Fibrinogen: a preliminary analysis of the amino acid sequences of the portion of the alpha, beta and gamma-chains postulated to form the interdomainal link between globular regions of the molecule

It has been suggested (Doolittle et al., 1977) that portions of the α-, β- and γ-chains of fibrinogen form a coiled-coil rope of α-helices and that this rope connects globular domains of the molecule. A fast Fourier transform analysis of the relevant amino acid sequences has shown that there is a significant 3.5-residue period in the linear disposition of the apolar residues in all three chains. This periodicity is characteristic of amino acid sequences of α-fibrous proteins, such as α-tropomyosin and α-keratin, where the tertiary structure is closely related to a coiled-coil of α-helices. However, a detailed study of the fibrinogen sequences shows that the structure is likely to contain several regions which do not have a simple secondary structure. The detailed conformation of the postulated rodlike region of fibrinogen is therefore complex and may approximate a coiled-coil only over relatively short lengths.An important question to emerge from this analysis is whether correct positioning of apolar residues in a pseudo-repeating heptad is sufficiently important to override low α-helix-favouring potential of other residues in the heptad.     

Structural organization of C-terminal parts of fibrinogen Aα-chains

Calorimetric studies of fibrinogen melting and of its early degradation products have shown that the C-terminal parts of both the Aα-chains form structural domains which strongly interact with each other in the native fibrinogen molecule.     

Designation of sequences involved in the “coiled-coil” interdomainal connections in fibrinogen: Construction of an atomic scale model

Primary structure studies on human fibrinogen (α₂β₂γ₂) have revealed certain unusual features which are compatible with the existence of a three-stranded set of supercoiled α-helices thought to be characteristic of the keratin family of fibrous proteins. In particular, each of the three non-identical chains has two characteristic braces of cysteines separated by 111 residues. The three chains are apparently bound together at these two junctures in unique six-cysteine rings. The amino acid sequences between these unusual cysteine pairs (themselves separated in all six cases by three residues) are helix-permissive over significant portions of their lengths. Moreover, the non-polar residues tend to vary rhythmically. To test the proposition that these sequences do indeed correspond to the “coiled-coils” long ago predicted on the basis of wide-angle X-ray diffraction studies, we constructed a detailed, atomic scale model of a part of this region. To this end, we fashioned three α-helical segments, each 29 residues long and corresponding to the designated sequences of the α, β and γ-chains, respectively. In each case we incorporated a pitch of approximately 200 Å. We were then able to fit the three helices together in the two possible combinations which yield a pseudo-3-fold axis. In either case all polar residues extend away from the parallel three-stranded rope, and almost all the non-polar side-chains are directed toward the interior. We also constructed a separate model showing how the six cysteines at each end of the proposed rod-like segment are best arranged. The co-ordinates from both models were collected and utilized in a computer-graphic Molecular Modeling System which can display features of the models selectively. Various projections were plotted automatically, some of which are reproduced here.     

The amino acid sequence of a 27-residue peptide released from the alpha-chain carboxy-terminus during the plasmic digestion of human fibrinogen.

The amino acid sequence of a 27-residue peptide released during the early stages of the plasmin digestion of human fibrinogen has been determined. The corresponding cyanogen bromide fragment has also been isolated from the purified α-chains of fibrinogen, although a separable fraction of those chains lack the fragment, evidently because of $\text{in}$$\text{vivo}$ degradation. The peptide is the carboxy-terminal segment of native α-chains.     

