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(SC15), 19-43 (SC25), and analogs of the N-terminal 15-residue sequence, where phosphoserines at positions 2 and 3 have been replaced by Ser (SNS15) and Asp (SNA15), respectively, were synthesized.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.     

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.     

Structural analysis of p36, a Ca2+/lipid-binding protein of the annexin family, by proteolysis and chemical fragmentation

Limited proteolysis of the core domain of the 36-kDa protein p36 by trypsin gives a first insight into the structural organization of the four annexin repeats. Trypsin opens only a single peptide bond, situated between residues 204 and 205. The two fragments (of 20 kDa and 15 kDa), each containing two annexin repeats, remain as a tight complex (nicked core), which binds phospholipids in a Ca2(+)-dependent manner. After denaturation by 9 M urea, the nicked core is again formed upon renaturation provided both fragments are present. If the fragments are separated by chromatography in urea prior to renaturation, they show different behaviour. The 15-kDa C-terminal repeats aggregate, while the 20-kDa N-terminal repeats stay in solution. In comparison to p36, fragments with two (20-kDa fragment) or one (N-terminal CNBr fragment) annexin repeats show a conformational alteration in CD spectroscopy and hydrodynamics and display an increased susceptibility to proteases. In line with these differences, their Ca2(+)-dependent affinity to phospholipids is more than 10-20-fold decreased. Thus the four annexin repeats form together an integrated domain with multiple contacts between the repeats. Although stable derivatives with less than four repeats can be obtained, their Ca2+/phospholipid binding affinities are noticeably reduced.     

Structures of the N-terminal and middle domains of E. coli Hsp90 and conformation changes upon ADP binding

Hsp90 is an abundant molecular chaperone involved in many biological systems. We report here the crystal structures of the unliganded and ADP bound fragments containing the N-terminal and middle domains of HtpG, an E. coli Hsp90. These domains are not connected through a flexible linker, as often portrayed in models, but are intimately associated with one another. The individual HtpG domains have similar folding to those of DNA gyrase B but assemble differently, suggesting somewhat different mechanisms for the ATPase superfamily. ADP binds to a subpocket of a large site that is jointly formed by the N-terminal and middle domains and induces conformational changes of the N-terminal domain. We speculate that this large pocket serves as a putative site for binding of client proteins/cochaperones. Modeling shows that ATP is not exposed to the molecular surface, thus implying that ATP activation of hsp90 chaperone activities is accomplished via conformational changes.     

Importance of local structures of second and third repeat fragments of microtubule-binding domain for tau filament formation

To investigate the importance of the seventh residue of the second and third repeat fragments (R2 and R3 peptides) of the microtubule-binding domain (MBD) for tau filamentous assembly, the residues Lys and Pro were substituted (R2-K7P and R3-P7K). The filament formations of the R2 and R3 peptides were almost lost due to their substitutions despite their overall conformational similarities. The NOE analyses showed the importance of the conformational flexibility for the R2 peptide and the coupled extended and helical conformations for the R3 peptide in their limited N-terminal regions around their seventh residues. The result shows that the filament formation of MBD is initiated from a short fragment region containing the minimal conformational or functional motif.     

Conformational aspects of the biological action of functional fragments of the p21ras oncoprotein family. 1. Fragments 1-11 and 9-16 of the polypeptide chain]

Using theoretical conformational analysis, spatial structures of the N-terminal undecapeptide, common to all p21 modifications, and of the 9-16 fragments of the protein's active and passive analogues have been investigated. The data obtained reveal an essential differences between the predominant backbone forms of the active and passive modifications of the oncoprotein.     

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.     

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.     

Spatial organization of the N-terminal beta-endorphin tridecapeptide

Using conformational analysis spatial structure and conformational properties of the N-terminal tridecapeptide--endorphine molecules were investigated. Calculations were based on the fragmental analysis using non-valent, electrostatic, torsional interactions and hydrogen bonds. It was shown that tridecapeptide could exist in several low-energetical conformational states. Enkephaline fragments structure depends on the most perspective structure of free metioninenkephaline. The results can be used for conformational analysis of endorphine molecules, for structure--function relations study.     

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.     

