Fluorescence,circular dichroism,NMR, and docking studies of the interaction of the alkaloid malbrancheamide with calmodulin

A new malbrancheamide analogue, isomalbrancheamide B (3), along with three known compounds, malbrancheamide (1), isomalbrancheamide (2), and premalbrancheamide (4), were isolated in higher yields from the alkaloid fraction of the fungus Malbranchea aurantiaca. The interaction of the alkaloids 1–4 with calmodulin (CaM) was analyzed using different enzymatic, fluorescence, spectroscopic, nuclear magnetic resonance (NMR), and molecular modelling techniques. On the basis of the enzymatic and fluorescence experiments, malbrancheamides 1–3 are classical CaM inhibitors. Compound 4, however, did not quench the extrinsic fluorescence of the CaM biosensor indicating that it could be a functional inhibitor. Circular dichroism, NMR, and molecular modelling studies revealed that 1 binds to CaM in the same hydrophobic pocket than the chlorpromazine and trifluoperazine, two classical CaM inhibitors. Thus, malbrancheamide and related monochlorinated analogues are compounds with a high potential for the development of new therapeutic agents, involving CaM as their molecular target.     

High-field NMR and circular dichroism solvent-dependent conformational studies of the bradykinin C-terminal tetrapeptide Ser-Pro-Phe-Arg

The conformational properties of the tetrapeptide Ser1-Pro2-Phe3-Arg4, the C-terminal fragment of the nonapeptide hormone bradykinin, have been studied by circular dichroism and two-dimensional NMR techniques. Measurements of coupling constants, NH temperature dependence rates and nuclear Overhauser effects (performed with rotating frame nuclear Overhauser spectroscopy, ROESY) in H2O and CD3OH/D2O (80/20, v/v) reveal different conformations in the corresponding solvent. In aqueous solution the molecule exists in a random conformation or as an average of several conformations in rapid exchange. In CD3OH/D2O, however, the conformation is well-defined. The backbone of the peptide is extended, and the side-chains of Phe3 and Arg4 exhibit unusual rigidity for a peptide of this size. Evidently, the secondary structure is stabilized by a charge interaction between the guanidino group of Arg4 and the terminal carboxyl group, since experiments at various pH's show clearly that the definition of conformation decreases strongly upon protonation of the carboxyl function. A NH3+(Ser1)-COO-(Arg4) salt bridge, as well as any form of turn stabilized by hydrogen bonds can be ruled out with certainty.     

Calcium-binding and its effect on circular dichroism of plant calmodulin

The Ca²⁺-binding properties of calmodulin purified from zucchini (Cucurbita pepo L.) has been determined. A value of 3.3 mol Ca²⁺ per mol of zucchini calmodulin was measured at pH 7.5 by equilibrium chromatography. The far-and near-UV circular-dichroic spectra of the Ca²⁺-and Mg²⁺-saturated as well as from the metal-free forms of zucchini calmodulin reveal that upon Ca²⁺-binding the -helix content increases. A comparison with the spectra of vertebrate calmodulin indicates that both calmodulin have a similar secondary structure, similar Ca²⁺-induced conformational changes and the same number of Ca²⁺-binding sites.Abbreviations CAPP 10-(3-aminopropyl)-2-chloro-phenothiazine - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - EDTA ethylenediaminetetraacetic acid Dedicated to Prof. Dr. Karl Decker on the occasion of his 60th birthday     

Conformations of alanine-based peptides in water probed by FTIR, Raman, vibrational circular dichroism, electronic circular dichroism, and NMR spectroscopy