Amelogenin supramolecular assembly in nanospheres defined by a complex helix-coil-PPII helix 3D-structure

Tooth enamel, the hardest material in the human body, is formed within a self-assembled matrix consisting mostly of amelogenin proteins. Here we have determined the complete mouse amelogenin structure under physiological conditions and defined interactions between individual domains. NMR spectroscopy revealed four major amelogenin structural motifs, including an N-terminal assembly of four α-helical segments (S9-V19, T21-P33, Y39-W45, V53-Q56), an elongated random coil region interrupted by two 3(10) helices (～P60-Q117), an extended proline-rich PPII-helical region (P118-L165), and a charged hydrophilic C-terminus (L165-D180). HSQC experiments demonstrated ipsilateral interactions between terminal domains of individual amelogenin molecules, i.e. N-terminal interactions with corresponding N-termini and C-terminal interactions with corresponding C-termini, while the central random coil domain did not engage in interactions. Our HSQC spectra of the full-length amelogenin central domain region completely overlapped with spectra of the monomeric Amel-M fragment, suggesting that the central amelogenin coil region did not involve in assembly, even in assembled nanospheres. This finding was confirmed by analytical ultracentrifugation experiments. We conclude that under conditions resembling those found in the developing enamel protein matrix, amelogenin molecules form complex 3D-structures with N-terminal α-helix-like segments and C-terminal PPII-helices, which self-assemble through ipsilateral interactions at the N-terminus of the molecule.     

Deep-etch visualization of proteins involved in clathrin assembly

Assembly proteins were extracted from bovine brain clathrin-coated vesicles with 0.5 M Tris and purified by clathrin-Sepharose affinity chromatography, then adsorbed to mica and examined by freeze-etch electron microscopy. The fraction possessing maximal ability to promote clathrin polymerization, termed AP-2, was found to be a tripartite structure composed of a relatively large central mass flanked by two smaller mirror-symmetric appendages. Elastase treatment quantitatively removed the appendages and clipped 35 kD from the molecule's major approximately 105-kD polypeptides, indicating that the appendages are made from portions of these polypeptides. The remaining central masses no longer promote clathrin polymerization, suggesting that the appendages are somehow involved in the clathrin assembly reaction. The central masses are themselves relatively compact and brick-shaped, and are sufficiently large to contain two copies of the molecule's other major polypeptides (16- and 50-kD), as well as two copies of the approximately 70-kD protease-resistant portions of the major approximately 105-kD polypeptides. Thus the native molecule seems to be a dimeric, bilaterally symmetrical entity. Direct visualization of AP-2 binding to clathrin was accomplished by preparing mixtures of the two molecules in buffers that marginally inhibit AP-2 aggregation and cage assembly. This revealed numerous examples of AP-2 molecules binding to the so-called terminal domains of clathrin triskelions, consistent with earlier electron microscopic evidence that in fully assembled cages, the AP's attach centrally to inwardly-directed terminal domains of the clathrin molecule. This would place AP-2s between the clathrin coat and the enclosed membrane in whole coated vesicles. AP-2s linked to the membrane were also visualized by enzymatically removing the clathrin from brain coated vesicles, using purified 70 kD, uncoating ATPase plus ATP. This revealed several brick-shaped molecules attached to the vesicle membrane by short stalks. The exact stoichiometry of APs to clathrin in such vesicles, before and after uncoating, remains to be determined.     

Domainal organization of the molecules of Lys-plasminogen

The intramolecular melting of the human Lys-plasminogen and its different fragments were studied by the differential scanning microcalorimetry method. Thermodynamical analysis of melting curves showed that the Lys-plasminogen molecule consists of 7 domains. Five of them are formed by five homologeus regions of the polypeptide chain (kringle), while two domains are formed by the part of the polypeptide chain corresponding to the plasmin light chain. The domains included in the fragments seem to be rather independent, since fragmentation does not lead to noticeable changes of their stability in comparison to that of the intact molecule. It has been shown also that plasminogen-plasmin conversion is accompanied by structural transformation of the molecule which results in the destabilization of one of the light chain domains.     