Conformational properties of gastrin fragments of increasing chain length

The conformational properties of a series of biologically active gastrin peptides of increasing chain length have been investigated in TEE solution by spectroscopic techniques. It was found that elongation of the glutamic acid sequence from 1 to 5 residues at the N-terminal portion of the molecules causes a cooperative change of the conformation of the peptide backbone. The environment of the biologically important C-terminal sequence-Trp-Nle-Asp-Phe-NH₂ monitored by the near-uv chiroptical propertical properties is alos affected by chain elongation. However, the change of the structure of the C-terminal portion does not parallel the conformational change of the peptide backbone. In fact, the final folded structure at the C-terminus is almost reached in the fragment with a sequence of 4 glutamic acid residues, while an additiona, relevent conformational change of the backbne is observed on further elongation of the chain to minigastrin and little gastrin. The ability of the fragments to fold into an ordered conformation on chain elongation parallels the increase of biolocical potency tested in vivo, reported in the literature, and suggests a correlation between these two facts. Ionization of the carboxyl side chains is without effect on the structure of the fragments with 2, 3, and 4 glutamic acid residues, while an effect is observed in minigastrin and little gastrin. From analysis of the CD properties and from their dependence upon side-chain ionization a structural model is proposed for the hormones minigastrin and little gastrin. This tentative model includes a β-bend located in the sequence Ala-Tyr-Gly-Trp- and a short helical section at the N-terminal portion of the hormones.     

Spatial structure of secretin molecule

By conformational analysis and circular dichroism the structure of peptide hormone secretin and its shortened N-terminal fragments in different solvents (water, aqueous solutions of alpha-L-phosphatidic acid and sodium dodecyl sulfate) have been studied. The results obtained by the two methods are compared.     

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.     

Studies on the lysine-binding sites of human plasminogen. The effect of ligand structure on the binding of lysine analogs to plasminogen

A method is described for measuring relative binding constants of lysine and analogs of lysine to plasminogen and plasminogen 'kringle' fragments. Plasminogen or kringle fragments adsorbed to lysine-Sepharose are eluted with increasing concentrations of lysine or other ligands, the concentration of ligand required to elute 50% of the protein being taken as a measure of the binding constant. The method is simple and is not dependent on monitoring conformational changes. We confirm earlier reports that the best ligands for the lysine binding sites of plasminogen are omega-amino acids containing five or six carbons. We show further that both Glu-plasminogen (the native form with N-terminal glutamic acid) and Lys-plasminogen (a degraded form with N-terminal lysine), as well as the heavy chain fragments, kringle 4 and kringle 1+2+3, have very similar properties with regard to binding specificity for omega-amino acids. For all species optimal binding is observed when the distance between the amino and carboxyl carbon is about 0.68 nm. The finding of ligands is decreased by the presence of polar atoms on the alpha and beta positions of the carbon chain of amino acids. Arginine binds relatively weakly at the lysine site and there does not appear to be a separate arginine binding site in plasminogen.     

Iron-binding fragments from the carboxyl-terminal region of hen ovotransferrin.

1. When iron-saturated hen ovotransferrin was treated with subtilisin the N-terminal half was digested at a faster rate than the C-terminal half, allowing the latter to be isolated as a single-chain fragment of mol.wt 35000. 2. In mildly acid conditions iron-ovotransferrin loses iron preferentially from its N-terminal binding site. Trypsin digestion of the resulting monoferric ovotransferrin also gave rise to a C-terminal fragment. 3. Comparison of the N-terminal fragment with the C-terminal fragments shows differences in composition, peptide 'maps', CNBr-cleavage patterns and antigenic structures. The C-terminal fragments carry the carbohydrate group of ovotransferrin. 4. Both N-terminal and C-terminal fragments donate their bound iron to rabbit reticulocytes.     

Conformational aspects of biological activity of functional fragments of the p21ras oncoproteins. 4. Address fragments of the receptor oncoproteins

Decapeptide fragments of the scr, fgr, fps, and yes oncoproteins were studied by theoretical conformational analysis, and the arrangement of ionized residues in these fragments was found to be complementary to the binding site of p21. The results demonstrated a similarity in conformational properties of these peptides and their structural complementarity to the address fragment of p21. On the basis of this computation, a model of interaction of the p21ras family of oncoproteins with their cellular receptors was suggested.     

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.     

The domain organization of streptokinase: nuclear magnetic resonance, circular dichroism, and functional characterization of proteolytic fragments.