We have used a combination of FTIR, VCD, ECD, Raman, and NMR spectroscopies to probe the solution conformations sampled by H-(AAKA)-OH by utilizing an excitonic coupling model and constraints imposed by the 3JCalphaHNH coupling constants of the central residues to simulate the amide I' profile of the IR, isotropic Raman, anisotropic Raman, and VCD spectra in terms of a mixture of three conformations, i.e., polyproline II, beta-strand and right-handed helical. The representative coordinates of the three conformations were obtained from published coil libraries. Alanine was found to exhibit PPII fractions of 0.60 or greater, mixed with smaller fractions of helices and beta-strand conformations. Lysine showed no clear conformational propensity in that it samples polyproline II, beta-strand, and helical conformations with comparable probability. This is at variance with results obtained earlier for ionized polylysine, which suggest a high polyproline II propensity. We reanalyzed previously investigated tetra- and trialanine by combining published vibrational spectroscopy data with 3JCalphaHNH coupling constants and obtained again blends dominated by PPII with smaller admixtures of beta-strand and right-handed helical conformations. The polyproline II propensity of alanine was found to be higher in tetraalanine than in trialanine. For all peptides investigated, our results rule out a substantial population of turn-like conformations. Our results are in excellent agreement with MD simulations on short alanine peptides by Gnanakaran and Garcia [(2003) J. Phys. Chem. B 107, 12555-12557] but at variance with multiple MD simulations particularly for the alanine dipeptide.     

Fluorescence and circular dichroism studies on human serum low density lipoprotein particles and lipid-depleted derivatives

The low density liporpotein from human serum, and derivitives prepared free of neutral lipids and total lipids, have been studied by fluorescence and circular dichorism methods. Removal of the neutral lipids had little effect on the tryptophan fluorescence at neutral pH. However, by the criteria of circular dichroism, over the range of 200 nm to 250 nm, there was a reduction in secondary structure of over 75%. Removal of the remaining phospholipids resulted in a qualitatively different structure by both fluorescence and circular dichroism criteria. Neutral lipids were removed from LDL in a step-wise fashion in order to determine the exact amount of neutral lipid required for the native circular dichroism spectrum. The circular dichroism band intensity was constant until approximately 10% of the total cholesterol (as cholesterol ester) remained. The intensity then abruptly dropped as more cholesterol was removed. We concluded that the two spectroscopic methods report on two distinct aspects of LDL structure. The tryptophan fluorescence appears to be sensitive to the presence of phospholipids. The circular dichroism, however, appears to be sensitive to the binding of a small amount of neutral lipid. These findings suggest that a functional and geometric separation of binding sites may exist for these two classes of lipids. Such a distinction is predicted by the icosohedral model of the quaternary structure of LDL. In this model, the phospholipids are located on the surface of the particle, in the holes of an icosohedrally symmetric surface network of protein subunits; the neutral lipids are located in the particle core. Finally, we suggest that functional significance may be attached to our finding that relatively few cholesterol ester molecles are needed to maintain the native secondary structure of LDL. This provides a mechanism whereby the amount of bound neutral lipid could be raised or lowered (for transport and transfer to cells) without affecting the protein in any structurally significant manner.     

Interaction of alamethicin with ether-linked phospholipid bilayers: oriented circular dichroism, 31P solid-state NMR, and differential scanning calorimetry studies

The arrangement of the antimicrobial peptide alamethicin was studied by oriented circular dichroism, 31P solid-state NMR, and differential scanning calorimetry in ether-linked phospholipid bilayers composed of 1,2-O-dihexadecyl-sn-glycero-3-phosphocholine (DHPC). The measurements were performed as a function of alamethicin concentration relative to the lipid concentration, and results were compared to those reported in the literature for ester-linked phospholipid bilayers. At ambient temperature, alamethicin incorporates into the hydrophobic core of DHPC bilayers but results in more lipid disorder than observed for ester-linked 1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayers. This orientational disorder appears to depend on lipid properties such as bilayer thickness. Moreover, the results suggest that alamethicin inserts into the hydrophobic core of the bilayers (at high peptide concentration) for both ether- and ester-linked lipids but using a different mechanism, namely toroidal for DHPC and barrel-stave for POPC.     

Protein engineering and NMR studies of calmodulin

Molecular and Cellular Biochemistry - The calcium regulatory protein calmodulin (CaM) plays a role as an on-off switch in the activation of many enzymes and proteins. CaM has a dumbbell shaped...     

