Physical and binding properties of large fragments of human serum albumin.

Three large fragments of human serum albumin were produced by peptic digestion of the native protein [Geisow & Beaven (1977) Biochem. J. 161, 619-625]. Fragment P44 represents residues 1-386 and fragments P29 and P31 represent residues 49-307 and residues 308-584 respectively of the albumin molecule. The large N-terminal fragment P44 has a similar percentage of alpha-helix to stored defatted albumin, although the alpha-helix content of all the fragments is significantly less than that of freshly prepared albumin. The fragment P44 appears to account for all the binding of the hydrophobic probe 8-anilinonaphthalene-1-sulphonate to albumin. N-Acetyl-L-tryptophan binds to this fragment and displaces one of the bound molecules of 8-anilinonaphthalene-1-sulphonate. Bilirubin binds to fragments P44 and P29, and the complexes show similar circular-dichroism spectra to that of the complex between bilirubin and whole albumin. These results are in agreement with affinity-labeling work on albumin with reactive ligands where substitution occurs in the N-terminal region of the molecule. The sharp conformational transitional transition in albumin which is observed between pH4 and 3.5 was absent from the fragments. This isomerization, usually called the N-F transition, probably occurs in intact albumin as a result of the unfolding or separation of the C-terminal third of the protein from the remainder of the molecule.     

Design of hydropathically complementary peptides for Big Endothelin affinity purification.

Molecular recognition between Big Endothelin (Big ET) and a computer generated peptide hydropathically complementary to Big ET[16-29] sequence has been studied by analytical high performance liquid affinity chromatography (HPLAC), circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Specific binding was observed between solid support immobilized complementary peptide and Big ET[1-38], [1-32], and [16-32], but not with Big ET fragments [1-21], [16-21], [22-32], and [22-38], obtained by chymotrypsin proteolytic degradation. Selectivity in the recognition process was clearly demonstrated by the ability of complementary peptide affinity column to purify the Big ET molecule from complex peptide mixtures, even when present in very low concentrations. Similar selectivity was evidenced with the Big ET fragment [16-32], [NH2-HLDIIWVNTPEHIVPYG-COOH] containing the entire hydropathically complementary sequence. Binding was followed by marked spectroscopic changes, as monitored by circular dichroism and one- and two-dimensional nuclear magnetic resonance experiments. The NMR spectra of the complementary peptides 1:1 mixture showed variations in the chemical shifts of proton resonances in several residues, both in the main chain (amide protons) and in the side chains (aliphatic and aromatic protons). These data support the hypothesis of a multilocalized type of interaction between complementary peptides, where many residues along the peptide chains participate in co-operative stabilizing contacts in the forming complex.     

Purification and characterization of peptic fragments derived from the carboxyl-terminal half of human albumin

Utilizing a combination of conventional and affinity-chromatographic procedures, we have purified four fragments of human albumin that were generated by controlled limited proteolysis with pepsin [0.3 mM albumin; 37°C; 10 min; pH 3.51; 4.2 mM octanoate; pepsin/albumin, 1:1000 (w/w)]. These fragments have a molecular weight range of 9200-17,000 Da. Amino acid compositions, N- and C-terminal sequences, molecular weights, and other internal markers were used to determine the location of these fragments within the parent molecule. All of the fragments were shown to be derived from the C-terminal half of human albumin. The presence of multiple pepsin-sensitive bonds near the C terminus of each fragment complicated the assignment of specific residue numbers to each fragment. Two pairs of similar peptides were identified: (A) those corresponding to a single-loop structure (residues 309–380 and 309–387) and (B) those containing multiple loops and intraloop cleavages [residues 309–(491–495) with 408–423 deleted]. Purification of these fragments without disulfide bond reduction confirms portions of the loop structure of human albumin and demonstrates increased susceptibility of two specific regions of the C-terminal half of the molecule to peptic digestion.     