Streptococcus equisimilis streptokinase (SK) is a bacterial protein of unknown tertiary structure and domain organization that is used extensively to treat acute myocardial infarction following coronary thrombosis. Six fragments of SK were generated by limited proteolysis with chymotrypsin and purified. NMR and CD experiments have shown that the secondary and tertiary structure present in the native molecule is preserved within all fragments, except the N-terminal fragment SK7. NMR spectra demonstrate the presence in SK of three structurally autonomous domains and a less structured C-terminal "tail." Cleavage within the N-terminal domain generates an N-terminal fragment, SK7, which remains noncovalently associated with the remainder of the molecule; in isolation, SK7 adopts an unfolded conformation. The abilities of these fragments to induce active site formation within human plasminogen upon formation of their heterodimeric complex were assayed. The lowest mass SK fragment exhibiting Plg-dependent activator activity was shown to be SK27 (mass 27,000, residues 147-380), which contains both central and C-terminal domains, although this activity was reduced approximately 6,000-fold relative to that of full-length SK. The activity of a 36,000 mass fragment, SK36 (residues 64-380), which differs from SK27 in possessing a portion of the N-terminal domain, was reduced to 0.1-1.0% of that of SK. Other fragments (masses 7,000, 11,000, 16,000, 17,000, 25,000, and 26,000), representing either single domains or single domains extended by portions of other domains, were inactive. However, SK7 (residues 1-63), at a 100-fold molar excess concentration, greatly potentiated the activities of SK27 and SK36, by up to 50- and > 130-fold, respectively. These findings demonstrate that all of SK's three domains are essential for native-like SK activity. The central and C-terminal domains mediate plasminogen-binding and active site-generating functions, whereas the N-terminal domain mediates an activity-potentiating function.     

Conformational Dynamics of Light-Harvesting Complex II in a Native Membrane Environment

Photosynthetic light-harvesting complexes (LHCs) of higher plants, moss, and green algae can undergo dynamic conformational transitions, which have been correlated to their ability to adapt to fluctuations in the light environment. Herein, we demonstrate the application of solid-state NMR spectroscopy on native, heterogeneous thylakoid membranes of Chlamydomonas reinhardtii (Cr) and on Cr light-harvesting complex II (LHCII) in thylakoid lipid bilayers to detect LHCII conformational dynamics in its native membrane environment. We show that membrane-reconstituted LHCII contains selective sites that undergo fast, large-amplitude motions, including the phytol tails of two chlorophylls. Protein plasticity is also observed in the N-terminal stromal loop and in protein fragments facing the lumen, involving sites that stabilize the xanthophyll-cycle carotenoid violaxanthin and the two luteins. The results report on the intrinsic flexibility of LHCII pigment-protein complexes in a membrane environment, revealing putative sites for conformational switching. In thylakoid membranes, fast dynamics of protein and pigment sites is significantly reduced, which suggests that in their native organelle membranes, LHCII complexes are locked in specific conformational states.     

Enzyme-linked immunosorbent assay for riboflavin carrier protein and modified protein: application for epitope analysis.

Rabbit antibodies to native riboflavin carrier protein (RCP), are to a large extent directed towards the conformational epitopes and antibodies to disulphide bond reduced carboxymethylated riboflavin carrier protein (RCM-RCP) to the sequential epitopes. Taking advantage of this premise and in order to map the epitopes of RCP recognized by the antibodies, enzyme-linked immunosorbent assays were validated for RCP and RCM-RCP using the Avidin-Biotin system. The usefulness of these assays were illustrated when antigenicity of peptides derived from RCM-RCP following trypsinization were examined. Two major (T1,T2) and one minor peptide (T3) fractions were obtained when the tryptic peptides were fractionated on DEAE-cellulose. RCP has a blocked N-terminal. Tryptic peptides (T1 and T2) on microsequencing revealed the absence of an N-terminal amino acid, indicating that these fragments emanate from the N-terminal region of RCP. In support of this observation is the finding that antipeptide antibody to cRCP (10-24) of cRCP interacted with T1 as well as T2 indicating the presence of the sequential epitope (10-24) of cRCP in these fragments. In RCP-ELISA, only T2 displaced RCP and peptides T1 and T2 displaced RCM-RCP in RCM-RCP ELISA. Differences in the ability of these fragments (T1 and T2) to displace RCP and RCM-RCP reflect the subtle changes in the spatial structures of these epitopes in RCP and RCM-RCP.