemble those from the liver fluke Fasciola hepatica and the mussel Trichomya hirsuta.     

Side chain interactions in aromatic dipeptides

A study of the 100-MHZ nuclear magnetic resonace spectra in D₂O solution was made of a series of linear dipeptides of the types L -phenylalanine-L -and-D -X, and L -phenylalanine-L -and-D -Y, where X comprised a group of amino acid residues with polar side chains (X = glutamine, glutamic acid, arginine, and N^ε-acetyllysine) and Y comprised amino acid residues with purely aliphatic side chains (Y = α-aminobutyric acid and norvaline). It was found that regardless of the side chain length, resonances due to the α-methylene protons in the X and Y side chains of the L -Phe-D -Y series consistently exhibited upfield shifts greater than any other protons in these side chains, when compared to the corresponding side chain resonances of the nonaromatic dipeptide series L -Ala-L -X and L -Ala-L -Y. The magnitudes of these shielding effects were consistently and considerably greater for the L -Phe-D -X series than for the L -Phe-D -Y series. An intramolecular complex–formed by association of armatic π-electrons with the positive end of the dipole in the polar side chains—was proposed as one plausible interpretation of the enhanced shielding effects. An increase in temperature from 32 to 70–80° was sufficient to overcome the enhanced shielding attributable to the suggested complex.     

Purification of adhesive proteins from mussels

The adhesive polyphenolic proteins from the mussels Mytilus chilensis and Choromytilus chorus have been purified based on their solubility in dilute perchloric acid and on differential precipitation with acetone containing about 0.3 N HCl. The specific activity of the proteins obtained was 0.16 mg of 3,4-dihydroxyphenylalanine per milligram of protein, or higher. The proteins have an apparent molecular weight of about 100,000 and they contain a high proportion of 3,4-dihydroxyphenylalanine, lysine, and proline.     

Chemoenzymatic synthesis of peptidyl 3,4-dihydroxyphenylalanine for structure-activity relationships in marine invertebrate polypeptides

An improved method for hydroxylating tyrosine-containing sequences in polypeptides to peptidyl 3,4-dihydroxyphenylalanine (DOPA) using mushroom tyrosinase at relatively high enzyme-to-substrate ratios is described. The new method involves incorporating borate into the reaction mixture to stop formation of the unwanted side product 3,4,5-trihydroxyphenylalanine. Using this method, a model for the palindromic central sequence for the antimicrobial peptide family, the styelins, Y*Y*KHKY*Y* (where Y* is DOPA), was successfully synthesized in high yield from YYKHKYY. This sequence represents a particularly challenging target because of the cluster of four precursor tyrosine residues are in close proximity. The method should be readily applied to larger polypeptides produced by either solid-phase synthesis or recombinant techniques and give greater insight into the roles of this unusual posttranslational modification in marine invertebrates such as mussels and ascidians.     

Self-assembly and adhesion of DOPA-modified methacrylic triblock hydrogels

Marine mussels anchor to a variety of surfaces by secreting liquid proteins that harden and form water-resistant bonds to a variety of surfaces. Studies have revealed that these mussel adhesive proteins contain an unusual amino acid, 3,4-dihydroxy-L-phenylalanine (DOPA), which is believed to be responsible for the cohesive and adhesive properties of these proteins. To separate the cohesive and adhesive roles of DOPA, we incorporated DOPA into the midblock of poly(methyl methacrylate)-poly(methacrylic acid)-poly(methyl methacrylate) (PMMA-PMAA-PMMA) triblock copolymers. Self-assembled hydrogels were obtained by exposing triblock copolymer solutions in dimethyl sulfoxide to water vapor. As water diffused into the solution, the hydrophobic end blocks formed aggregates that were bridged by the water-soluble midblocks. Strong hydrogels were formed with polymer weight fractions between 0.01 and 0.4 and with shear moduli between 1 and 5 kPa. The adhesive properties of the hydrogels on TiO2 surfaces were investigated by indentation with a flat-ended cylindrical punch. At pH values of 6 and 7.4, the fully protonated DOPA groups were highly adhesive to the TiO2 surfaces, giving values of approximately equal to 2 J/m2 for the interfacial fracture energy, which we believe corresponds to the cohesive fracture energy of the hydrogel. At these pH values, the DOPA groups are hydrophobic and have a tendency to aggregate, so contact times of 10 or 20 min are required for these high values of the interfacial strength to be observed. At a pH of 10, the DOPA groups were hydrophilic and highly swellable, but less adhesive gels were formed. Oxidation of DOPA groups, a process that is greatly accelerated at a pH of 10, decreased the adhesive performance of the hydrogels even further.     

