Hierarchic organization of globular proteins: Experimental studies on thermolysin

Previous studies from this laboratory have shown that the thermolysin fragment 121–316, comprising entirely the“all-α” COOH-terminal structural domain 158–316, as well as fragment 206–316 (fragment FII) are able to refold into a native-like, stable structure independently from the rest of the protein molecule. The present report describes conformational properties of fragments 228–316 and 255–316 obtained by chemical and enzymatic cleavage of fragment FII, respectively. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultra-violet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. Melting curves of the secondary structure of the fragments show cooperativity with a temperature of half-denaturationT _mof 65–66°C. The results of this study provide evidence that it is possible to isolate stable supersecondary structures (folding units) of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain 158–316 of thermolysin.     

Folding of thermolysin fragments. Correlation between conformational stability and antigenicity of carboxyl-terminal fragments

Circular dichroism (CD) and immunochemical measurements have been used to examine conformational properties of COOH-terminal fragments 121-316, 206-316 and 225(226)-316 of thermolysin, and to compare these properties to those of native thermolysin and thermolysin S, the stable partially active two-fragment complex composed of fragments 5-224(225) and 225(226)-316. In aqueous solution at neutral pH, all the COOH-terminal fragments attain a native-like conformation, as judged both by the content of secondary structure deduced from far-ultraviolet CD spectra and by the recognition of rabbit polyclonal antibodies specific for the COOH-terminal region in native thermolysin. The three fragments showed reversible cooperative unfolding transitions mediated by both heat and guanidine hydrochloride (Gdn X HCl). The phase transition curves were analyzed for Tm (temperature of half-denaturation) and Gibbs free energies (delta GD) of unfolding from native to denatured state. The observed order of thermal stability is 225(226)-316 less than or equal to 206-316 less than 121-316 less than thermolysin S less than thermolysin. The ranking of delta GD values for the three fragments correlates with the size of each fragment. Competitive binding studies by radioimmunoassay using 14C-labeled thermolysin and affinity purified antibodies specific for native antigenic determinants in segment 206-316 of native thermolysin indicate that the COOH-terminal fragments adopt native-like conformations which are in equilibrium with non-native conformations. These equilibria are shifted towards the native state as the fragment size increases from 225(226)-316, to 206-316, to 121-316. Fragment 225(226)-316, when combined with fragment 5-224(225) in the thermolysin S complex, adopts a more stable native-like conformation and becomes much more antigenic. It has been shown that the degree of antigenicity of COOH-terminal fragments towards thermolysin antibodies correlates directly with their conformational stability. The results of this study are discussed in relation to the recently proposed correlation between antigenicity and segmental mobility of globular proteins.     

Synthesis and conformational studies of peptides encompassing the carboxy-terminal helix of thermolysin

The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val- Gly-Val-Lys, corresponding to sequence 296-316 of thermolysin and thus encompassing the COOH-terminal helical segment 301-312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296-316 was then cleaved with trypsin at Lys307 and Staphylococcus aureus V8 protease at Glu302, producing the additional fragments 296-307, 308-316, 296-302, and 303-316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296-316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301-312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296-316 attains in 90% aqueous trifluoroethanol the same percentage (approximately 58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296-316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule.     

Folding of thermolysin fragments. Hydrodynamic properties of isolated domains and subdomains

Sedimentation analysis in the analytical ultracentrifuge has been used to characterize the size and shape of thermolysin and a number of its fragments obtained by chemical or enzymatic cleavage of the protein. Four fragments (121-316, 206-316, 225/226-316 and 255-316) originate from the C-terminal domain, and two (1-155 and 1-205) from the N-terminal domain of the intact molecule. In aqueous solution at neutral pH the hydrodynamic properties of the C-terminal fragments, except 255-316, are consistent with compact homogeneous monomers. Fragment 255-316 is a monomeric species below 0.08 mg/ml concentration and forms a dimer above this concentration. Dimerization does not lead to changes in fragment conformation, as determined by far-ultraviolet circular dichroic measurements, but to an increase of 5.6 degrees C (to 68.2 degrees C at 1.0 mg/ml) in the temperature for thermal unfolding and a corresponding increase of 4.6 kJ/mol in the free energy of unfolding. Fragments derived from the N-terminal domain show a strong tendency to form high-molecular-mass aggregates. Previous experiments utilizing circular dichroic measurements and antibody binding data suggested that the C-terminal fragments listed above are able to refold in aqueous solution at neutral pH into a stable conformation of native-like characteristics [Dalzoppo, D., Vita, C. & Fontana, A. (1985) J. Mol. Biol. 182, 331-340] (and references cited therein). Present data establish that all these C-terminal fragments are globular monomeric species in solution (at concentrations approximately 0.1 mg/ml) and thus represent 'isolated' domains (or subdomains) with intrinsic conformational stability typical of small globular proteins.     