Chromatin core particle obtained by selective cleavage of histones H3 and H4 by clostripain

A new chromatin core particle characterized by a half-proteolyzed octamer is obtained by controlled digestion of the native core particle by clostripain. The proteolyzed histones correspond to four polypeptide fragments which are tentatively assigned to H2A[4-129], H2B[1-125], H3[27-135] and H4[18-102] on the basis of electrophoretic evidence and the known specificity of clostripain for arginyl residues. Despite the loss of the N-terminal regions of histones H3 and H4, the partially proteolyzed core particle retains the structural conformation of the native one as shown by circular dichroism. As expected, this half-proteolyzed core particle presents an intermediate accessibility to polycations, such as spermidine, in comparison with that observed with the native core particle and a fully proteolyzed core particle. The latter includes the polypeptide fragments H2A[12-129], H2B[21-125], H3[27-135] and H4[20-102].     

Characterization of fragments of human albumin purified by Cibacron blue F3GA affinity chromatography.

Controlled limited proteolysis of human plasma albumin (0.3 mM; 37 degrees C; 15 min; pH 3.7) with pepsin [pepsin/albumin, 1:1000 (w/w)] in the presence of octanoic acid (4.2 mM) yields at least 14 fragments in the range of 5000--56000 Da. By utilizing a combination of conventional and affinity-chromatographic procedures, two fragments with mol. wts. 25000 and 27000 were purified to more than 99% homogeneity. The larger fragment consists of a continuous polypeptide chain and has been shown to contain the primary bilirubin-binding site. The small fragment contains an internal cleavage site. On the basis of amino acid compositions, N-terminal sequences, C-terminal sequences, molecular weights and other internal markers the locations of these fragments within the known sequence of human albumin were determined to be residues 49--308 for the 27000 Da peptide and 309--585 for the 25000 Da peptide. Peptide 309--585 contains an internal cleavage site and appears to be missing residues 408--423. These non-overlapping fragments should be useful for investigations of individual ligand-binding sites and for the determination of antigenic sites.     

RNA polymerase and gal repressor bind simultaneously and with DNA bending to the control region of the Escherichia coli galactose operon.

The Escherichia coli galactose operon contains an unusual array of closely spaced binding sites for proteins governing the expression from the two physically overlapping gal promoters. Based on studies of two gal promoter-up mutants we have previously suggested RNA-polymerase-induced DNA bending of gal promoter DNA. Here we present new evidence confirming and extending this interpretation. It was obtained by the circular permutation assay of gel electrophoretic mobility [Wu and Crothers (1984), Nature, 308, 509-513] applied to three analogous series of circularly permuted fragments derived from wild-type and two promoter-up mutant DNAs. The same circularly permuted DNA fragments have further been used to study the binding of gal repressor to its operator sites by electrophoretic mobility shift and by DNase I footprinting techniques. The main results are: (i) complexes carrying repressor either exclusively at the upstream operator O1 or at the downstream operator O2 exhibit different electrophoretic mobilities; (ii) binding to either one of the operators results in protein-induced DNA bending by the criteria of the circular permutation mobility assay; and (iii) occupation of both gal operators by gal repressor does not prevent cAMP-CRP-independent binding of RNA polymerase to the gal promoters, as judged by DNase I protection and gel retardation assays. The latter finding imposes constraints on any attempt to model the regulation of gal expression by assumed DNA-protein and protein-protein interactions.     

Structural characterization of two genetic variants of human serum albumin

In the present paper we report the structural characterization of two genetic mutants of human serum albumin: albumin Vanves, a very rare, electrophoretically fast variant of French origin, and albumin Verona, a slow-migrating variant which is the most frequently observed in Italy and which possesses the same electrophoretic mobility as albumin B. Both variants were isolated from the sera of healthy heterozygous subjects. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585) in both cases. The modified fragments were then isolated on a preparative scale by HPLC and subjected to tryptic digestion. Sequential analysis of the abnormal tryptic peptide, purified by HPLC, established the mutation responsible for albumin Vanves as 574 Lys----Asn and the molecular defect of albumin Verona as 570 Glu----Lys, both probably due to point mutations in the structural genes. The amino-acid substitutions found in albumins Verona and Vanves are consistent with the electrophoretic mobilities observed for the native proteins at pH 8.6.     

Two alloalbumins with identical electrophoretic mobility are produced by differently charged amino acid substitutions.