Polyphosphoprotein from the adhesive pads of Mytilus edulis

Achieving a satisfactory biochemical explanation for the opportunistic underwater adhesion of marine invertebrates such as mussels and barnacles requires a detailed characterization of proteins extracted from holdfast structures produced by these organisms. Mefp-5 is an adhesive protein derived from the foot of the common mussel, Mytilus edulis, and deposited into the byssal attachment pads. Purification and primary structure of mefp-5 was determined by peptide mapping and cDNA sequencing. The protein is 74 residues long and has a mass of about 9500 Da. Mefp-5 composition shows a strong amino acid bias: aromatic amino acids, lysine, and glycine represent 65 mol % of the composition. More than a third of all the residues in the protein are posttranslationally modified by hydroxylation or phosphorylation. The conversion of tyrosine to 3, 4-dihydroxyphenyl-L-alanine (DOPA) and serine to O-phosphoserine accounts for the hydroxylation and phosphorylation, respectively. Neither modification is complete since variations in the extent of phosphorylation and hydroxylation can be detected by mass spectrometry. More than 75% of the DOPA is adjacent to basic residues, e.g., Lys-DOPA and DOPA-Lys. Phosphoserine occurs in sequences strikingly reminiscent of acidic mineral-binding motifs that appear in statherin, osteopontin, and others. This may be an adaptation for adhesion to the most common substrata for mussels, i.e., calcareous materials.     

The phylogeny and chemical diversity of quinone-tanned glues and varnishes

1. 3,4-Dihydroxyphenyl-L-alanine (DOPA)-containing proteins are widely distributed throughout the animal kingdom and appear to serve chiefly as waterproof adhesives and varnishes. 2. The unique chemical and physical stability of these adhesives and varnishes is imparted by quinone-tanning, an oxidative process that leads to the polymerization of DOPA-containing and other proteins. 3. Recent advances in the biochemistry of DOPA-containing proteins suggest that most consist of tandemly repeated sequence motifs. Each motif contains DOPA, a basic amino acid (usually lysine), and abundant glycine or proline. 4. The DOPA residues undergo catechol oxidase-catalyzed conversion to o-quinones at the onset of quinone-tanning. 5. The complexity of quinone chemistry is discussed with regard to quinone-tanning.     

Structure of the SPXX motif.

To understand the structure of the DNA-binding SPXX motif, an analysis of Ser1-Pro2-X3-X4 and Thr1-Pro2-X3-X4 structures observed in proteins is presented. About half (43-46%) of the (S or T) PXX sequences fold into a beta-turn of type (I) or one of a few closely related turn structures. The turn structure has either or both of two compatible hydrogen bonds, one between CO of (Ser or Thr) and NH of X4 (a standard beta-turn type), and the other between OH of (Ser or Thr) and NH of X3 (which we name the sigma type). Within the beta-turn of the TPXX sequence, another type of hydrogen bond (which we name the tau type) occurs between OH of Thr and NH of X4 with the frequency of 72%. These observations support a previous proposal that the (S or T) PXX sequences of DNA-binding proteins fold into a compact beta-turn stabilized by a side-chain-main-chain interaction, which may be suitable to fit into the groove of DNA.     

Domain coupling in a multimodular cellobiohydrolase CbhA from Clostridium thermocellum

Cellobiohydrolase A (CbhA) from Clostridium thermocellum is composed of an N-terminal carbohydrate-binding domain 4 (CBD4), an immunoglobulin-like domain (Ig), a glycoside hydrolase 9 (GH9), X1(1) and X1(2) domains, a CBD3, and a dockerin domain. All domains, except the Ig, bind Ca2+. The following constructs were made: X1(2), X1(1)X1(2), CBD3, X1(1)X1(2)-CBD3, Ig, GH9, Ig-GH9, Ig-GH9-X1(1)X1(2), and Ig-GH9-X1(1)X1(2)-CBD3. Interactions between domains in (1) buffer, (2) with Ca2+, or (3) ethylenediaminetetraacetic acid (EDTA) were studied by differential scanning calorimetry. Thermal unfoldings of all constructs were irreversible. Calcium increased T(d) and cooperativity of unfolding. Multi-domain constructs exhibited more cooperative unfolding in buffer and in the presence of EDTA than did individual domains. They denatured by mechanism simpler than expected from their modular architecture. The results indicate that domain coupling in thermophilic proteins constitutes a significant stabilizing factor.     