Plasminogen-binding centers of molecules of fibrinogen, fibrin and products of their proteolysis

Using affinity chromatography, the binding of Lys-plasminogen to fibrinogen, fibrin and the consecutively formed products of their proteolysis was studied. The optimal conditions for this binding were elaborated, and the quantitative parameters of Lys-plasminogen binding to fibrinogen-Sepharose were determined. It was found that the interaction of Lys-plasminogen with fibrinogen- and fibrin-Sepharose is provided for by the lysine-binding sites of the proenzyme molecule. After partial hydrolysis of fibrinogen by plasmin, the amount of adsorbed plasminogen increases and the type of binding changes; part of the proenzyme molecules bind in the presence of 0.003 M 6-aminohexanoic acid, i.e., when lysine-binding sites appear to be blocked. A comparative study of plasminogen binding to fibrinogen fragments was carried out. The resistance of the complexes formed to the effect of 6-aminohexanoic acid and arginine competing for the binding sites was determined. The data obtained testify to the appearance of additional plasminogen-binding sites in the fibrinogen molecule during proteolysis. These sites are complementary for both lysine-and arginine-binding sites of the plasminogen molecule and are localized in the peripheral domains of the fibrinogen molecule.     

Mass spectrometric analysis of rat hemoglobin by FAB-overlapping: Primary structure of the α-major and of four β constitutive chains

• 1.1. The globin chain components of Sprague-Dawley rat hemoglobin were obtained by reverse-phase HPLC which showed the presence of two α-chain and four β-chains.
• 2.2. The accurate molecular weight of each globin chain was determined by means of electrospray mass spectrometry. Extensive mass spectrometric analysis on several enzymatic digests by fast atom bombardment mass spectrometry (FAB-overlapping) meant to determine the complete sequence of the α-major and of the four β-globins.
• 3.3. The primary structure of the α-major globin was found in agreement with literature data (Garrick et al., 1975 Biochem. J.149, 245–258; Chua et al., 1987).
• 4.4. Sequence analysis of the four β -globin chains showed that amino acid differences are restricted to two protein portions: the region 22–25 and 123–125, the remaining portions of the molecule being unchanged in the four globins. Furthermore, all the amino acid replacements correspond to single point DNA mutations and (with the exception of the substitution Asp 22 → Asn in the β₂-globin) involve uncharged substitutions.

     

Effect of α-MSH 11–13 (lysine-proline-valine) on fever in the rabbit

In previous experiments, α-MSH (1–13) and ACTH (1–24), which contains the α-MSH 1–13 amino acid sequence, were found to reduce fever after central and peripheral administration of low, non-hypothermic doses. Shorter molecules, including α-MSH 1–10, had no effect. The idea that the 11–13 amino acid sequence is important to the effect of the parent molecule was tested by giving lysine-proline-valine both centrally and peripherally to rabbits made febrile by IV administration of leukocytic pyrogen. The tripeptide reduced fever after both central (0.5–2.0 mg) and peripheral (2–200 mg) administration. It appears that the 11–13 sequence is part of the message sequence of α-MSH with regard to antipyretic activity. However, the lower potency relative to that of the parent molecule suggests that other portions of the molecule are essential to full expression of the antipyretic effect.     

Sequence of amino acids comprising the single intra-chain disulfide loop in the alpha-chain of human fibrinogen.

Amino acid sequence studies have revealed that the largest cyanogen bromide fragment from the α-chain of human fibrinogen contains two cysteine residues which are situated thirty residues apart and near the carboxy-terminal end of that fragment. In contrast to a recent report, no other cysteines exist in this region of the α-chain. The sequence has been compared to disulfide loops in the β- and γ-chains and some very marginal homology suggested.     

11S storage globulin from pumpkin seeds: Regularities of proteolysis by papain

Limited proteolysis of the α- and β-chains and deep cleavage of the αβ-subunits by the cooperative (one-by-one) mechanism was observed in the course of papain hydrolysis of cucurbitin, an 11S storage globulin from seeds of the pumpkin Cucurbita maxima. An independent analysis of the kinetics of the limited and cooperative proteolyses revealed that the reaction occurs in two successive steps. In the first step, limited proteolysis consisting of detachments of short terminal pep-tides from the α- and β-chains was observed. The cooperative proteolysis, which occurs as a pseudo-first order reaction, started at the second step. Therefore, the limited proteolysis at the first step plays a regulatory role, impacting the rate of deep degradation of cucurbitin molecules by the cooperative mechanism. Structural alterations of cucurbitin induced by limited proteolysis are suggested to generate its susceptibility to cooperative proteolysis. These alterations are tentatively discussed on the basis of the tertiary structure of the cucurbitin subunit pdb|2EVX in comparison with previously obtained data on features of degradation of soybean 11S globulin hydrolyzed by papain.     

