The structure-function organization of neurokinin A and neurokinin B molecules. II. Theoretical conformational analysis of neurokinin B

The spatial structure of a neurokinin B molecule was investigated by the method of theoretical conformational analysis. The conformational analysis of this molecule indicated that the possible structure of neurokinin B under polar conditions may be described by five families of low-energy conformations possessing a conformationally relatively rigid C-terminal heptapeptide and variable N-terminal fragments.     

Conformational features of bradykinin. A circular dichroism study of some peptide fragments and structural analogues of bradykinin.

The vasoactive hormone bradykinin, its N-and C-terminal fragments and some structural analogues were studied by Circular Dichroism. Conformational features of the peptide can be detected by comparative analysis of the various CD spectra recorded as a function of aqueous pH, solvent and temperature. It is shown that the two biologically essential arginine residues (Arg1 and Arg9) are important for the specific folded bradykinin conformation. Differences between bradykinin, its fragments and analogues become clearly established in conformational terms, and are discussed in relation to the biological activity of these peptides.     

Properties of human neutral bronchial mucins after modification of the peptide or the carbohydrate moieties.

Trypsin and pronase treatment of purified human neutral bronchial mucins released small fragments from the C-terminal end of these molecules and resulted in slight increases in their sedimentation coefficient presumably reflecting conformational changes. The antigenic determinant of neutral bronchial mucins which appears to be located on this C-terminal fragment is destroyed by pronase or by treatments such as periodate oxidation or galactose oxidase-bromine oxidation which modify the carbohydrate moieties. Thus, both amino acid and carbohydrate residues are involved in the structure of the antigenic determinant.     

Studies on cytochrome c. XI. Circular dichroism studies on synthetic peptides related to the C-terminal region of baker's yeast iso-1-cytochrome c

Circular dichroism studies on synthetic peptides related to the C-terminal region of yeast iso-1-cytochrome c were carried out and compared with conformational studies on horse cytochrome c fragments. Evidence is presented for a weaker predisposition for ordered structure in the former peptides when compared with the corresponding region in horse cytochrome c. These findings agree with theoretical predictions and with observations that yeast and other mammalian type cytochromes c differ in several minor respects.     

Solution conformational study of Scyliorhinin I analogues with conformational constraints by two-dimensional NMR and theoretical conformational analysis.

Two analogues of Scyliorhinin I (Scyl), a tachykinin with N-MeLeu in position 8 and a 1,5-disubstituted tetrazole ring between positions 7 and 8, introduced in order to generate local conformational constraints, were synthesized using the solid-phase method. Conformational studies in water and DMSO-d6 were performed on these peptides using a combination of the two-dimensional NMR technique and theoretical conformational analysis. The algorithm of conformational search consisted of the following three stages: (i) extensive global conformational analysis in order to find all low-energy conformations; (ii) calculation of the NOE effects and vicinal coupling constants for each of the low energy conformations; (iii) determining the statistical weights of these conformations by means of a nonlinear least-squares procedure, in order to obtain the best fit of the averaged simulated spectrum to the experimental one. In both solvents the three-dimensional structure of the analogues studied can be interpreted only in terms of an ensemble of multiple conformations. For [MeLeu8]Scyl, the C-terminal 6-10 fragment adopts more rigid structure than the N-terminal one. In the case of the analogue with the tetrazole ring in DMSO-d6 the three-dimensional structure is characterized by two dominant conformers with similar geometry of their backbones. They superimpose especially well (RMSD = 0.28 A) in the 6-9 fragments. All conformers calculated in both solvents superimpose in their C-terminal fragments much better than those of the first analogue. The results obtained indicate that the introduction of the tetrazole ring into the Scyl molecule rigidifies its structure significantly more than that of MeLeu.     

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.     

Monoclonal antibody studies of creatine kinase. The ART epitope: evidence for an intermediate in protein folding.