We describe the amino acid substitutions of albumins Sondrio and Paris 2, two slow moving variants of human serum albumin, which show an identical electrophoretic mobility on cellulose acetate at three different pH values. These variants have been found in several instances in a wide geographic area including Northern Italy and France. Both alloalbumins were isolated from the sera of heterozygous subjects. Isoelectric focusing analysis of CNBr fragments from the purified variants allowed us to localize the mutation of albumin Sondrio in fragment CNBr V (residues 330-446) and that of albumin Paris 2 in CNBr VII (residues 549-585). Sequential analysis of the variant CNBr VII established the molecular defect of albumin Paris 2 as 563 Asp----Asn. Fragments CNBr V from normal and Sondrio albumins were isolated on a preparative scale and subjected to tryptic and V8 proteinase digestion. Sequence determination of the abnormal tryptic and V8 peptides revealed that the variant arises from the substitution of glutamic acid 333 by lysine. Thus, a +1 change in the C-terminal region of the albumin molecule produces a variant with the same electrophoretic mobility as an alloalbumin with a +2 substitution in the central domain, suggesting a higher degree of exposure to the solvent of the C-terminal tailpiece. Both amino acid substitutions are consistent with a G----A transition in the first position of the corresponding codon in the structural gene.     

Binding of a high reactive heparin to human apolipoprotein E: identification of two heparin-binding domains

Ligand-blotting and dot-blotting procedures were used to investigate the binding of [125I]-heparin to apolipoprotein E, its thrombin fragments E22 (residues 1-191) and E12 (residues 192-299), and to nine apolipoprotein E synthetic fragments. E22 and E12 bound [125I] heparin indicating multiple heparin-binding domains. Synthetic peptides of apoE corresponding to residues 129-169, 139-169, and 144-169, but not 148-169, bound [125I] heparin suggesting that residues 144-147 (Leu-Arg-Lys-Arg) in E22 are important for binding. Peptide 202-243 and 211-243 but not 219-243 bound [125I] heparin suggesting that residues 211-218 (Gly-Glu-Arg-Leu-Arg-Ala-Arg-Met) comprise a portion of the E12 heparin-binding domain.     

The dimerization of half-molecule fragments of transferrin.

Partial proteolysis was used to prepare half-molecule fragments of hen ovotransferrin. N-Terminal and C-terminal fragments associate to form an N-terminal fragment-C-terminal fragment dimer. Variant forms of the N- and C-terminal fragments can be prepared in which a few amino acid residues are lacking from the C-terminal ends of the fragments. These variant fragments are partially or completely unable to associate; the suggestion that the molecular recognition sites are located in these C-terminal stretches of the N-terminal half-molecule (320-332) and of the C-terminal half-molecule (683-686) is in agreement with X-ray-crystallography data for human lactotransferrin [Anderson, Baker, Dodson, Norris, Rumball, Waters & Baker (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1769-1773].     

The semisynthesis of fragments corresponding to residues 66-104 of horse heart cytochrome c.

We describe the N epsilon-acetimidylation of horse heart cytochrome c with retention of biological activity, the cleavage of the modified protein by CNBr, the separation of the fragments, and their further side-chain protection. We describe the manipulation of the amino acid sequences of the fragments by stepwise semisynthetic methods. We have prepared fragments corresponding to residues 66-78 and 66-79 of the protein, as well as the [Asp66] analogue of fragment 66-79. We have prepared the natural sequence and the [o-fluoro-Phe82] analogue of the fragment corresponding to residues 81-104 of the protein, and the [N epsilon-trifluoroacetyl-Lys79], the [N epsilon-dinitrophenyl-Lys79] and the [S-acetamidomethyl-Cys79] analogues of fragment 79-104, and the [N epsilon-Cbz-Lys81] analogue of fragment 80-104. We have coupled back the fragments of natural sequence to form a semisynthetic fragment corresponding to residues 66-104 of the protein. Modified fragments were also coupled to give analogues of the 66-104-residue sequence. In every case the homoserine residue representing methionine-80 was removed from the C-terminus of the 66-80-residue fragment and replaced by methionine on the N-terminus of the 81-104 residue fragment during the preparation of the fragments for coupling. The semisynthetic fragments are ready for specific deprotection and further coupling. We have coupled one such fragment to the (1-65)-peptide to produce semisynthetic [Hse65]cytochrome c. The product has satisfactory characteristics on chemical analysis, and on assay of its biological activity.     

