Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments

Many alpha-helical proteins that form two-chain coiled coils possess a 13-residue trigger motif that seems to be required for the stability of the coiled coil. However, as currently defined, the motif is absent from intermediate filament (IF) protein chains, which nevertheless form segmented two-chain coiled coils. In the present work, we have searched for and identified two regions in IF chains that are essential for the stability necessary for the formation of coiled-coil molecules and thus may function as trigger motifs. We made a series of point substitutions with the keratin 5/keratin 14 IF system. Combinations of the wild-type and mutant chains were assembled in vitro and in vivo, and the stabilities of two-chain (one-molecule) and two-molecule assemblies were examined with use of a urea disassembly assay. Our new data document that there is a region located between residues 100 and 113 of the 2B rod domain segment that is absolutely required for molecular stability and IF assembly. This potential trigger motif differs slightly from the consensus in having an Asp residue at position 4 (instead of a Glu) and a Thr residue at position 9 (instead of a charged residue), but there is an absolute requirement for a Glu residue at position 6. Because these 13 residues are highly conserved, it seems possible that this motif functions in all IF chains. Likewise, by testing keratin IF with substitutions in both chains, we identified a second potential trigger motif between residues 79 and 91 of the 1B rod domain segment, which may also be conserved in all IF chains. However, we were unable to find a trigger motif in the 1A rod domain segment. In addition, many other point substitutions had little detectable effect on IF assembly, except for the conserved Lys-23 residue of the 2B rod domain segment. Cross-linking and modeling studies revealed that Lys-23 may lie very close to Glu-106 when two molecules are aligned in the A(22) mode. Thus, the Glu-106 residue may have a dual role in IF structure: it may participate in trigger formation to afford special stability to the two-chain coiled-coil molecule, and it may participate in stabilization of the two-molecule hierarchical stage of IF structure.     

Modeling alpha-helical coiled-coil interactions: the axial and azimuthal alignment of 1B segments from vimentin intermediate filaments

Attempts at predicting the relative axial alignments of fibrous protein molecules in filamentous structures have relied upon representing the (multichain) molecular structure by a one-dimensional sequence of amino acids. Potential intermolecular ionic and apolar interactions were counted and determined as a function of the relative axial stagger between the molecules. No attempts were made to consider the azimuthal aspect of the interacting molecules and neither were apolar or ionic energy terms used. Surprisingly, this simple approach proved remarkably informative and yielded accurate predictions of the axial periods present. However, a more comprehensive analysis involving the energetics of aggregation taking due regard for the relative azimuths of the molecules as well as their separation should decrease the noise level in the calculations and reveal other pertinent information. Toward that end, we have modeled the interaction between two alpha-helical coiled-coil segments in intermediate filament molecules (1B segments from human vimentin). The relative axial alignment and polarity of the molecules is already known from detailed crosslinking studies and this provides a criterion against which the success (or otherwise) of the modeling can be judged. The results confirm that an antiparallel alignment of two 1B segments is preferred over any of the parallel options (as observed experimentally). The calculated axial alignment, however, is not identical to that observed from detailed crosslinking studies indicating that other parts of the molecule (probably the head and tail domains as well as other coiled-coil segments) have a crucial role in determining the precise mode of axial aggregation. The results also show that the apolar interactions seem to be significantly less important in the alignment process than the ionic ones. This is consistent with the observation of a well-defined period in the linear disposition of the charged (but not apolar) residues along the length of the outer surface of the vimentin molecule.     

Sequence comparisons of intermediate filament chains: evidence of a unique functional/structural role for coiled-coil segment 1A and linker L1