Kinetic studies of the interaction of methyl orange with poly(L-lysine) by stopped-flow with circular dichroism detection

The interaction of methyl orange with poly(L -lysine) was studied kinetically by the stopped-flow technique with CD detection, as well as by static CD titration experiments. In the static experiments, the differences observed in the polymer-to-dye ratio dependences of the CD spectra and absorption spectra suggested at least two kinds of bound states of the methyl orange attached to the polymer. The kinetic experiments using the stopped-flow apparatus, however, revealed four distinct reaction processes. The reaction mechanism was elucidated from the concentration dependence of the time constant for each process as follows: the first process was attributed to the bimolecular binding step of methyl orange to the side chain of poly(L -lysine), the second and third process were ascribed to the intramolecular reaction of the polymer–dye complex, and the fourth process was found to be the intermolecular aggregation of the polymer–dye complex. The origin of the stacking of methyl orange on poly(L -lysine) is discussed on the basis of the characteristics of signal amplitudes obtained from the kinetic experiments for these processes.     

Characterization of proteolysis fragments of aspartokinase I: homoserine dehydrogenase I. Fluorescence and circular dichroism studies

Proteolysis of native aspartokinase-homoserine dehydrogenase by chymotrypsin, subtilisin, clostripain, and V8 protease yields active dehydrogenase fragments. Fluorescence and near-UV circular dichroism measurements demonstrate that the bulk of the spectroscopic signal observed in the native protein originates in the residual fragments. Kinetic studies and far-UV CD spectra further distribute the fragments into two groups. Even though the remaining dehydrogenase activity is no longer inhibited by L-threonine, ultrafiltration binding studies and far-UV CD spectra clearly demonstrate that one of the two sets of inhibitor-binding sites is still intact. Computer analysis of the far-UV CD data of the native protein and the isolated fragments in the presence and absence of L-threonine has been used to resolve contributions from helix, beta, turn, and aperiodic components. This analysis indicates that the binding of the inhibitor induces decreases in helix content and generation of aperiodic structure within the molecule. The changes observed are similar in the native molecule and the fragments.     

Bicelle membranes and their use for hydrophobic peptide studies by circular dichroism and solid state NMR

Mixtures of dicaproyl- (DC), dimyristoyl- (DM) and 1-tetradecanoyl-2-biphenylbutanoyl-(TBB) phosphatidylcholine (PC) in water produce bicelle membranes that are oriented by magnetic fields. DMPC/DCPC systems orient such that their membrane plane is parallel to the magnetic field, whereas for TBBPC/DCPC, the plane is perpendicular to the field. Partial temperature-composition-hydration diagrams are established using solid-state 31P-NMR. DMPC/DCPC bicelles exist on a large range of composition but on a narrow temperature domain (25-45 degrees C). At converse, TBBPC/DCPC form bicelles on a narrow compositional range but over a large temperature span (10-70 degrees C). The TBBPC/DCPC bicelles are shown to be a very powerful potential tool to study the orientation of hydrophobic helices in membranes using wide line 15N-NMR. The DMPC/DCPC system that undergoes a micelle-to-bicelle transition on going from 10 degrees C to 40 degrees C may be used with circular dichroism to study the state of association of hydrophobic helices within the membrane. Results suggest that the transmembrane fragment of the neu/erbB-2 receptor is monomeric in micellar medium and dimeric/multimeric in bicelle membranes.     

A circular dichroism study of charged polypeptides interaction with salts.

The effect of salts on the experimental circular dichroism spectra of polypeptides is presented using poly-L-lysine as the main model. Salt effects are analyzed into: (a) shielding at low (less than 0.5 M) concentrations of all salts; (b) binding to positively charged and some neutrally charged side-chains by certain anions (e.g., CCl3COO-, CF3C00-, ClO4-), with induction of helicity; (c) binding of these same anions, at high concentration, to the backbone leading toward random structure; (d) binding of high concentration of denaturing cations (La+3, Ca++, Li+) to the backbone, with La+3 and Ca++ leading to collapsed random structure (R) while Li+ tends to leave the polypeptide somewhat extended; (e) indirect interaction of salting-out salts (NaH2PO4, (NH4)2SO4, NH4F), at high concentration, leading toward complete alpha helicity, probably by competition with the polypeptide and the anion for available water. Effects of changing the temperature from 5 degrees to 50 degrees on the circular dishroism spectra of different polypeptide-salt solutions throughout the region from extended (LES) to alpha helical conformation are analyzed in terms of introduction of randomness (R) at high temperature. Applications to effects of salt on protein structures are considered.     