Analysis of the membrane-interacting domain of the sea urchin sperm adhesive protein bindin

We have investigated the domain of the bindin polypeptide that selectively associates with gel-phase phospholipid vesicles. We found that small trypsin fragments of bindin retain the ability to selectively associate with gel-phase vesicles. The primary amino acid sequence of bindin suggests that these peptides are derived from the central portion of the polypeptide between residues 77 and 126, which is the most hydrophobic region of bindin. We have also employed 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine (TID) and novel, radioiodinated, photoactivatable derivatives of the polar head group of phosphatidylethanolamine (ASD-PE and ASA-PE) to identify membrane-associated polypeptide segments after the transfer of radiolabel from the probe to the bindin polypeptide. After photolysis, bindin was selectively labeled only from probes incorporated in gel-phase vesicles. The labeling of bindin was much more efficient from the head group probes ASA-PE and ASD-PE (8 and 2% of the total label, respectively) in comparison to the hydrophobic probe TID (less than 0.02% of the total label), suggesting that bindin is localized within the polar part of the bilayer. Protease mapping experiments with V8 protease, trypsin, and endoprotease Lys-C suggest that some of the probe label is distributed along the amino-terminal portion of bindin between residues 1 and 76 and the rest of the label is restricted to the segments between residues 77 and 126 which also selectively bind to gel-phase vesicles. The carboxyl-terminal portion of bindin between residues 127 and 236 is not labeled.     

Immune response of rabbits to native G-actins

Actins are highly conserved proteins and are therefore claimed to be not very immunogenic without prior denaturation or chemical modification. We have obtained in rabbits high-titered antibodies to "native" G-actins from chicken and man, and assayed their cross-reaction using an enzyme immunoassay, Western blotting and immunohistochemistry. The antigens differ in their ability to induce antibody formation (chicken gizzard actin [(beta), gamma] greater than chicken skeletal actin [alpha] = human platelet actin [beta, (gamma)]). Antibodies to skeletal actin [alpha] are muscle-specific and mainly directed against the homologous region comprising the N-terminus (residues 1-226). Antibodies to gizzard actin [(beta), gamma] cross-react, to a lesser extent, with the alpha and beta, (gamma) isoforms. They show no regional specificity within the homologous antigen. Antibodies to the tryptic core fragment (residues 69-374) of skeletal actin react with fragments comprising the C-terminal part of muscular actins. Antibodies to platelet actin [beta, (gamma)] cross-react with muscular actins, recognizing not the native, but slightly degraded molecules. Platelet actin induces the formation of high-titered albumin antibodies for hitherto unknown reasons.     

Shape-selective recognition of a model Okazaki fragment by geometrically-constrained bis-distamycins

Okazaki fragments represent interesting targets for the design of anticancer drugs because of their selective occurrence during DNA replication, a process often elevated in aggressive malignancies. Structural studies have indicated a bend occurs in the helical axis at the junction region (JR) that joins the DNA duplex region (DDR) and the RNA-DNA hybrid duplex region (HDR) of model Okazaki fragments. To identify a structural motif that provides a shape complementary to the Okazaki fragment minor groove, we have investigated the binding of geometrically-constrained bis-distamycins to a model Okazaki fragment, [OKA], with a sequence derived from the genome of simian virus 40 (SV40). Both the JR and the DDR of [OKA] contain consecutive A/T base pairs that could accommodate distamycin binding. Of the six bis-distamycins selected for analysis, the two with a para configuration of the distamycins on the benzene or pyridine scaffold bound [OKA] tightly (Kd approximately 10(-6) M from gel-shift assays; Kd approximately 10(-8) M from deltaT(M)) while the four with a meta orientation did not bind. The two mono-distamycins studied also did not bind [OKA]. Molecular modeling of the complex between the para bis-distamycin MT-9 and [OKA] revealed MT-9 adopted an S- shape complementary to the minor groove of the model Okazaki fragment.     