Fibrin gel structure: influence of calcium and covalent cross-linking on the elasticity

Calcium ion causes the development of a higher elastic modulus in fibrinogen solutions clotted by thrombin. By also measuring the development of covalent crosslinks introduced by activated Factor XIII, we find that (a) calcium ion causes an overall increase of the modulus, (b) covalent crosslinking of α-chains causes an increase of the modulus, while (c) covalent crosslinking of γ-chains does not.     

Primary structure of the hemolglobin β-chain of Rose-ringed Parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin β-chain was established, completing the analysis of this hemoglobin. Comparisons with other avian β-chains show variations smaller than those for the corresponding α-chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform β-chain, and a total of 35 positions are affected by differences among all avian β-chains analyzed (versus 61 for the α-chains). At three positions, the Psittacula β-chain has residues unique to this species. Three α₁β₁ contacts are modified, by substitutions at positions β51, β116, and β125.     

Staining of glycoproteins in polyacrylamide and agarose gels with fluorescent lectins.

We describe a simple and sensitive method for staining of the carbohydrate moiety of glycoproteins in polyacrylamide or agarose electrophoretic gels. Gels are incubated in a solution of fluorescein-labeled concanavalin A. Following destaining with a neutral buffer, glycoproteins exhibit fluorescence under long-range ultraviolet light. Thus, the glucose/mannose containing β- and γ-chains of human fibrinogen give fluorescent bands, whereas the carbohydrate-free α-chain does not react. Less than 100 ng of hexose bound to fibrinogen β- or γ-chains could be detected. The procedure is suitable for staining of other carbohydrate residues in glycoproteins, which can be recognised by specific agglutinins, as shown by binding of fluorescein-labeled lectins from Ricinus communis to galactose residues of fibrinogen.     

The structure of the Cys-rich terminal domain of Hydra minicollagen, which is involved in disulfide networks of the nematocyst wall

The minicollagens found in the nematocysts of Hydra constitute a family of invertebrate collagens with unusual properties. They share a common modular architecture with a central collagen sequence ranging from 14 to 16 Gly-X-Y repeats flanked by polyproline/hydroxyproline stretches and short terminal domains that show a conserved cysteine pattern (CXXXCXXXCXXX-CXXXCC). The minicollagen cysteine-rich domains are believed to function in a switch of the disulfide connectivity from intra- to intermolecular bonds during maturation of the capsule wall. The solution structure of the C-terminal fragment including a minicollagen cysteine-rich domain of minicollagen-1 was determined in two independent groups by 1H NMR. The corresponding peptide comprising the last 24 residues of the molecule was produced synthetically and refolded by oxidation under low protein concentrations. Both presented structures are identical in their fold and disulfide connections (Cys2-Cys18, Cys6-Cys14, and Cys10-Cys19) revealing a robust structural motif that is supposed to serve as the polymerization module of the nematocyst capsule.     