Independent folding of the carboxyl-terminal fragment 228-316 of thermolysin

The COOH-terminal fragment 206-316 of thermolysin was shown previously to maintain a stable folded structure in aqueous solution comparable to that of the corresponding region in native thermolysin and thus to possess protein domain characteristics [Fontana, A., Vita, C., & Chaiken, I. M. (1983) Biopolymers 22, 69-78]. In order to study the effect of polypeptide chain length on folding and stability of an isolated domain, the 111 amino acid residue fragment was shortened on the NH2-terminal side by removal of a 22-residue segment. Treatment of fragment 206-316 with hydroxylamine under alkaline conditions permitted selective cleavage of the Asn227-Gly228 peptide bond, and from the reaction mixture fragment 228-316 was isolated in homogeneous form. This fragment appeared to attain in aqueous solution the folding properties of the corresponding segment in the intact protein, as indicated by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunological properties. Thus, double-immunodiffusion analyses showed that fragment 228-316 is able to recognize and precipitate anti-thermolysin antibodies raised in rabbits with native thermolysin as immunogen. The fragment displayed fully reversible and cooperative conformational transitions mediated by pH, heat, and guanidine hydrochloride (Gdn.HCl), as expected for a globular protein species. Thermal denaturation of the fragment in aqueous solution at pH 7.8 showed a Tm of 66 degrees C and the Gdn.HCl-mediated unfolding a midpoint transition at 2.2 M denaturant concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Domain characteristics of the carboxyl-terminal fragment 206-316 of thermolysin: immunochemical studies

The extent of nativeness of the stable conformation of the thermolysin fragment containing the carboxyl-terminal third of the protein (from residues 206 to 316, denoted fragment FII) was examined by its immunogenic and antigenic characteristics. Antisera elicited in rabbits by either intact thermolysin or fragment FII were fractionated serially on two affinity columns, containing either the isolated fragment or intact protein. Both sera gave rise to substantial antibody populations which recognized the fragment FII region in native thermolysin. The relative affinities of these specific antibodies for isolated fragment FII and intact thermolysin were evaluated by radioimmunoassay, by assessing the relative extents of competition by these for binding of either 14C-labeled thermolysin or 14C-labeled fragment FII to each antibody population. Competition by fragment FII was substantial, though generally weaker than that for intact thermolysin, for antibody binding of both labeled antigens. The data demonstrate that the stable structure of fragment FII as observed spectroscopically likely is one which possesses conformational features similar to those of this region in intact thermolysin, but with perhaps less conformational rigidity. The results support the view that the region of thermolysin composed primarily of residues 206-316 is a conformational domain of the intact protein and that isolated fragment FII retains domain-like characteristics of stable and native-like conformation.     

Domain characteristics of the carboxyl-terminal fragment 206-316 of thermolysin

The existence, location, and characteristics of protein domains have been investigated by studying the structural properties of the carboxyl-terminal cyanogen bromide fragment 206–316 of thermolysin. As judged by far-uv CD measurements in aqueous solution under neutral conditions, the fragment attains a substantial degree of α-helical structure comparable to that exhibited by the corresponding region in native thermolysin. By radioimmunoassay techniques, a considerable degree of nativeness of fragment conformation has been deduced from comparison of the relative affinities of thermolysin and fragment 206–316 for antibodies specific for the 206–316 region in the intact protein. The fragment shows noteworthy stability to protein denaturants. The overall spectroscopic and immunochemical data suggest that fragment 206–316 is able to refold into a stable, nativelike structure independently from the rest of the molecule, thus providing support for the view that this fragment may contain a substantial part, if not all, of a protein domain structure.     