Chemical cleavage at cysteine residues with nitrothiocyanobenzoic acid shows that the last 98 amino acids of the 380-amino-acid sequence of chick muscle creatine kinase are sufficient for binding of the monoclonal antibody CK-ART. Removal of the last 30 amino acids by cleavage at methionine residues with CNBr results in loss of CK-ART binding. CK-ART binding is also lost when these C-terminal methionine residues are oxidized to sulphoxide, but binding is regained on reduction. Proteinase K 'nicks' native CK at a single site near the C-terminus and two fragments of 327 amino acides and 53 amino acids can be separated by subsequent SDS or urea treatment. CK-ART still binds normally to 'nicked' CK, which is enzymically inactive. After treatment with either urea (in a competition enzyme-linked immunosorbent assay) or SDS (on Western blots), however, CK-ART binds to neither of the two fragments, although these treatments do not affect binding to intact CK. This suggests that parts of both CK fragments contribute to the CK-ART epitope. CK-ART is both species- and isoenzyme-specific, binding only to chick M-CK. The only C-terminal regions containing chick-specific sequences are residues 300-312 and residues 368-371, the latter group being close to the essential methionine residues. We suggest that one, or possibly both, of these regions is involved in forming the conformational epitope on the surface of the CK molecule which CK-ART recognizes. Native CK is resistant to trypsin digestion. The C-terminal half of urea-treated and partly-refolded CK is also resistant to trypsin digestion, whereas the N-terminal half is readily digested. The results suggest a C-terminal region which can refold more rapidly than the rest of the CK molecule and provide evidence for an intermediate in CK refolding.     

Structure of Cry3A delta-endotoxin within phospholipid membranes.

Interaction of delta-endotoxin and its proteolytic fragments with phospholipid vesicles was studied using electron microscopy, scanning microcalorimetry, and limited proteolysis. It was shown that native protein destroys liposomes. The removal of 4 N-terminal alpha-helices and the extreme 56 C-terminal amino acid residues did not affect this ability. The results obtained by limited proteolysis of delta-endotoxin bound to lipid vesicles show essential conformational changes in three or four N-terminal helices and in the C-terminal region. The calorimetric method used in this study provides a unique possibility for the validation of existing models of protein binding and for a more accurate determination of the regions where conformational changes take place. It was found that the binding of the protein to model liposomes does not alter its structure in the regions starting with the fourth alpha-helix of domain I. This can be concluded from the fact that the activation energy of denaturation of the protein remains unchanged upon its binding to the phospholipid membranes. A new structural model has been proposed which agrees with the data obtained.     

Effect of the C-terminal actin-binding sites of caldesmon on the interaction of actin with myosin

TRITC-phalloidin or FITC-labeled F-actin of ghost muscle fibers was bound to tropomyosin and C-terminal recombinant fragments of caldesmon CaDH1 (residues 506-793) or CaDH2 (residues 683-767). After that the fibers were decorated with myosin subfragment 1. In the absence of caldesmon fragments, subfragment 1 interaction with F-actin caused changes in parameters of polarized fluorescence, that were typical of "strong" binding of myosin heads to F-actin and of the "switched on" conformational state of actin. CaDH1 inhibited, whereas CaDH2 activated the effect of subfragment 1. It is suggested that C-terminal part of caldesmon may modulate the transition of F-actin subunits from the "switched on" to the "switched off" state.     

Investigating the conformational coupling between the transmembrane and cytoplasmic domains of a single-spanning membrane protein. A 1H-NMR study

PMP1 is a 38-residue single-spanning membrane protein whose C-terminal cytoplasmic domain, Y25-F38, is highly positively charged. The conformational coupling between the transmembrane span and the cytoplasmic domain of PMP1 was investigated from 1H-nuclear magnetic resonance data of two synthetic fragments: F9-F38, i.e. 80% of the whole sequence, and Y25-F38, the isolated cytoplasmic domain. Highly disordered in aqueous solution, the Y25-F38 peptide adopts a well-defined conformation in the presence of dodecylphosphocholine micelles. Compared with the long PMP1 fragment, this structure exhibits both native and non-native elements. Our results make it possible to assess the influence of a hydrophobic anchor on the intrinsic conformational propensity of a cytoplasmic domain.     

Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin.

In an attempt to delineate potential folding initiation sites for different protein structural motifs, we have synthesized series of peptides that span the entire length of the polypeptide chain of two proteins, and examined their conformational preferences in aqueous solution using proton nuclear magnetic resonance and circular dichroism spectroscopy. We describe here the behavior of peptides derived from a simple four-helix bundle protein, myohemerythrin. The peptides correspond to the sequences of the four long helices (the A, B, C and D helices), the N- and C-terminal loops and the connecting sequences between the helices. The peptides corresponding to the helices of the folded protein all exhibit preferences for helix-like conformations in solution. The conformational ensembles of the A- and D-helix peptides contain ordered helical forms, as shown by extensive series of medium-range nuclear Overhauser effect connectivities, while the B- and C-helix peptides exhibit conformational preferences for nascent helix. All four peptides adopt ordered helical conformations in mixtures of trifluoroethanol and water. The terminal and interconnecting loop peptides also appear to contain appreciable populations of conformers with backbone phi and psi angles in the alpha-region and include highly populated hydrophobic cluster and/or turn conformations in some cases. Trifluoroethanol is unable to drive these peptides towards helical conformations. Overall, the peptide fragments of myohemerythrin have a marked preference towards secondary structure formation in aqueous solution. In contrast, peptide fragments derived from the beta-sandwich protein plastocyanin are relatively devoid of secondary structure in aqueous solution (see accompanying paper). These results suggest that the two different protein structural motifs may require different propensities for formation of local elements of secondary structure to initiate folding, and that there is a prepartitioning of conformational space determined by the local amino acid sequence that is different for the helical and beta-sandwich structural motifs.     

Reconstitution of active octameric mitochondrial creatine kinase from two genetically engineered fragments.

Creatine kinase (CK) has been postulated to consist of two flexibly hinged domains. A previously demonstrated protease-sensitive site in M-CK (Morris & Jackson, 1991) has directed our attempts to dissect mitochondrial CK (Mi-CK) into two protein fragments encompassing amino acids [1-167] and [168-380]. When expressed separately in Escherichia coli, the two fragments yielded large amounts of insoluble inclusion bodies, from which the respective polypeptides could be purified by a simple two-step procedure. In contrast, co-expression of the two fragments yielded a soluble, active, and correctly oligomerizing enzyme. This discontinuous CK showed nearly full specific activity and was virtually indistinguishable from native Mi-CK by far- and near-UV CD. However, the positive cooperativity of substrate binding was abolished, suggesting a role of the covalent domain linkage in the crosstalk between the substrate binding sites for ATP and creatine. The isolated C-terminal fragment refolded into a native-like conformation in vitro, whereas the N-terminal fragment was largely unfolded. Prefolded [168-380] interacted in vitro with [1-167] to form an active enzyme. Kinetic analysis indicated that the fragments associate rapidly and with high affinity (1/K1 = 17 microM) and then isomerize slowly to an active enzyme (k2 = 0.12 min-1; k-2 = 0.03 min-1). Our data suggest that the C-terminal fragment of Mi-CK represents an autonomous folding unit, and that the folding of the C-terminal part might precede the conformational stabilization of the N-terminal moiety in vivo.     

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].     

A novel beta-defensin structure: a potential strategy of big defensin for overcoming resistance by Gram-positive bacteria

Big defensin is a 79-residue peptide derived from hemocytes of the Japanese horseshoe crab. It has antimicrobial activities against Gram-positive and -negative bacteria. The amino acid sequence of big defensin can be divided into an N-terminal hydrophobic half and a C-terminal cationic half. Interestingly, the trypsin cleaves big defensin into two fragments, the N-terminal and C-terminal fragments, which are responsible for antimicrobial activity against Gram-positive and -negative bacteria, respectively. To explore the antimicrobial mechanism of big defensin, we determined the solution structure of mature big defensin and performed a titration experiment with DPC micelles. Big defensin has a novel defensin structure; the C-terminal domain adopts a beta-defensin structure, and the N-terminal domain forms a unique globular conformation. It is noteworthy that the hydrophobic N-terminal domain undergoes a conformational change in micelle solution, while the C-terminal domain remains unchanged. Here, we propose that the N-terminal domain achieves its antimicrobial activity in a novel fashion and explain that big defensin has developed a strategy different from those of other beta-defensins to suppress the growth of Gram-positive bacteria.     