A comprehensive analysis of the sequences of all types of intermediate filament chains has been undertaken with a particular emphasis on those of segment 1A and linker L1. This has been done to assess whether structural characteristics can be recognized in the sequences that would be consistent with the role of each region in the recently proposed "swinging head" hypothesis. The analyses show that linker L1 is the most flexible rod domain region, that it is the most elongated structure (on a per residue basis), and that it is the most variable region as regards sequence and length. Segment 1A has one of the two most highly conserved regions of sequence in the rod domain (the other being at the end of segment 2B), with seven particular residues conserved across all chain types. It also contains one of the very few potential interchain ionic interactions that could be conserved across all chain types. However, the aggregation of chains in segment 1A is specified less precisely overall by interchain ionic interactions than are the other coiled-coil segments. The apolar residue contents in positions a and d of the heptad substructure are the highest of any coiled-coil segment in the intermediate filament family. Segment 1A also displays an amino acid composition atypical of not only coiled-coil segments 1B and 2B, but indeed of two-stranded coiled coils in general. Nonetheless, molecular modeling based on the crystal structure of the monomeric 1A fragment from human vimentin shows that coiled-coil formation is plausible. The most extensive regions of apolar/aromatic residues lie at the C-terminal end of segment 2B in the helix termination motif and in segment 1A in and close to the helix initiation motif. The predicted stability of the individual alpha-helices in segment 1A is greater than in those comprising segments 1B and 2B, though potential intrachain ionic interactions are either lacking or are minimal in number. Analysis of the 1A sequence and those regions immediately N- and C-terminal to it has shown that the capping residues are near optimal close to the previously predicted ends, thus adding to the likely stability of the alpha-helical structure. However, a second terminating sequence is predicted in 1A (about 10 residues back from the C-terminus). This allows the possibility of some unwinding of the alpha-helical structure of 1A immediately adjacent to linker L1 when the head domains no longer stabilize the coiled-coil structure. All of these data are consistent with the concept of a flexible hinge at L1 and with the ability of the two alpha-helical coiled-coil strands to separate under appropriate conditions and partly unwind at their C-terminal ends to allow the head domains a greater degree of mobility, thus facilitating function.     

Keratin intermediate filament chains in the European common wall lizard (Podarcis muralis) and a potential keratin filament crosslinker

On the basis of sequence homology with mammalian α-keratins, and on the criteria that the coiled-coil segments and central linker in the rod domain of these molecules must have conserved lengths if they are to assemble into viable intermediate filaments, a total of 28 Type I and Type II keratin intermediate filament chains (KIF) have been identified from the genome of the European common wall lizard (Podarcis muralis). Using the same criteria this number may be compared to 33 found here in the green anole lizard (Anole carolinensis) and 25 in the tuatara (Sphenodon punctatus). The Type I and Type II KIF genes in the wall lizard fall in clusters on chromosomes 13 and 2 respectively. Although some differences occur in the terminal domains in the KIF chains of the two lizards and tuatara, the similarities between key indicator residues – cysteine, glycine and proline – are significant. The terminal domains of the KIF chains in the wall lizard also contain sequence repeats commonly based on glycine and large apolar residues and would permit the fine tuning of physical properties when incorporated within the intermediate filaments. The H1 domain in the Type II chain is conserved across the lizards, tuatara and mammals, and has been related to its role in assembly at the 2–4 molecule level. A KIF-like chain (K80) with an extensive tail domain comprised of multiple tandem repeats has been identified as having a potential filament-crosslinking role.     

Towards a pharmacophore for amyloid

Diagnosing and treating Alzheimer's and other diseases associated with amyloid fibers remains a great challenge despite intensive research. To aid in this effort, we present atomic structures of fiber-forming segments of proteins involved in Alzheimer's disease in complex with small molecule binders, determined by X-ray microcrystallography. The fiber-like complexes consist of pairs of β-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. The structures suggest that apolar molecules drift along the fiber, consistent with the observation of nonspecific binding to a variety of amyloid proteins. In contrast, negatively charged orange-G binds specifically to lysine side chains of adjacent sheets. These structures provide molecular frameworks for the design of diagnostics and drugs for protein aggregation diseases.     

cDNA and deduced primary structure of rat protein B23, a nucleolar protein containing highly conserved sequences

Protein B23 (Mr/pI = 38,000/5.1) is a major RNA-associated nucleolar phosphoprotein which contains highly acidic segments and has a high affinity for silver ions. Using synthetic oligonucleotides as probes cloned cDNAs encoding protein B23 were isolated and characterized. One of the cDNAs, obtained from a rat brain library, contained an insert of 1232 base pairs of DNA encoding a polypeptide of 292 amino acid residues. Segments of the protein sequence were confirmed by partial sequencing of CNBr fragments from rat hepatoma protein B23. The protein contains a methionine-rich amino-terminal sequence and two highly acidic segments in the center of the sequence. The first acidic segment, in which 11 of the 13 residues are acidic, begins at residue 120 and contains a major phosphorylation site. In the second segment (residues 159-187) there are four copies of the sequence Asp-Asp-Glu, and all but two of the 29 residues have acidic side chains. When the sequence of the rat protein was compared with available sequences from other species a high degree of conservation was found; the 77-residue carboxyl-terminal sequence is identical with that of human protein B23 (Chan, P. K., Chan, W.-Y., Yung, B. Y. M., Cook, R. G., Aldrich, M. B., Ku, D., Goldknopf, I. L., and Busch, H. (1986) J. Biol. Chem. 261, 14335-24341), and about 63% of the residues are identical when the rat B23 sequence is compared with protein N038 from Xenopus laevis (Schmidt-Zachmann, M. S., Hügle-D?rr, B., and Franke, W. (1987) EMBO J. 6, 1881-1890). Except for the presence of highly acidic regions no significant similarities were found with protein C23 (nucleolin), the other major nucleolar protein.     