Semisynthesis of carboxy-terminal fragments of thermolysin

Enzyme-catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparing COOH-terminal fragments of the metallo-protease thermolysin. Fragment 205-316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively with Staphylococcus aureus V8 protease at the level of the single Glu302 residue into fragments 205-302 and 303-316. Upon incubation for 2-5 days of fragment 205-302 with a 5-fold excess of peptide 303-316, prepared by solid phase synthesis, with V8-protease in 0.1 M ammonium acetate, pH 6.0, containing 50% glycerol as organic cosolvent, enzyme-catalyzed reformation of the peptide bond was achieved in yields up to approximately 90% (based on fragment 205-302). The same procedure was used to prepare also the thermolysin fragments 205-315 and 205-311 by enzymatic coupling of fragment 205-302 to peptide 303-315 or 303-311, these last prepared by proteolytic digestion of the synthetic peptide 303-316. This procedure of semisynthesis opens up an approach for the site-directed modification of the tetrahelical COOH-terminal fragment 205-316 of thermolysin at the level of its helical segment encompassing residues 301-312 in the native, intact protein. Such analogs will be useful for examining structure-folding-stability relationships in this folded fragment possessing domain-like characteristics.     

Characterization of a 48-kDa nucleic-acid-binding fragment of nucleolin

Nucleolin (C23 or 100 kDa) is an abundant single-stranded-nucleic-acid-binding nucleolar protein proposed to be involved in the early stages of ribosome assembly. A stable 48-kDa fragment of the protein was produced either by proteolytic activity present in nucleolar extracts or by added trypsin. The hydrodynamic and DNA-binding properties of the 48-kDa fragment were compared with the parent molecule. Protein sequencing indicated that the fragment begins at residue 282; amino acid composition of the fragment including 10-12 methylated arginine residues suggested that the fragment contains the entire COOH-terminal two-thirds of the protein. The 48-kDa fragment was more globular than nucleolin, as indicated by a lower frictional coefficient (1.3 vs. 2.0 for nucleolin) and a similar sedimentation coefficient (4.1-4.3S) in spite of the reduction in molecular mass. Although the 48-kDa fragment retained single-stranded-DNA-binding activity, the binding capacity and the ability to reassociate DNA were about fivefold and sixfold lower, respectively, than nucleolin. Similarly, tenfold higher concentrations of the 48-kDa fragment were required to form nucleoprotein aggregates. These results suggest that nucleolin contains a globular COOH-terminal domain for nucleic-acid binding and a NH2-terminal region which is involved in protein-protein interactions and modulating nucleic-acid-binding activity.     

Substructure of the myosin molecule. 3. Preparation of single-headed derivatives of myosin

Native myosin has two globular regions attached to an a-helical rod. Papain is able to cleave the globular “heads” from the rod, leading to the formation of a variety of single-headed molecules. Among these subfragments are isolated globules (HMM S-1) and single globules attached to helical rods of lengths varying from 500 to 1400 Å. These subfragments can be separated from the other products of the proteolytic digestion by salt elution from a DEAE-cellulose column. Some of the properties of single-headed heavy meromyosin and myosin have been determined by hydrodynamic methods, and shadow-cast preparations of these subfragments have been directly visualized by electron microscopy. In addition to providing further evidence for the presence of two similar halves in myosin, these new subfragments can be used in studies related to the question of why myosin has two active “heads”.     