Synthesis and assembly of flagellar components by Caulobacter crescentus motility mutants

Cultures of wild-type Caulobacter crescentus and strains with fla mutations representing 24 genes were pulse-labeled with 14C-amino acids and analyzed by immunoprecipitation to study the synthesis of flagellar components. Most fla mutants synthesize flagellin proteins at a reduced rate, suggesting the existence of some mechanism to prevent the accumulation of unpolymerized flagellin subunits. Two strains contain deletions that appear to remove a region necessary for this regulation. The hook protein does not seem to be subject to this type of regulation and, in addition, appears to be synthesized as a faster-sedimenting precursor. Mutations in a number of genes result in the appearance of degradation products of either the flagellin or the hook proteins. Mutations in flaA, -X, -Y, or -Z result in the production of filaments (stubs) that contain altered ratios of the flagellin proteins. In some flaA mutants, other flagellin-related proteins were assembled into the stub structures in addition to the flagellins normally present. Taken together, these analyses have begun to provide insight into the roles of individual fla genes in flagellum biogenesis in C. crescentus.     

A histidine-rich protein from the vitellaria of the liver fluke Fasciola hepatica

The vitellaria are an extensive network of glandular cells and ducts distributed throughout the peripheral tissues of the liver fluke Fasciola hepatica. Eggshell precursor proteins are produced and stockpiled in the vitelline cells of mature flukes. Vitelline protein C has an extraordinary composition: the amino acid 3,4-dihydroxyphenyl-L-alanine (DOPA) and histidine each comprise about 20% of the residues, while glycine represents 41-42% in all variants of what appears to be a microheterogeneous protein family. Protein C has an apparent molecular weight of 16,000-17,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although the protein appears homogeneous following polyacrylamide gel electrophoresis in Tris-glycine with SDS and a acetic acid-urea, electrophoresis in borate, however, suggests that the vitelline protein consists of four or more closely related proteins weighing from 16,000 to 18,500. Isoelectric focusing of the protein family in the presence of 8 M urea resolved only two species having pI values of 6.89 and 6.99. A single N-terminus having the sequence H-H-W-D-G-DOPA-G-DOPA-G was detected. The primary structure of vitelline protein C is characterized by a repeated motif consisting of (G-X)n, where X is Ser, DOPA, or His. Most of the His occurs as G-H repeats in a pepsin-resistant fragment of the protein. Previously, a 31-kDa protein, representing up to 6% of the total protein in the fluke, was reported [Waite, J. H., & Rice-Ficht, A (1987) Biochemistry 26, 7819-7825] to contain significant levels of DOPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mass spectral data for 64 eluted peptides and structural modeling define peptide binding preferences for class I alleles in two chicken MHC-<Emphasis Type="Italic">B</Emphasis> haplotypes associated with opposite responses to Marek's disease

In the chicken, resistance to lymphomas that form following infection with oncogenic strains of Marek's herpesvirus is strongly linked to the major histocompatibility complex (MHC)-B complex. MHC-B21 haplotype is associated with lower tumor-related mortality compared to other haplotypes including MHC-B13. The single, dominantly expressed class I gene (BF2) is postulated as responsible for the MHC-B haplotype association. We used mass spectrometry to identify peptides and structural modeling to define the peptide binding preferences of BF2*2101 and BF2*1301 proteins. Endogenous peptides (8-12 residues long) were eluted from affinity-purified BF2*2101 and BF2*1301 proteins obtained from transduced cDNA expressed in RP9 cells, hence expressed in the presence of heterologous TAP. Sequences of individual peptides were identified by mass spectrometry. BF2*2101 peptides appear to be tethered at the binding groove margins with longer peptides arching out but selected by preferred residues at positions P3, P5, and P8: X-X-[AVILFP]-X((1-5))-[AVLFWP]-X((2-3))-[VILFM]. BF2*1301 peptides appear selected for residues at P2, P3, P5, and P8: X-[DE]-[AVILFW]-X((1-2))-[DE]-X-X-[ED]-X((0-4)). Some longer BF2*1301 peptides likely also arch out, but others are apparently accommodated by repositioning of Arg83 so that peptides extend beyond the last preferred residue at P8. Comparisons of these peptides with earlier peptides derived in the presence of homologous TAP transport revealed the same side chain preferences. Scanning of Marek's and other viral proteins with the BF2*2101 motif identified many matches, as did the control human leukocyte antigen A*0201 motif. The BF2*1301 motif is more restricting suggesting that this allele may confer a selective advantage only in infections with a subset of viral pathogens.     