Large fragments of human serum albumin.

Large fragments of human serum albumin were produced by treatment of the native protein with pepsin at pH3.5. Published sequences of human albumin [Behrens, Spiekerman & Brown (1975) Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 591; Meloun, Moravek & Kostka (1975) FEBSLett.58, 134-137]were used to locate the fragments in the primary structure. The fragments support both the sequence and proposed disulphide-linkage pattern (Behrens et al., 1975). As the pH of a solution of albumin is lowered from pH4 to pH3.5, the protein undergoes a reversible conformational change known as the N-F transition. The distribution of large fragments of human albumin digested with pepsin in the above pH region was critically dependent on pH. It appeared that this distribution was dependent on the conformation of the protein at low pH, rather than the activity of pepsin. The yields of the large fragments produced by peptic digestion at different values of pH suggested that the C-terminal region of albumin unfolds or separates from the rest of the molecule during the N-F transition. The similarity of peptic fragments of human and bovine albumin produced under identical conditions supports the proposed similar tertiary structure of these molecules.     

The effects of Ca2+ and Cd2+ on the secondary and tertiary structure of bovine testis calmodulin. A circular-dichroism study.

Near-u.v. and far-u.v. c.d. spectra of bovine testis calmodulin and its tryptic fragments (TR1C, N-terminal half, residues 1-77, and TR2C, C-terminal half, residues 78-148) were recorded in metal-ion-free buffer and in the presence of saturating concentrations of Ca2+ or Cd2+ under a range of different solvent conditions. The results show the following: if there is any interaction between the N-terminal and C-terminal halves of calmodulin, it has not apparent effect on the secondary or tertiary structure of either half; the conformational changes induced by Ca2+ or Cd2+ are substantially greater in TR2C than they are in TR1C; the presence of Ca2+ or Cd2+ confers considerable stability with respect to urea-induced denaturation, both for the whole molecule and for either of the tryptic fragments; a thermally induced transition occurs in whole calmodulin at temperatures substantially below the temperature of major thermal unfolding, both in the presence and in the absence of added metal ion; the effects of Cd2+ are identical with those of Ca2+ under all conditions studied.     

Conformational analysis of the Galpha(s) protein C-terminal region.

The C-terminal domain of the heterotrimeric G protein a-subunits plays a key role in selective activation of G proteins by their cognate receptors. Several C-terminal fragments of Galpha(s) (from 11 to 21 residues) were recently synthesized. The ability of these peptides to stimulate agonist binding was found to be related to their size. Galpha(s)(380-394) is a 15-mer peptide of intermediate length among those synthesized and tested that displays a biological activity surprisingly weak compared with that of the corresponding 21-mer peptide, shown to be the most active. In the present investigation, Galpha(s)(380-394) was subjected to a conformational NMR analysis in a fluorinated isotropic environment. An NMR structure, calculated on the basis of the data derived from conventional 1D and 2D homonuclear experiments, shows that the C-terminal residues of Galpha(s)(380-394) are involved in a helical arrangement whose length is comparable to that of the most active 21 -mer peptide. A comparative structural refinement of the NMR structures of Galpha(s)(380-394) and Galpha(s)(374-394)C379A was performed using molecular dynamics calculations. The results give structural elements to interpret the role played by both the backbone conformation and the side chain arrangement in determining the activity of the G protein C-terminal fragments. The orientation of the side chains allows the peptides to assume contacts crucial for the G protein/receptor interaction. In the 15-mer peptide the lack as well as the disorder of some N-terminal residues could explain the low biological activity observed.     