Reconstitution of a native-like SH2 domain from disordered peptide fragments examined by multidimensional heteronuclear NMR

The N-terminal SH2 domain from the p85alpha subunit of phosphatidylinositol 3' kinase is cleaved specifically into 9- and 5-kD fragments by limited proteolytic digestion with trypsin. The noncovalent SH2 domain complex and its constituent tryptic peptides have been investigated using high-resolution heteronuclear magnetic resonance (NMR). These studies have established the viability of the SH2 domain as a fragment complementation system. The individual peptide fragments are predominantly unstructured in solution. In contrast, the noncovalent 9-kD + 5-kD complex shows a native-like (1)H-(15)N HSQC spectrum, demonstrating that the two fragments fold into a native-like structure on binding. Chemical shift analysis of the noncovalent complex compared to the native SH2 domain reveals that the highest degree of perturbation in the structure occurs at the cleavage site within a flexible loop and along the hydrophobic interface between the two peptide fragments. Mapping of these chemical shift changes on the structure of the domain reveals changes consistent with the reduction in affinity for the target peptide ligand observed in the noncovalent complex relative to the intact protein. The 5-kD fragment of the homologous Src protein is incapable of structurally complementing the p85 9-kD fragment, either in complex formation or in the context of the full-length protein. These high-resolution structural studies of the SH2 domain fragment complementation features establish the suitability of the system for further protein-folding and design studies.     

Domain characteristics of the carboxyl-terminal fragment 206–316 of thermolysin: pH and ionic strength dependence of conformation

The pH and ionic strength dependence of conformation of the COOH-terminal fragment 206–316 (fragment FII) of thermolysin was monitored by far-uv CD and difference absorption measurements. This fragment was shown previously to possess the properties of a protein domain, i.e., able to refold into a stable nativelike structure [Fontana, A., Vita, C. & Chaiken, I. M. (1983) Biopolymers 22 , 69–78]. Analysis of the CD spectra in the pH range of 1–12 indicated that near pH 1, the conformation of fragment FII appears to be in an intermediate state (H) between the fully unfolded one (U) [the guanidine hydrochloride (Gdn · HCl)-induced unfolded state] and the nativelike state (N—that attained at neutral pH). Quantitative analysis of secondary structure from CD spectra revealed that state H at 4°C is characterized by some 30% α-helical structure, compared to 47% for state N. The heat- and Gdn · HCl-mediated unfolding transitions of state H were fully reversible and characterized by little cooperativity, which is taken as an indication that state H corresponds to several species possessing different, and low, conformational stabilities. The midpoint transition from state H to N occurs near pH 2.5, implying that the acid transition results from the titration of carboxyl groups of the fragment with anomalously low pK, as would be expected for groups involved in specific salt bridges. Fragment FII at pH 1 (state H) may be induced to exhibit nearly the same degree of helicity of state N simply by increasing the ionic strength of the solution, thus reducing the repulsive interactions between positive charges within the highly charged fragment at pH 1. The results obtained emphasize the role of electrostatic interactions in the folding and stability of fragment FII and suggest a mechanism of folding of the fragment from U to N involving an intermediate state characterized by an assembly of fluctuating α-helices.     