The effects of stabilizing mutations in the central part of the α-chain of tropomyosin on the structural and functional properties of αβ-tropomyosin heterodimers

The effects of the D137L/G126R double mutation in the central part of the tropomyosin α-chain via the simultaneous replacement of two highly conserved non-canonical residues, viz., Asp137 and Gly126, by canonical residues Leu and Arg, respectively, on the properties of the αβ-tropomyosin heterodimer have been studied. It has been shown using circular dichroism that this mutation substantially increases the thermal stability of αβ-tropomyosin heterodimers, which, nevertheless, remains lower than that of αα-tropomyosin homodimers with these mutations in both α-chains. The stability of tropomyosin complexes with F-actin has also been studied by measuring the temperature dependences of their dissociation, which is detected by a decrease in light scattering. It has been revealed that αβ-tropomyosin heterodimers carrying the D137L/G126R mutation in the α-chain dissociate from the surface of actin filaments at a higher temperature than ββ-homodimers but at a lower temperature than αα-homodimers with these mutations in both α-chains. It has also been shown using the in vitro motility assay that D137L/G126R substitution in the α-chain increases the sliding velocity of regulated actin filaments in the case of αα-homodimers, while it noticeably decreases the velocity in the case of αβ-tropomyosin heterodimers. Thus, we can conclude that mutations in one of the chains of the tropomyosin dimeric molecule may have different effects on the properties of tropomyosin homodimers and heterodimers.     

Differences in the Subunit Structure of Human Fibrin formed by the Action of Arvin,Reptilase and Thrombin

INTEREST has focused recently on the clinical use of proteolytic enzymes similar in properties to thrombin which can directly cleave fibrinogen. Potentially the most important are arvin, derived from the venom of Agkistrodon rhodostoma and reptilase, isolated from the venom of Bothrops atrox. These only release fibrinopeptide A from fibrinogen^1–3, whereas thrombin cleaves fibrinopeptides A and B from fibrinogen to form fibrin. Thrombin also activates fibrin stabilizing factor (FSF) which introduces amide bonds between the subunits of soluble fibrin⁴. FSF rapidly forms covalent links between pairs of γ(C)-chains giving γ(C)-dimers and in a slower reaction α(A)-chains are linked to produce high molecular weight polymers⁵. Although reptilase, like thrombin, activates FSF⁶, arvin apparently does not, which would explain why the fibrin formed by arvin seems to be more friable than that produced by thrombin or reptilase⁷.     

Domains in human plasminogen

Calorimetric studies of intramolecular melting of human plasminogen and of its fragments under various solvent conditions show that the intact plasminogen molecule consists of seven compact co-operative subunits, which can be regarded as structural domains. Five of these domains are formed by the homologous regions, the kringles, two domains are formed by the C-terminal part of the polypeptide chain that is split at activation, forming the light chain in plasmin, while the initial 76 amino acid residue peptide does not form any compact co-operative structure. The specific influence of epsilon-aminocaproic acid on the stability of the first, the fourth and, to a lesser extent, on the second kringle domain, provides evidence that these three domains in plasminogen possess lysine-binding ability. The first four kringle domains are almost independent in the molecule, while the fifth interacts with that part of the light chain not included in either of the two domains of this chain. These two domains are of different size and co-operate strongly in plasminogen, but at its activation into plasmin they decooperate and the stability of the smaller domain, which is formed by the N-terminal part of the light chain, decreases significantly. Since the light chain is responsible for the proteolytic activity of plasmin, it becomes clear that the active site of this protein is composed of two domains, as is the case for other serine proteases.     

The pK of the amino terminal groups of carbonmonoxy- and deoxyhemoglobin measured by dinitrophenylation in phosphate buffers

The rate of reaction of the terminal valines of the α- and β-chains of hemoglobin with 1-fluoro-2,4-dinitrobenzene was followed spectrophotometrically at 353 nm. The variation with pH of the rate of dinitrophenylation of these groups was measured for both carbonmonoxy- and deoxyhemoglobin. In carbonmonoxyhemoglobin the results indicated a pK near 6.7 and 7.7 for the amino terminal groups of the two kinds of subunils, and were attributed to the α- and β-chains respectively. Removal of ligands produced an increase of 0.1 in both pK values and a decrease of 40% of the pH-independent kinetic constant for dinitrophenylation of the β-subunits. These modifications are due to the conformational changes associated with ligand binding in the system. In phosphate buffers the contribution to the Bohr effect of the amino terminal residues of either chains is negligible.