Proton Magnetic Resonance Studies of the Interactions of Histones F1 and F2B with DNA

THE determination of sequences of histones F2A1(refs. 1 and 2) and F2B (ref. 3) and the amino-acid compositions of peptides of histone F1 (ref. 4) has shown that the distribution of residues along the polypeptide chains is extremely irregular. In particular, different regions of the molecule have markedly different characters. Thus the amino half of F2A1 has a ratio of basic to acidic residues of 15.6: 1 while for the carboxyl half of the molecule it is 1.5 : 1; for F2B this ratio is 13 : 1 for the amino quarter of the molecule and 1.9: 1 for the remainder. In the case of F1 this “polarity” is reversed: the carboxyl half of the molecule has a ratio of basic to acidic residues of 15 : 1, the amino half of 1.2 : 1. Further, regions rich in basic residues also contain a high proportion of helix destabilizing residues, proline in F1 and F2B and glycine in F2A1. In contrast, the non-basic regions contain a higher proportion of the apolar residues regarded as helix stabilizers and higher proportions of acidics, aromatics and threonine and serine residues. The characterization of two different regions led to the suggestion^1–4 that the basic regions of the polypeptide chains are the primary sites of interaction with DNA, while the non-basic regions have the potential for the formation of definite conformations and might be involved in specific interactions other than with DNA.     

Keratin intermediate filaments: Differences in the sequences of the Type I and Type II chains explain the origin of the stability of an enzyme-resistant four-chain fragment

Previous studies have shown that a strong interaction exists between oppositely directed 1B molecular segments in the intermediate filaments of trichocyte keratins. A similar interaction has been identified as having a significant role in the formation of unit-length filaments, a precursor to intermediate filament formation. The present study is concerned with the spatial relationship of these interacting segments and its dependence on differences in the amino acid sequences of the two-chain regions that constitute the 1B molecular segment. It is shown that along a particular line of contact both chain segments possess an elevated concentration of residues with a high propensity for dimer formation. The transition from the reduced to the oxidized state involves a simple axial displacement of one molecular segment relative to the other, with no attendant rotation of either segment. This changes the inter-relationship of the two 1B molecular segments from a loosely packed form to a more compact one. After the slippage eight of the cysteine residues in the dimer are precisely aligned to link up and form the disulfide linkages as observed. The two remaining cysteine residues are located on the outside of the dimer and are presumably involved in inter-dimer bonding. The existence of a unique line of contact requires that two chains in the molecule have different amino acid compositions with the clustering of dimer-favoring residues phased by half the pitch length of the coiled coil.     

Packed protein bilayers in the 0.90 A resolution structure of a designed alpha helical bundle.

A 12-residue peptide designed to form an alpha-helix and self-associate into an antiparallel 4-alpha-helical bundle yields a 0.9 A crystal structure revealing unanticipated features. The structure was determined by direct phasing with the "Shake-and-Bake" program, and contains four crystallographically distinct 12-mer peptide molecules plus solvent for a total of 479 atoms. The crystal is formed from nearly ideal alpha-helices hydrogen bonded head-to-tail into columns, which in turn pack side-by-side into sheets spanning the width of the crystal. Within each sheet, the alpha-helices run antiparallel and are closely spaced (9-10 A center-to-center). The sheets are more loosely packed against each other (13-14 A between helix centers). Each sheet is amphiphilic: apolar leucine side chains project from one face, charged lysine and glutamate side chains from the other face. The sheets are stacked with two polar faces opposing and two apolar faces opposing. The result is a periodic biomaterial composed of packed protein bilayers, with alternating polar and apolar interfaces. All of the 30 water molecules in the unit cell lie in the polar interface or between the stacked termini of helices. A section through the sheet reveals that the helices packed at the apolar interface resemble the four-alpha-helical bundle of the design, but the helices overhang parts of the adjacent bundles, and the helix crossing angles are less steep than intended (7-11 degrees rather than 18 degrees).     

Secondary structure of component 8c-1 of alpha-keratin. An analysis of the amino acid sequence.