Enhanced Protein Thermostability by Ala --> Aib Replacement

The introduction into peptide chains of alpha-aminoisobutyric acid (Aib) has proven to stabilize the helical structure in short peptides by restricting the available range of polypeptide backbone conformations. In order to evaluate the potential stabilizing effect of Aib at the protein level, we have studied the conformational and stability properties of Aib-containing analogs of the carboxy-terminal subdomain 255-316 of thermolysin. Previous NMR studies have shown that this disulfide-free 62-residue fragment forms a dimer in solution and that the global 3D structure of each monomer (3 alpha-helices encompassing residues 260-274, 281-295, and 301-311) is largely coincident with that of the corresponding region in the X-ray structure of intact thermolysin. The Aib analogs of fragment 255-316 were prepared by a semisynthetic approach in which the natural fragment 255-316 was coupled to synthetic analogs of peptide 303-316 using V8-protease in 50% (v/v) aqueous glycerol [De Filippis, V., and Fontana, A. (1990) Int. J. Pept. Protein Res. 35, 219-227]. The Ala residue in position 304, 309, or 312 of fragment 255-316 was replaced by Aib, leading to the singly substituted fragments Ala304Aib, Ala309Aib, and Ala312Aib. Moreover, fragment Ala304Aib/Ala309Aib with a double Ala --> Aib exchange in positions 304 and 309 was produced. Far- and near-UV circular dichroism measurements demonstrated that both secondary and tertiary structures of the natural fragment 255-316 are fully retained upon Ala --> Aib substitution(s). Thermal unfolding measurements, carried out by recording the ellipticity at 222 nm upon heating, showed that the melting temperatures (Tm) of analogs Ala304Aib and Ala309Aib were 2.2 and 5.4 °C higher than that of the Ala-containing natural species (Tm = 63.5 °C), respectively, whereas the Tm of the Ala312Aib analog was lowered by -0.6 °C. The enhanced stability of the Ala304Aib analog can be quantitatively explained on the basis of a reduced backbone entropy of unfolding due to the restriction of the conformational space allowed to Aib in respect to Ala, while the larger stabilization observed for the Ala309Aib analog can be accounted for by both entropic and hydrophobic effects. In fact, whereas Ala304 is a surface residue, Ala309 is shielded from the solvent, and thus the enhanced stability of fragment Ala309Aib is also due to the burial of an additional -CH3 group with respect to the natural fragment. The slightly destabilizing effect of the Ala --> Aib exchange in position 312 appears to derive from unfavorable strain energy effects, since phi and psi values for Ala312 are out of the allowed angles for Aib. Of interest, the simultaneous incorporation of Aib at positions 304 and 309 leads to a significant and additive increase of +8 °C in Tm. The results of this study indicate that the rational incorporation of Aib into a polypeptide chain can be a general procedure to significantly stabilize proteins.     

Sulfatide-binding domain of the laminin A chain

A sulfatide-binding site on the globular end region of the long arm of laminin has been identified. Following proteolytic digestion with thermolysin, an intact fragment of the laminin A chain carboxyl-terminal domain exhibiting sulfatide-binding activity was isolated using gel filtration and heparin affinity chromatography. This fragment is composed of two peptides that are covalently linked by at least one disulfide bond and encompass the carboxyl-terminal 394 amino acids of the A chain. The clusters of charged residues in the primary structure of these fragments are sufficient for heparin-binding activity but not sulfatide binding since reduction and alkylation of the fragments abolished sulfatide binding under conditions in which heparin binding was retained. Thus, sulfatide binding requires an intact three-dimensional structure. The iodinated fragment bound to A2058 melanoma and T47D breast carcinoma cells and could be displaced by the unlabeled fragment. Based on incorporation of [35S] sulfate, both cell lines synthesize sulfated glycolipids that bind to laminin. In agreement with previous data that indicate a synergistic interaction of the sulfatide-binding domain with other laminin-binding sites on melanoma cells during attachment, the isolated sulfatide-binding fragment significantly inhibited interaction of labeled intact laminin with melanoma and breast carcinoma cells in direct binding assays.     

Serological homologies between H1 degrees and H5 include the carboxyl-terminal domain

We have reported previously that antibodies to chicken H5 and antibodies to H1 both cross-react with mammalian H1 degree (Mura, C. V., and Stollar, B. D. (1981) J. Biol. Chem. 256, 9767-9769). The antigenic sites in H1 degree recognized by these antibodies were analyzed using immunoblotting. Peptides of H1 degree were prepared by partial digestion with acetic acid and tested for reactivity with: 1) antibodies induced by H5 alone, which reacted primarily with the central globular region of H5; 2) antibodies induced by H5 X RNA complexes, which reacted with this domain as well as the basic COOH-terminal domain; and 3) antiserum to calf thymus H1. Anti-H5 antibodies (anti-globular region) cross-reacted with H1 degree peptides that co-migrated with peptides of H5 that contain the globular region, but did not cross-react with H1. Anti-H5/RNA antibodies (anti-globular + anti-COOH-terminal) cross-reacted with these peptides and, in addition, with a lysine-rich H1 degree peptide that co-migrated with the basic COOH-terminal H5 peptide. This H1 degree peptide, but not the putative globular H1 degree peptides, was also recognized by an antiserum to calf H1 which was primarily reactive with the large, COOH-terminal N-bromosuccinimide fragment of calf H1. A weaker cross-reaction between this antiserum and the carboxyl-terminal domain of H5 could be visualized when large quantities of H5 were used in immunoblots. The results indicate that structural homologies between H5 and H1 degree extend beyond the globular region and into the lysine-rich carboxyl-terminal domain. Antigenic homologies between H1 degree and H1 are also at least partially localized in this domain. H1 degree is serologically intermediate between H5 and H1.     