Identification of the bile acid-binding region in the soy glycinin A1aB1b subunit

Soy glycinin has five major subunits which are classified into two groups according to their homology in amino acid sequences (group I, A1aB1b, A1bB2 and A2B1a; group II, A3B4 and A5A4B3). It has been reported that the peptide fragments derived from the A1a and A2 chains of the A1aB1b and A2B1a subunits had bile acid-binding ability and that the region of 114-161 residues of the A1a chain was responsible for this bile acid-binding ability. In this study, we constructed A1a, A3 and 9 deletion mutants of A1a lacking various numbers of residues at the C-terminus, and evaluated their bile acid-binding ability by a cholic acid-conjugated column and fluorescence analysis. The bile acid-binding ability of A1a was higher than that of A3 and there was a remarkable decrease in the bile acid-binding ability between the delta[138-291] and delta[130-291] mutants. The 130-138 region is rich in hydrophobic residues. In this regard, when we constructed the delta[129-134] mutant lacking six contiguous hydrophobic residues (VAWWMY) and evaluated its bile acid-binding ability, a similar remarkable decrease in the bile acid-binding ability was observed. These results indicate that the 129-134 residue region (VAWWMY) with high hydrophobicity was important for bile acid-binding of A1a.     

Two amino acids within the alpha4 helix of Galphai1 mediate coupling with 5-hydroxytryptamine1B receptors.

We previously reported that residues 299-318 in Galphai1 participate in the selective interaction between Galphai1 and the 5-hydroxytryptamine1B (5-HT1B) receptor (Bae, H., Anderson, K., Flood, L. A., Skiba, N. P., Hamm, H. E., and Graber, S. G. (1997) J. Biol. Chem. 272, 32071-32077). The present study more precisely defines which residues within this domain are critical for 5-HT1B receptor-mediated G protein activation. A series of Galphai1/Galphat chimeras and point mutations were reconstituted with Gbetagamma and Sf9 cell membranes containing the 5-HT1B receptor. Functional coupling to 5-HT1B receptors was assessed by 1) [35S]GTPgammaS binding and 2) agonist affinity shift assays. Replacement of the alpha4 helix of Galphai1 (residues 299-308) with the corresponding sequence from Galphat produced a chimera (Chi22) that only weakly coupled to the 5-HT1B receptor. In contrast, substitution of residues within the alpha4-beta6 loop region of Galphai1 (residues 309-318) with the corresponding sequence in Galphat either permitted full 5-HT1B receptor coupling to the chimera (Chi24) or only minimally reduced coupling to the chimeric protein (Chi25). Two mutations within the alpha4 helix of Galphai1 (Q304K and E308L) reduced agonist-stimulated [35S]GTPgammaS binding, and the effects of these mutations were additive. The opposite substitutions within Chi22 (K300Q and L304E) restored 5-HT1B receptor coupling, and again the effects of the two mutations were additive. Mutations of other residues within the alpha4 helix of Galphai1 had minimal to no effect on 5-HT1B coupling behavior. These data provide evidence that alpha4 helix residues in Galphai participate in directing specific receptor interactions and suggest that Gln304 and Glu308 of Galphai1 act in concert to mediate the ability of the 5-HT1B receptor to couple specifically to inhibitory G proteins.     

Stabilization of 30 S ribosomal subunits of Bacillus subtilis W168 by spermidine and magnesium ions

Purified prothrombin fragments 1 derived from normal (10-carboxyglutamyl) and dicoumarol-induced 7-, 5-, 2-, 1-, and 0-carboxyglutamyl prothrombins contained the same number of gamma-carboxyglutamyl residues as their respective parent molecules. The effect of gamma-carboxyglutamyl residues was more pronounced on the fragments 1 than on the prothrombins. Consequently, the pI values of the fragments 1 were very well differentiated, with normal fragment 1 focusing at pH 3.58, 7-carboxyglutamyl fragment 1 at 3.79, 5- at 3.97, and 2- at pH 4.29. Similarly, by agar gel electrophoresis, normal fragment 1 was the most mobile, followed by 7-, 5-, 2-, 1- and lastly 0-carboxyglutamyl fragment 1. Because of Ca2+ being bound to the carboxyglutamyl residues, the electrophoretic mobility of normal fragment 1, in the presence of Ca2+, was reduced the most, followed by 7-, 5- and then 2-carboxyglutamyl fragment 1, while the mobilities of the 1- and 0-carboxyglutamyl fragments 1 were not affected. In contrast to their parent molecules, all of the fragments 1 in the presence of EDTA gave negative immunoprecipitation reactions against antibodies produced against normal prothrombin. In the presence of Ca2+, conversely, the fragments 1 containing comparable amounts of antigenic activity all gave positive reactions. However, the intensity of the immunoprecipitates varied, as normal fragment 1 gave the most prominent immunoprecipitation reaction, consecutively followed by 7-, 5-, 2-, 1- and lastly 0-carboxyglutamyl fragment 1 where the precipitation was so faint that it was hardly visible.     