The amino acid sequence of component 8c-1 from alpha-keratin was analysed by using secondary-structure prediction techniques, homology search methods, fast Fourier-transform techniques to detect regularities in the linear disposition of amino acids, interaction counts to assess possible modes of chain aggregation and assessment of hydrophilicity distribution. The analyses show the following. The molecule has two lengths of coiled-coil structure, each about 20 nm long, one from residues 56-202 with a discontinuity from about residue 91 to residue 101, and the other from residues 219-366 with discontinuities from about residue 238 to residue 245 and at about residue 306. The acidic and basic residues in the coiled-coil segment between residues 102 and 202 show a 9,4-residue structural period in their linear disposition, whereas between residues 246 and 366 a period of 9.9 residues is observed in the positioning of ionic residues. Acidic and basic residues are out of phase by 180 degrees. Similar repeats occur in corresponding regions of other intermediate-filament proteins. The overall mean values for the repeats are 9.55 residues in the N-terminal region and 9.85 residues in the C-terminal region. The regions at each end of the protein chain (residues 1-55 and 367-412) are not alpha-helical and contain many potential beta-bends. The regions specified in have a significant degree of homology mainly due to a semi-regular disposition of proline and half-cystine residues on a three-residue grid; this is especially apparent in the C-terminal segment, in which short (Pro-Cys-Xaa)n regions occur. The coiled-coil segments of component 8c-1 bear a striking similarity to corresponding segments of other intermediate-filament proteins as regards sequence homology, structural periodicity of ionic residues and secondary/tertiary-structure predictions. The assessments of the probabilities that these homologies occurred by chance indicate that there are two populations of keratin filament proteins. The non-coiled-coil regions at each end of the chain are less hydrophilic than the coiled-coil regions. Ionic interactions between the heptad regions of components 8c-1 and 7c from the microfibrils of alpha-keratin are optimized when a coiled-coil structure is formed with the heptad regions of the constituent chains both parallel and in register.     

Mapping the functional domains of nucleolar protein B23

Protein B23 is a multifunctional nucleolar protein whose cellular location and characteristics strongly suggest that it is a ribosome assembly factor. The protein has nucleic acid binding, ribonuclease, and molecular chaperone activities. To determine the contributions of unique polypeptide segments enriched in certain classes of amino acid residues to the respective activities, several constructs that produced N- and C-terminal deletion mutant proteins were prepared. The C-terminal quarter of the protein was shown to be necessary and sufficient for nucleic acid binding. Basic and aromatic segments at the N- and C-terminal ends, respectively, of the nucleic acid binding region were required for activity. The molecular chaperone activity was contained in the N-terminal half of the molecule, with important contributions from both nonpolar and acidic regions. The chaperone activity also correlated with the ability of the protein to form oligomers. The central portion of the molecule was required for ribonuclease activity and possibly contains the catalytic site; this region overlapped with the chaperone-containing segment of the molecule. The C-terminal, nucleic acid-binding region enhanced the ribonuclease activity but was not essential for it. These data suggest that the three activities reside in mainly separate but partially overlapping segments of the polypeptide chain.     

Residues in the 1A rod domain segment and the linker L2 are required for stabilizing the A11 molecular alignment mode in keratin intermediate filaments

Both analyses of x-ray diffraction patterns of well oriented specimens of trichocyte keratin intermediate filaments (IF) and in vitro cross-linking experiments on several types of IF have documented that there are three modes of alignment of pairs of antiparallel molecules in all IF: A11, A22 and A12, based on which parts of the major rod domain segments are overlapped. Here we have examined which residues may be important for stabilizing the A11 mode. Using the K5/K14 system, we have made point mutations of charged residues along the chains and examined the propensities of equimolar mixtures of wild type and mutant chains to reassemble using as criteria: the formation (or not) of IF in vitro or in vivo; and stabilities of one- and two-molecule assemblies. We identified that the conserved residue Arg10 of the 1A rod domain, and the conserved residues Glu4 and Glu6 of the linker L2, were essential for stability. Additionally, conserved residues Lys31 of 1A and Asp1 of 2A and non-conserved residues Asp/Asn9 of 1A, Asp/Asn3 of 2A, and Asp7 of L2 are important for stability. Notably, these groups of residues lie close to each other when two antiparallel molecules are aligned in the A11 mode, and are located toward the ends of the overlap region. Although other sets of residues might theoretically also contribute, we conclude that these residues in particular engage in favorable intermolecular ionic and/or H-bonding interactions and thereby may play a role in stabilizing the A11 mode of alignment in keratin IF.     