Superoxide and hydrogen peroxide formation during enzymatic oxidation of DOPA by phenoloxidase

Generation of superoxide anion and hydrogen peroxide during enzymatic oxidation of 3-(3,4-dihydroxyphenyl)-DL-alanine (DOPA) has been studied. The ability of DOPA to react with O2*- has been revealed. EPR spectrum of DOPA-semiquinone formed upon oxidation of DOPA by O2*- was observed using spin stabilization technique of ortho-semiquinones by Zn2+ ions. Simultaneously, the oxidation of DOPA by O2*- was found to produce hydrogen peroxide (H2O2). The analysis of H2O2 formation upon oxidation of DOPA by O2*- using 1-hydroxy-3-carboxy-pyrrolidine (CP-H), and SOD as competitive reagents for superoxide provides consistent values of the rate constant for the reaction between DOPA and O2*- being equal to (3.4+/-0.6)x10(5) M(-1) s(-1).The formation of H2O2 during enzymatic oxidation of DOPA by phenoloxidase (PO) has been shown. The H2O2 production was found to be SOD-sensitive. The inhibition of H2O2 production by SOD was about 25% indicating that H2O2 is produced both from superoxide anion and via two-electron reduction of oxygen at the enzyme. The attempts to detect superoxide production during enzymatic oxidation of DOPA using a number of spin traps failed apparently due to high value of the rate constant for DOPA interaction with O2*-.     

Determination of residue specificity in the EF-hand of troponin C for Ca2+ coordination, by genetic engineering.

Utilizing protein engineering of troponin C (TnC), combined with the physiology of skinned fibers, the present study sought to delineate the mechanisms for metal ion coordination and sensitivity in the sites (EF-hands) that execute the Ca2+ switch for contraction. A total TnC-encoding gene comprising multiple target sequences for restriction enzymes was synthesized, furnishing a pliant molecular handle to manipulate sites I and II in the NH2 terminus of the protein. Of the six positions (X, Y, Z, -Y, -X, and -Z) essential for metal ion chelation in a typical EF-hand, invariably the X position has aspartate, and -Z position has glutamate. In the X position of site II, mutation of aspartate for either glutamate (gamma-carboxylate) or asparagine (same side chain length as aspartate) yielded functionally inactive proteins with concomitantly diminished Ca2+ binding capacity. Similarly, in -Z position (site I), neither aspartate nor glutamine were compatible in exchange for the conserved glutamate. In contrast, for the Y coordinate of site II, a preference for asparagine comparable to that for wild-type aspartate was detected, but glutamate was impermissible. Evidently, physicochemical and steric factors both are critical in governing the mechanism for metal ion chelation in TnC in a physiological milieu. Furthermore, the findings manifest that the quaternary structure of hydrated TnC restrains the EF-hands during on-off operation of the Ca2+ switch.     

The adhesive protein of Choromytilus chorus (Molina, 1782) and Aulacomya ater (Molina, 1782): a proline-rich and a glycine-rich polyphenolic protein

The adhesive polyphenolic proteins from Aulacomya ater and Choromytilus chorus with apparent molecular masses of 135000 and 105000, respectively, were digested with trypsin and the peptides produced resolved by reversed phase liquid chromatography. About 5 and 12 major peptides were obtained from the protein of A. ater and C. chorus, respectively. The major peptides were purified by reverse-phase chromatography and the amino acid sequence indicates that both polyphenolic proteins consisted of repeated sequence motifs in their primary structure. The major peptides of A. ater contain seven amino acids corresponding to the consensus sequence AGYGGXK, whereas the tyrosine was always found as 3, 4-dihydroxyphenylalanine (Dopa), the X residue in position 6 was either valine, leucine or isoleucine, and the carboxy terminal was either lysine or hydroxylysine. On the other hand, the major peptides of C. chorus ranged in size from 6 to 21 amino acids and the majority correspond to the consensus sequence AKPSKYPTGYKPPVK. Both proteins differ markedly in the sequence of their tryptic peptides, but they share the common characteristics of other adhesive proteins in having a tandem sequence repeat in their primary structure.     