Structural characterization of a chain termination mutant of human serum albumin

Mutant forms of human serum albumin have been detected on the basis of their abnormal electrophoretic mobility which is either faster or slower than that of normal albumin. In the present work we have studied the structure of a slow variant, referred to as albumin Ge/Ct, in order to define the cause of its genetic abnormality. The protein was isolated from the serum of a young healthy woman homozygous for the variant. Analysis of CNBr fragments by isoelectric focusing allowed us to localize the mutation to the COOH-terminal region of the molecule (residues 549-585). This fragment was isolated on a preparative scale and subjected to tryptic digestion. All tryptic peptides were purified by reverse-phase high performance liquid chromatography and characterized. Sequential analysis of three abnormal peptides revealed that albumin Ge/Ct has a shortened chain with the following COOH-terminal sequence: Leu-Val-Ala-Ala-Ser-Lys580-Leu-Pro. The presence of an additional lysine residue accounts for the electrophoretic behavior of the variant. It is likely that the variant may be caused by a single base deletion in the structural gene, a Cyt in mRNA codon 580, and the consequent shift in reading frame.     

Studies on actin fragments obtained by digestion with thrombin,BNPS-skatole and nitrothiocyanobenzoic acid

We have isolated fragments of actin prepared by thrombic digestion (residues 40–374 [I], and 114–374 [II]), BNPS-skatole cleavage (residues 87–339 [III]) and nitrothiocyanobenzoic acid treatment (residues 10–216 [IV]) using preparative electrophoretic and chromatographic techniques. Analysis of the filament-forming and tropomyosin-binding properties of demonstrably homogeneous fragments revealed that only fragments I and II oculd form F-actin-like filaments after attempted renaturation, and that only fragment I could bind to tropomyosin. These results in addition to our previous studies on actin fragments and chemically-modified intact actin suggest that residues 1–86 and 340–374 are not required for F-actin filament formation, whereas residues 70–86 and/or 340–374 are essential for tropomyosin-binding activity.     

Fragmentation and reduction of bovine secretory component. Preparation of a biologically active fragment and some evidence for a multiple-domain structure.

A tryptic fragment (A) of Mr 25000 was prepared from bovine secretory component. The fragment binds polymeric immunoglobulin, although 9 times less effectively than secretory component on a molar basis. The fragment has four buried half-cystine residues and two exposed half-cystine residues. It gives rise to two fragments of Mr 11000-13000 on prolonged digestion with trypsin, and these do not bind polymeric immunoglobulin. It is proposed that fragment A consists of two immunoglobulin-like domains. Bovine secretory component was found to have 9-11 buried half-cystine residues and four exposed half-cystine residues. Reduction and alkylation of the exposed residues decreases the binding of polymeric immunoglobulin by 3-fold. Initial tryptic cleavage of bovine secretory component gives a fragment (Q) disulphide-bridged to a further fragment (T). Fragment Q is similar in size to a three-domain immunoglobulin fragment, and fragment T is similar in size to a two-domain immunoglobulin fragment. The two-domain fragment A is derived from fragment Q by further tryptic cleavage. The results are compatible with the proposal by Mostov, Friedlander & Blobel [(1984) Nature (London) 308, 37-43] that secretory component consists of multiple immunoglobulin-like domains. The results also indicate that optimal binding of polymeric immunoglobulin involves several domains stabilized by an exposed disulphide bridge.