The CorA Mg(2+) transport protein of Salmonella typhimurium. Mutagenesis of conserved residues in the second membrane domain

Salmonella typhimurium CorA is the archetypal member of the largest family of Mg(2+) transporters of the Bacteria and Archaea. It contains three transmembrane segments. There are no conserved charged residues within these segments indicating electrostatic interactions are not used in Mg(2+) transport through CorA. Previous mutagenesis studies of CorA revealed a single face of the third transmembrane segment that is important for Mg(2+) transport. In this study, we mutated hydroxyl-bearing and other conserved residues in the second transmembrane segment to identify residues involved in transport. Residues Ser(260), Thr(270), and Ser(274) appear to be important for transport and are oriented such that they would also line a face of an alpha-helix. In addition, the sequence (276)YGMNF(280), found in virtually all CorA homologues, is critical for CorA function because even conservative mutations are not tolerated at these residues. Finally, mutations of residues in the second transmembrane segment, unlike those in the third transmembrane segment, revealed cooperative behavior for the influx of Mg(2+). We conclude that the second transmembrane segment forms a major part of the Mg(2+) pore with the third transmembrane segment of CorA.     

Both a short hydrophobic domain and a carboxyl-terminal hydrophilic region are important for signal function in the Escherichia coli leader peptidase

Leader peptidase, typical of inner membrane proteins of Escherichia coli, does not have an amino-terminal leader sequence. This protein contains an internal signal peptide, residues 51-83, which is essential for assembly and remains as a membrane anchor domain. We have employed site-directed mutagenesis techniques to either delete residues within this domain or substitute a charged amino acid for one of these residues to determine the important properties of the internal signal. The deletion analysis showed that a very small apolar domain, residues 70-76, is essential for assembly, whereas residues that flank it are dispensable for its function. However, point mutations with charged amino acid residues within the polar sequence (residues 77-82) slow or abolish leader peptidase membrane assembly. Thus, a polar region, Arg-Ser-Phe-Ile-Tyr-Glu, is important for the signal peptide function of leader peptidase, unlike other signals identified thus far.     

Computation of 3D structure and distribution of helical parameters for D-period segments of human fibrillar collagen III

The structures of D-period segments of collagen (234 amino residues or ～1/4 of whole length) are established by methods of molecular mechanics and geometry analysis. Each D-period segment proves to have a unique spatial structure. The distributions of local helical parameters along the molecule are calculated. It is found that a second hydrogen bond is formed in every case when the second residue in the tripeptide G-X-Y is an amino acid. With such a combined H-bond network, all the peptide CO groups of glycines and of the third residues in tripeptides have quasi-equivalent positions on the surface of the collagen molecule. The local deformations of the polyproline II helix in the triple complex give rise to the observed modulation of structure at the macromolecular level, which may be important for the mutual orientation of collagen molecules during fibrillogenesis.     

Collagen XXIV,a vertebrate fibrillar collagen with structural features of invertebrate collagens: selective expression in developing cornea and bone

Tissue-specific assembly of fibers composed of the major collagen types I and II depends in part on the formation of heterotypic fibrils, using the quantitatively minor collagens V and XI. Here we report the identification of a new fibrillar-like collagen chain that is related to the fibrillar alpha1(V), alpha1(XI), and alpha2(XI) collagen polypeptides and which is coexpressed with type I collagen in the developing bone and eye. The new collagen was designated the alpha1(XXIV) chain and consists of a long triple helical domain flanked by typical propeptide-like sequences. The carboxyl propeptide is classic, with 8 conserved cysteine residues. The amino-terminal peptide contains a thrombospodin-N-terminal-like (TSP) motif and a highly charged segment interspersed with several tyrosine residues, like the fibril diameter-regulating collagen chains alpha1(V) and alpha1(XI). However, a short imperfection in the triple helix makes alpha1(XXIV) unique from other chains of the vertebrate fibrillar collagen family. The triple helical interruption and additional select features in both terminal peptides are common to the fibrillar chains of invertebrate organisms. Based on these data, we propose that collagen XXIV is an ancient molecule that may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development.     