Copper binding to plant ozone-inducible proteins (OI2-2 and OI14-3)

Ozone-inducible proteins (OI2-2 and OI14-3) from Atriplex canescens whose structure and function are unknown are rich in glycine intercepted with histidine and tyrosine with putative signal peptides at the N-terminus. OI2-2 and OI14-3 contain 8 and 10 tandem repeats of YGHGGG, respectively. In order to study whether these proteins bind Cu(2+), circular dichroism (CD), and nuclear magnetic resonance (NMR) were measured for four synthetic peptides corresponding to sections of the sequences of these proteins; 1 (HGGGY), 2 (HGGGYGH), 3 (YGHGGGY), and 4 (YGHGGGYGHGGGY), where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH(2) group at the C-terminus. Visible CD spectra of the four peptides show positive peaks near 580 and 340nm, which were observed at pH 7.4 but not pH 6.0, indicating clearly that the four peptides bind Cu(2+). The NMR spectra indicate that the addition of small amounts of CuSO(4) to 3 (Y1-G2-H3-G4-G5-G6-Y7) causes significant broadening of resonances of the side chain protons (C(beta)H, C(epsilon1)H, and C(delta2)H) of His3 and the side chain C(beta)H of Tyr1 at pH 7.4. In addition, the backbone C(alpha)H resonances of Gly2 and Gly4 were broadened more strongly than those of Gly5 and Gly6. CD titration experiment suggested that two repeats of YGHGGG comprise the fundamental Cu(2+) binding unit. Thus, the ozone-inducible proteins are capable of binding at least four or five copper ions per protein. These copper-binding proteins would function as active oxygen scavengers.     

The influence of the side-chain sequence on the structure-activity correlations of immunomodulatory branched polypeptides. Synthesis and conformational analysis of new model polypeptides

New branched polypeptides were synthesized for a detailed study of the influence of the side-chain structure on the conformation and biological properties. The first subset of polypeptides were prepared by coupling of tetrapeptides to poly[L-Lys]. These polymers contain either DL-Ala3-X [poly[Lys-(X-DL-Ala3)n]] or X-DL-Ala3 [poly[Lys-(DL-Ala3-X)n] (n less than or equal to 1)] tetrapeptide side chains. Another group of branched polymers comprise a mixture of DL-Alam and of DL-Alam-X oligomeric branches in a random distribution [poly[Lys-(DL-Alam-Xi)] (i less than 1, m approximately 3)]. In each subset the X = Leu or Phe derivatives were prepared. The N-protected tetrapeptides were synthesized by conventional liquid phase methods and were coupled as active esters. The degree of racemization was found relatively high both for active esters and coupled derivatives, when optically active amino acids were in the C-terminal position of the tetrapeptides. In the case of the poly[Lys-(Leu-DL-Ala3)n] derivative, comparative experiments were carried out using various methodical alterations. The highest stereochemical homogeniety could be achieved when the tetrapeptide active ester was synthesized by the "backing off" method. CD spectra of poly[Lys-(Xi-DL-Alam)] (i less than 1, m approximately 3) and of poly[Lys-(X-DL-Ala3)n] were analyzed and compared to those of poly[Lys-(DL-Alam-Xi)] and of poly[Lys-(DL-Ala3-X)n]. All measurements were performed in water solutions of varying pH values and ionic strengths. The data obtained suggest that branched polypeptides containing a mixture of two different types of oligomeric side chains (DL-Alam and DL-Alam-Xi or Xi-DL-Alam) distributed randomly adopt an almost identical conformation to those that comprise only the respective tetrapeptide (DL-Ala3-X or X-DL-Ala3) branches. The results also indicate that the tendency to form an ordered structure is determined by the identity and the position of the chiral amino acid X (Phe or Leu) in the side chain.     