Salivary statherin. Dependence on sequence, charge, hydrogen bonding potency, and helical conformation for adsorption to hydroxyapatite and inhibition of mineralization.

The structural domains of salivary statherin that are partly responsible for the protection and recalcification of tooth enamel were examined with respect to charge, sequence, hydrophobicity, hydrogen bonding potential, and conformation. Several fragments of statherin, 1-15 (SN15), 5-15 (SN11), 15-29 (SM15), 29-43 (SC15), 19-43 (SC25), and analogs of the N-terminal 15-residue sequence, where phosphoserines at positions 2 and 3 have been replaced by Ser (SNS15) and Asp (SNA15), respectively, were synthesized. The abilities of these fragments to adsorb at hydroxyapatite (HAP) surfaces and to inhibit its mineralization in supersaturated solutions were determined and compared with those of the whole statherin molecule, reported previously. The conformational preferences of the fragments both in aqueous and nonaqueous solutions were examined by circular dichroism. The highly charged N-terminal SN15 fragment has the greatest adsorption to HAP as compared with statherin and all other fragments. Its mineralization inhibitory activity is significantly greater than those of other fragments and comparable with that of the whole molecule. The dephosphorylated N-terminal fragment SNS15 shows a decreased tendency to adhere to and inhibit the formation of HAP, as compared with SN15. However, the substitution of Asp residues in place of phosphoserines (SNA15), restores the binding affinity and crystal growth inhibition properties, suggesting that the negative charge density at the N-terminal rather than any specific interaction of the phosphate group is important for HAP surface interactions. The C-terminal SC15 and SC25 fragments elicit a much higher affinity for HAP surface than that of the middle sequence (SM15), indicating that hydrogen bonding potential of the C-terminal sequence also contributes to the interaction of statherin with HAP. CD studies provide evidence that the N-terminal SN15 fragment has a strong tendency to adopt an ordered helical conformation, whereas the shorter N-terminal sequence, middle, and C-terminal fragments are structurally flexible and prefer to adopt scattered turn structures or unordered random conformations in organic and aqueous solutions. Collectively, the data indicate that the negative charge density, sequence (1-15), and helical conformation at the N-terminal region of statherin are important for its surface interaction with HAP.     

A study on the C-terminal membrane anchoring of Escherichia coli penicillin-binding protein 5.

Escherichia coli penicillin-binding protein 5 (PBP5) anchors to the inner membrane in a pH-dependent manner via a C-terminal amphiphilic alpha-helix. Low pH was found to enhance both levels of PBP5 membrane anchoring and levels of alpha-helicity in an aqueous PBP5 C-terminal homologue, which led to the suggestion that levels of PBP5 membrane anchoring are related to levels of PBP5 C-terminal alpha-helicity. Here we have used Fourier-transformed infrared spectroscopy (FTIR) and a peptide homologue of the PBP5 C-terminal sequence to investigate the effect of pH on the conformational behavior of this sequence at a lipid interface and on its ability to interact with lipid. Our results suggest that the membrane-anchoring mechanism of PBP5 is unlikely to involve conformational change in the protein's C-terminal region and may therefore involve conformational changes in the protein's ectomembranous domain.     

Structural dynamics and functional domains of the fur protein

Proteolytic enzymes were used to detect metal-induced conformational changes in the ferric uptake regulation (Fur) protein of Escherichia coli K12. Metal binding results in enhanced cleavage of the N-terminal region of Fur by trypsin and chymotrypsin. Activation of both trypsinolysis sensitivity and DNA binding have similar metal ion specificity and concentration dependencies, suggesting that the conformational change detected is required for operator DNA binding. Isolation and characterization of biochemically generated fragments of Fur as well as other data indicate that the N-terminal region is necessary for the interaction of the repressor with DNA and that a C-terminal domain is sufficient for binding to metal ions.