A structural model of human erythrocyte protein 4.1

Limited proteolysis and specific chemical cleavage methods have enabled a detailed structural characterization of human erythrocyte protein 4.1. This protein is composed of two chemically very similar polypeptide chains (a and b) with apparent molecular masses of 80,000 and 78,000 daltons. Cleavage of protein 4.1 at cysteine residues by 2-nitro-5-thiocyanobenzoic acid produces a series of doublets which differ by approximately 2,000 daltons and have identical peptide maps. Alignment of these peptides by mapping analysis has localized 4 cysteine residues within a 17,000-dalton segment on both a and b polypeptides. Mild chymotryptic treatment at 0 degrees C cleaves protein 4.1 primarily in three central locations and generates two families of unrelated peptides. Analysis of these fragments in two-dimensional gels and by peptide mapping reveals an unusual polarity in protein 4.1 structure in that each polypeptide chain contains two segments, one relatively acidic the other basic, that are segregated at opposite ends of the molecule. The basic region is digested into a cysteine-rich 30,000-dalton domain which resists further breakdown while the acidic region is readily degraded into smaller fragments. The peptides derived from the acidic region all appear as doublets suggesting that protein 4.1 a and b polypeptides differ close to the terminus of the acidic end. Similar phosphorylation sites occur on both polypeptides within a segment some 24,000-34,000 daltons from the acidic terminus.     

A proposed structure of bacteriophage T4 gene product 22--a major prohead scaffolding core protein

Gene 22 of bacteriophage T4 encodes a major prohead scaffolding core protein of 269 amino acid residues. From its nucleotide sequence the gene product (gp) 22 has a predicted Mr of 29.9 and a pI of 4.3. The protein is rich in charged residues (glutamic acid and lysine) and contains low amounts of proline and glycine and no cysteine residues. We suggest that gp22 undergoes limited proteolytic processing which eliminates the short C-terminal piece from the molecule during the early steps of prohead assembly. Most amino acid residues of the gp22 polypeptide chain (80%) have an alpha-helical conformation and form seven peculiar alpha-helices. A model suggesting the spatial organization of gp22 is presented. Three long alpha-helices numbered 1 (1A and 1B), 3, and 5 (5A and 5B) are packed in an antiparallel fashion along the major axis of the road-shaped molecule. Two rather short alpha-helices (2 and 4) are located at the distal and proximal ends of the protein molecule, respectively. Helix number 2, which is a proteolytic fragment of gp22 found in mature T4 heads, is packed with helices 1A and 3, similar to a novel element of supersecondary structure, the alpha alpha-corner. Helix number 4 probably interacts with the gp20 connector of the prohead. The implications of the structure of the gp22 molecule for the assembly of the prohead core are discussed.     

The hemoglobins of the bullfrog Rana catesbeiana. The structure of the beta chain of component C and the role of the alpha chain in the formation of intermolecular disulfide bonds

The adult bullfrog Rana catesbeiana has two major hemoglobin components, B and C. Component C polymerizes by disulfide bond formation between tetramers but component B does not. The amino acid sequence of the first 112 residues of the beta chain of component C has been reported (Baldwin, T. O., and Riggs, A. (1974) J. Biol. Chem. 249, 6110-6118). We have completed the sequence of the beta chain of component C by determining the last 28 residues. This segment contains the 2 cysteinyl residues of the chain. Examination of models indicates that neither of these is in a readily accessible position for the formation of intertetramer disulfide bonds. Reactive sulfhydryl groups of the alpha chains are shown to be responsible for the initial formation of disulfide bonds between tetramers. The beta chains within the tetramers form disulfide bonds only when the hemoglobin molecules are subjected to prolonged incubation at 37 degrees C under oxygen. The beta chains of components B and C appear to be identical; the alpha chains are clearly quite different. This suggests that the alpha B and alpha C subunits interact in the association of the deoxygenated tetramers B and C to form what appears to be a BC2 molecule.     

The cysteine residues in the carboxy terminal domain of tropoelastin form an intrachain disulfide bond that stabilizes a loop structure and positively charged pocket.

Analysis of purified bovine tropoelastin with Ellman's reagent and [14C]iodoacetamide demonstrated that the only two cysteine residues in the molecule form an intrachain disulfide bond. Molecular modeling suggests that the cysteine residues are juxtaposed as the result of a tight turn that produces an antiparallel beta structure. Protruding from the C-terminal end of the turn is the sequence Arg-Lys-Arg-Lys which forms the floor of a positively charged pocket created by the extension of the arginine and lysine side chains on opposite sides of the peptide chain perpendicular to the plane of the turn. The side chain of a conserved lysine residue in the disulfide-bonded loop forms the top of the pocket. This positively charged pocket may define a binding site for acidic microfibrillar proteins that mediate elastic fiber assembly.