Adsorption of chemically synthesized mussel adhesive peptide sequences containing DOPA on stainless steel

The adsorption of proteins at solid–liquid interfaces is important in biosensor and biomaterial applications. Marine mussels affix themselves to surfaces using a highly cross‐linked, protein‐based adhesive containing a high proportion of L‐3,4‐dihydroxyphenylalanine (DOPA) residues. In this work, the effect of DOPA residues on protein adhesion on stainless steel surfaces was studied using a quartz crystal microbalance with dissipation system. The adsorption of two repetitive peptide motifs, KGYKYYGGSS and KGYKYY, from the mussel Mytilus edulis foot protein 5 on stainless steel was studied before and after chemo‐enzymatic modification of tyrosine residues to DOPA using mushroom tyrosinase. Conversion from tyrosine to DOPA, evaluated by HPLC, was in the range 70–99%. DOPA‐modified sequences showed fourfold greater adhesion than unmodified M. edulis foot protein 5 motifs. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Adhesion a la moule

Mussels owe their sessile way of life in the turbulent intertidalzone to adaptive adjustments in the process and biochemistryof permanent attachment. These have understandably attractedscientific interest given that the attachment is rapid, versatile,tough and not subverted by the presence of water. The adhesivepads of mussel byssus contain at least six different proteinsall of which possess the peculiar amino acid 3, 4-dihydroxyphenylalanine(DOPA) at concentrations ranging from 0.1 to 30 mol %. Studiesof protein distribution in the plaque indicate that proteinswith the highest levels of DOPA, such as mefp-3 (20 mol %) andmefp-5 (30 mol %), appear to predominate at or near the interfacebetween the plaque and substratum. Although the presence ofDOPA in proteins has traditionally been associated with cross-linkingvia chelate-mediated or covalent coupling, recent experimentswith natural and synthetic DOPA-containing polypeptides suggestthat cross-link formation is not the only fate for DOPA. IntactDOPA, particularly near the interface, may be essential forgood chemisorption to polar surfaces. Uniformly high DOPA oxidationto cross-links leads to interfacial failure but high cohesivestrength, while low DOPA oxidation results in better adhesionat the expense of cohesion. Defining the adaptations involvedin balancing these two extremes is crucial to understandingmarine adhesion.     

Immunolocalization of keratin‐associated beta‐proteins (beta‐keratins) in the regenerating lizard epidermis indicates a new process for the differentiation of the epidermis in lepidosaurians

The process of keratinocyte differentiation was analyzed in the regenerating epidermis of the lizard Anolis carolinensis, where the genes coding for beta‐proteins (beta‐keratins) are known. The regenerating epidermis forms all epidermal layers found in normal scales (Oberhäutchen‐, beta‐, mesos‐, and alpha‐layer). Three specific proteins representing the larger families of beta‐proteins, glycine‐rich (HgG5, 28% glycine, 3.6% cysteine), glycine‐cysteine medium‐rich (HgGC10, 13% glycine, 14.5% cysteine), and glycine‐cysteine rich (HgGC3, 30.4% glycine, 8.7% cysteine) have been immunolocalized at the ultrastructural level. HgG5 is only present in differentiating beta‐cells, a weak or no labeling is observed in Oberhäutchen and is absent in alpha‐cells. The protein is located in the pale corneous material forming the compact beta‐layer but is absent in mature Oberhäutchen cells. HgGC10 is present among beta‐packets in Oberhäutchen and beta‐cells but disappears in more compact and electron‐pale corneous material. The labeling disappears in mesos‐cells and is present with variable intensity in alpha‐cells, whereas lacunar and clear‐cells are low labeled to unlabeled. HgGC3 is sparse or absent in beta‐cells but is lightly present in the darker corneous material of differentiating and mature alpha‐cells, lacunar‐cells, and clear‐cells. The study suggests that while glycine‐rich proteins (electron‐pale) are specifically used for building the resistant and hydrophobic beta‐layer, cysteine–glycine rich proteins (electron‐denser) are used to form the pliable corneous material present in the Oberhäutchen and alpha‐cells. The differential accumulation of beta‐proteins on the alpha‐keratin cytoskeleton scaffold and not the alternance of beta‐ with alpha‐keratins allow the differentiation of different epidermal layers. © 2012 Wiley Periodicals, Inc.