Differences in domain structure between human fibronectins isolated from plasma and from culture supernatants of normal and transformed fibroblasts. Studies with domain-specific antibodies

The domain structure of human fibronectins isolated from plasma and from the conditioned medium of normal and transformed fibroblasts was analyzed by limited proteolysis and S-cyanylation followed by immunostaining of released fragments with five kinds of antibodies, each specific for one functional domain. The results indicate that all three human fibronectins are composed of the same set of functional domains aligned in the same topological order. However, the following clear differences were found in specific fragments released from plasma fibronectin (pFN) and those released from fibronectin of normal (N-cFN) and transformed fibroblasts (T-cFN). Two fragments (Mr = 70,000 and 60,000) were released from the COOH-terminal region of pFN by cathepsin D. These fragments represent the COOH-terminal heparin-binding (Hep-2) and fibrin-binding (Fib-2) domains. The corresponding fragments released from both N-cFN and T-cFN by cathepsin D had much larger molecular weights (Mr = 100,000 and 83,000-74,000) than those from pFN. The fragments from the Fib-2 domain alone, however, did not show any difference among all three FNs. The internal region, from the gelatin-binding (Gel) domain through the Hep-2 domain, of N-cFN and T-cFN was released as a Mr = 210,000 fragment upon mild trypsin digestion. The corresponding fragment from pFN was released as a Mr = 185,000 fragment. The COOH-terminal half, including the Hep-2 domain, of both N-cFN and T-cFN was released by S-cyanylation as Mr = 160,000-145,000 fragments, which are 25,000-20,000 larger than the corresponding fragments of pFN. These results clearly indicate that the Hep-2 domain of N-cFN and T-cFN is 30,000-20,000 daltons larger than the same domain of pFN. Although various fragments released from N-cFN and T-cFN showed a similar pattern, there were minor differences. Thermolysin fragments derived from the Hep-2 domain of N-cFN were clearly distinguishable from those from T-cFN. Three groups of fragments with Mr = 40,000, 35,000-32,000, and 30,000 were released from N-cFN, while only the 35,000-32,000 fragment was released from T-cFN. The Mr = 44,000/60,000 thermolysin fragments representing the Gel domain and the Mr = 210,000/165,000 tryptic fragments representing the internal domains of T-cFN were slightly, but consistently, larger than those of N-cFN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Further characterization of a conserved actin-binding 27-kDa fragment of actinogelin and alpha-actinins and mapping of their binding sites on the actin molecule by chemical cross-linking

A conserved actin-binding domain (Mr = 27,000) of rat hepatic actinogelin, rat skeletal muscle, and chicken gizzard alpha-actinins (Mimura, N., and Asano, A. (1986) J. Biol. Chem. 261, 10680-10687) was separated into two components having different isoelectric points (peptides A and B) by chromatofocusing. Thermolysin digestion of peptide A generated peptide B with concomitant loss of peptide A. Amino acid compositions and tryptic maps of peptides A and B also demonstrated that peptide A is a precursor of peptide B upon thermolysin digestion. All of peptides A and B retained the activity to bind with F-actin competitively to each other. By the gel-filtration method, it was also shown that the native actin-binding 27-kDa fragments are monomeric and globular. The non-actin-binding 50- or 53.5-kDa fragment of actinogelin/alpha-actinins was, however, found to be asymmetric and dimeric in the native state. Chemical cross-linking of the 27-kDa fragment with F-actin with a water-soluble carbodiimide produced at least four different complexes (I-IV). Chemical cleaving analysis of the cross-linked products (complexes I and II) indicated that the 27-kDa fragment possesses two possible binding sites on actin at the NH2-terminal residues 1-12 (for complex I) and at residues spanning 86-119 or 123 (for complex II).     