Conformations of bombolitins I and III in aqueous solutions: circular dichroism, 1H NMR, and computer simulation studies

The heptadecapeptides bombolitin I and bombolitin III are two of a series of peptides postulated to be biologically active within a membrane environment. In the preceding paper [Bairaktari, E., Mierke, D.F., Mammi, S., & Peggion, E. (1990) Biochemistry (preceding paper in this issue)] the conformational preferences of these peptides in the presence of SDS surfactant micelles, a mimetic for biological membranes, were examined. During these studies the conformations of these peptides were investigated in aqueous solutions by circular dichroism and nuclear magnetic resonance. A large difference was observed for the two peptides. Bombolitin I lacks any observable secondary structure in aqueous solution, independent of temperature, pH, and concentration. In striking contrast, bombolitin III adopts a well-defined alpha-helix at concentrations greater than 1.3 mM. This is indeed surprising given the great similarity of the two peptides. The alpha-helix of bombolitin III is pH dependent, with a great decrease in the observed secondary structure at pH values below 3.5. This observation could only be due to the protonation of the Asp residue at the fifth position. These findings suggest that the secondary structure arises from molecular aggregation of bombolitin III through the formation of a salt bridge involving the Asp side chain. The alpha-helix observed at "high" concentration (greater than 2.5 mM) has been characterized by CD and by the NOE's measured throughout a majority of the peptide. The experimentally determined structure has been energy refined with restrained molecular dynamics. The conformational results from this study are then compared with the conformations found in the presence of surfactant micelles.     

Induced circular dichroism of the interaction between pinacyanol and algal alginates

The interaction between pinacyanol chloride and sodium alginate or guluronate-rich alginate is found to effect profound changes in the visible absorbance and circular dichroism spectra. Two different types of aggregates are observed depending on the relative dye/alginate concentrations. With a dye/alginate ratio at 1:1, a complex is deduced based on an analysis of Job’s method and conductometric titrations. Another complex forms at 1:10 dye/alginate ratio and only in the presence of alginate or guluronate-rich alginate. The two aggregates are in dynamic equilibrium according to the presence of isosbestic points in the visible spectra. The effects of pH and divalent cations on the spectra are studied. The 1:10 complex is damaged by addition of hydrochloric acid and divalent cations; however, at low concentration of these agents the spectra indicate conversion of the complex into the 1:1 aggregate. Models for the two complexes are proposed taking into account the preference of guluronate binding sites for chelating ions.     

Conformational prediction and circular dichroism studies on the lac repressor

Using the protein predictive model of Chou & Fasman (1974b), the secondary structure of the lac repressor has been elucidated from its amino acid sequence of 347 residues. The conformation is predicted to contain 37% α-helix and 35% β-sheet for the repressor, and 29% helix and 41% β-sheet for the trypsin-resistant core (residues 60 to 327). Circular dichroism studies indicate that native lac repressor contains 40% helix and 42% β-sheet, while the core has 16% helix and 54% β-sheet, in general agreement with the predicted conformation. The sharp reduction in helicity for the trypsinized lac repressor could be due to the loss of two long helical regions, 26–45 and 328–344, predicted at both terminals. There are extensive β-sheets predicted in the 215–324 region, which may be responsible for tetrameric stabilization found in both the lac repressor and the core. Residues 17 to 33 were previously predicted by Adler et al. (1972) to be helical and were proposed to bind in the major groove of DNA. However, the present analysis shows that there are two anti-parallel β-sheet regions: 4–7 and 17–24 at the N-terminal as well as 315–318 and 321–324 at the C-terminal of the lac repressor. These β-sheet pairs may assume the twisted “polypeptide double helix” conformation (Carter & Kraut, 1974) and bind to complementary regions in the major groove of DNA. The OH groups of Tyr at the N-terminal and those of Thr and Ser side chains, in both β-sheets at the N and C-terminal ends, could form hydrogen bonds to specific sites on the lac operator. There are 23 reverse β-turns predicted that may control the tertiary folding of the lac repressor, which is essential for operator binding. The behavior of several lac repressor mutants can be satisfactorily explained in terms of polar to non-polar group replacements as well as conformational changes in light of the present predicted model.