Limited proteolysis of lactose permease from Escherichia coli

Escherichia coli lactose permease (also referred to as lactose carrier) is an integral protein of the cytoplasmic membrane. Using lactose permease either radiolabeled biosynthetically in plasmid-bearing E. coli minicells or radioalkylated post-synthetically by chemical modification, we have determined sites on the membrane-bound protein accessible to proteolytic attack and we have characterized several high-molecular-mass products. The most prominent polypeptide obtained from lactose permease radiolabeled biosynthetically is observed after digestion with different proteases. The fragment produced by thermolysin was shown to contain the intact N-terminus and to extend into the region around amino acid residue 140 which, according to secondary structure models, is presumed to be less tightly folded than the rest of the molecule. Evidence is presented that the corresponding fragments obtained after digestion with several other proteases also originate from the N-terminal part of the protein. This N-terminal segment of the lactose carrier is resistant to proteolytic digestion even in the presence of non-ionic detergents and it may represent a tightly folded domain. Additional proteolytic cleavage sites located C-terminal of the Cys148 residue can be inferred.     

Mammalian heterogeneous nuclear ribonucleoprotein A1. Nucleic acid binding properties of the COOH-terminal domain

A1 is a core protein of the eukaryotic heterogeneous nuclear ribonucleoprotein complex and is under study here as a prototype single-stranded nucleic acid-binding protein. A1 is a two-domain protein, NH2-terminal and COOH-terminal, with highly conserved primary structure among vertebrate homologues sequenced to date. It is well documented that the NH2-terminal domain has single-stranded DNA and RNA binding activity. We prepared a proteolytic fragment of rat A1 representing the COOH-terminal one-third of the intact protein, the region previously termed COOH-terminal domain. This purified fragment of 133 amino acids binds to DNA and also binds tightly to the fluorescent reporter poly(ethenoadenylate), which is used to access binding parameters. In solution with 0.41 M NaCl, the equilibrium constant is similar to that observed with A1 itself, and binding is cooperative. The purified COOH-terminal fragment can be photochemically cross-linked to bound nucleic acid, confirming that COOH-terminal fragment residues are in close contact with the polynucleotide lattice. These binding results with isolated COOH-terminal fragment indicate that the COOH-terminal domain in intact A1 can contribute directly to binding properties. Contact between both COOH-terminal domain and NH2-terminal domain residues in an intact A1:poly(8-azidoadenylate) complex was confirmed by photochemical cross-linking.     

Domain structure of the HSC70 cochaperone, HIP.

The domain structure of the HSC70-interacting protein (HIP), a 43-kDa cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor, has been probed by limited proteolysis and biophysical and biochemical approaches. HIP proteolysis by thrombin and chymotrypsin generates essentially two fragments, an NH2-terminal fragment of 25 kDa (N25) and a COOH-terminal fragment of 18 kDa (C18) that appear to be well folded and stable as indicated by circular dichroism and recombinant expression in Escherichia coli. NH2-terminal amino acid sequencing of the respective fragments indicates that both proteases cleave HIP within a predicted alpha-helix following the tetratricopeptide repeat (TPR) region, despite their different specificities and the presence of several potential cleavage sites scattered throughout the sequence, thus suggesting that this region is particularly accessible and may constitute a linker between two structural domains. After size exclusion chromatography, N25 and C18 elute as two distinct and homogeneous species having a Stokes radius of 49 and 24 A, respectively. Equilibrium sedimentation and sedimentation velocity indicate that N25 is a stable dimer, whereas C18 is monomeric in solution, with sedimentation coefficients of 3.2 and 2.3 S and f/f(o) values of 1.5 and 1.1 for N25 and C18, respectively, indicating that the N25 is elongated whereas C18 is globular in shape. Both domains are able to bind to the ATPase domain of HSC70 and inhibit rhodanese aggregation. Moreover, their effects appear to be additive when used in combination, suggesting a cooperation of these domains in the full-length protein not only for HSC70 binding but also for chaperone activity. Altogether, these results indicate that HIP is made of two structural and functional domains, an NH2-terminal 25-kDa domain, responsible for the dimerization and the overall asymmetry of the molecule, and a COOH-terminal 18-kDa globular domain, both involved in HSC70 and unfolded protein binding.