Structure, dynamics, and topological orientation of the polyether, ionophore antibiotic monensin, in a micellar environment

The structure and dynamics of the ionophoric antibiotic monensin in the presence of micelles have been determined. The conformation of monensin was derived from 50 nuclear Overhauser enhancement (NOE) derived distance restraints and metric-matrix based distance geometry calculations. The conformation was further refined with extensive NOE restrained molecular dynamics simulations carried out in a biphasic simulation cell. From the addition of doxylstearate and monitoring of the induced relaxation of the nmr signals, the relative topological orientation of the molecule within the micelle was ascertained. The results indicate two dihedral angles that act as hinge regions allowing the molecule to adopt a wide range of conformations. Considering the biological activity of monensin, i.e., the capture and transport of cations across cell membranes, an open and closed form of monensin have been postulated. The identification of these hinge regions, which are only observed in the membrane-like environment of the detergent micelles, provides insight into the mechanism of action and can serve as targets for modification to alter the biological profile of monensin.     

Solution conformation of salmon calcitonin in sodium dodecyl sulfate micelles as determined by two-dimensional NMR and distance geometry calculations

The 32 amino acid hormone salmon calcitonin was studied at pH 3.7 and 7.4 by two-dimensional NMR in sodium dodecyl sulfate (SDS) micelles at 310 K. The spectrum was fully assigned, and the secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHN alpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 260 interproton distances, derived from NOESY, and hydrogen-bond constraints, obtained from analysis of the amide exchange, were used. From the initial random conformations, 13 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In SDS, at both pHs, the main conformational feature of the hormone is an alpha-helix from Thr6 through Tyr22, thus including the amphipathic 8-22 segment and two residues of the Cys1-Cys7 N-terminal loop. The C-terminal decapeptide forms a loop folded back toward the helix. The biological significance of this conformation is discussed.     

Solution structure of neurotoxin I from the sea anemone Stichodactyla helianthus. A nuclear magnetic resonance, distance geometry, and restrained molecular dynamics study

The three-dimensional structure of the sea anemone polypeptide Stichodactyla helianthus neurotoxin I in aqueous solution has been determined using distance geometry and restrained molecular dynamics simulations based on NMR data acquired at 500 MHz. A set of 470 nuclear Overhauser enhancement values was measured, of which 216 were used as distance restraints in the structure determination along with 15 dihedral angles derived from coupling constants. After restrained molecular dynamics refinement, the eight structures that best fit the input data form a closely related family. They describe a structure that consists of a core of twisted, four-stranded, antiparallel beta-sheet encompassing residues 1-3, 19-24, 29-34, and 40-47, joined by three loops, two of which are well defined by the NMR data. The third loop, encompassing residues 7-16, is poorly defined by the data and is assumed to undergo conformational averaging in solution. Pairwise root mean square displacement values for the backbone heavy atoms of the eight best structures are 1.3 +/- 0.2A when the poorly defined loop is excluded and 3.6 +/- 1.0A for all backbone atoms. Refinement using restrained molecular dynamics improved the quality of the structures generated by distance geometry calculations with respect to the number of nuclear Overhauser enhancements violated, the size of the total distance violations and the total potential energies of the structures. The family of structures for S. heliathus neurotoxin I is compared with structures of related sea anemone proteins that also bind to the voltage-gated sodium channel.     

Unique helical conformation of the fourth cytoplasmic loop of the CB1 cannabinoid receptor in a negatively charged environment

The proximal portion of the C-terminus of the CB(1) cannabinoid receptor is a primary determinant for G-protein activation. A 17 residue proximal C-terminal peptide (rodent CB1 401-417), the intracellular loop 4 (IL4) peptide, mimicked the receptor's G-protein activation domain. Because of the importance of the cationic amino acids to G-protein activation, the three-dimensional structure of the IL4 peptide in a negatively charged sodium dodecyl sulfate (SDS) micellar environment has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR) spectroscopy and distance geometry calculations. Unambiguous proton NMR assignments were carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints were used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures which were refined using restrained energy minimization and dynamics. In water, the IL4 peptide prefers an extended conformation, whereas in SDS micelles, 3(10)-helical conformation is induced. The predominance of 3(10)-helical domain structure in SDS represents a unique difference compared with structure in alternative environments, which can significantly impact global electrostatic surface potential on the cytoplasmic surface of the CB(1) receptor and might influence the signal to the G-proteins.     

The combined use of NMR, distance geometry, and restrained molecular dynamics for the conformational study of a cyclic somatostatin analogue

A cyclic peptide analogue of somatostatin, including the o-aminomethylphenylacetic acid spacer, was studied by the combined use of two-dimensional nmr spectroscopy, distance geometry, and restrained molecular dynamics. Analysis of distances determined from nuclear Overhauser effect (NOE) buildup rates revealed that these were inconsistent with a unique backbone conformation near the spacer. Assuming that the conformational heterogeneity is localized to the spacer, the NOE distances measured for the remaining part of the molecule were used to generate a large number of structures with the distance geometry algorithm, which were then refined by restrained energy minimization. Four classes of structures emerged, which together account for all observed NOEs. A representative structure of each class was further refined with the restrained molecular dynamics technique, and shown to be stable on a 20-ps time scale. The flexibility of the spacer was examined by simulating interconversions induced by an appropriate restraining potential. As a result, the explanation for the lack of somatostatin activity of the analogue studied was reconsidered.     

Refinement of the solution structure of the ribonucleotide 5'r(GCAUGC)2: combined use of nuclear magnetic resonance and restrained molecular dynamics

The solution structure of the self-complementary hexamer 5'r(GCAUGC)2 is investigated by means of nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The proton resonances are assigned in a sequential manner, and a set of 110 approximate interproton distance restraints are derived from the two-dimensional nuclear Overhauser enhancement spectra. These distances are used as the basis of a structure refinement by restrained molecular dynamics in which the experimental restraints are incorporated into the total energy function of the system in the form of effective potentials. Eight restrained molecular dynamics simulations are carried out, four starting from a structure with regular A-type geometry and four from one with regular B-type geometry. The atomic root mean square (rms) difference between the initial structures is 3.2 A. In the case of all eight simulations, convergence is achieved both globally and locally to a set of very similar A-type structures with an average atomic rms difference between them of 0.8 +/- 0.2 A. Further, the atomic rms differences between the restrained dynamics structures obtained by starting out from the same initial structures but with different random number seeds for the assignment of the initial velocities are the same as those between the restrained dynamics structures starting out from the two different initial structures. These results suggest that the restrained dynamics structures represent good approximations of the solution structure. The converged structures exhibit clear sequence-dependent variation in some of the helical parameters, in particular helix twist, roll, slide, and propellor twist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution structure of the tachykinin peptide eledoisin

Both the aqueous and the lipid-induced structure of eledoisin, an undecapeptide of mollusk origin, have been studied by two-dimensional proton nuclear magnetic resonance spectroscopy and distance geometry calculations. Unambiguous nuclear magnetic resonance assignments of protons have been made with the aid of correlation spectroscopy experiments and nuclear Overhauser effect spectroscopy experiments. The distance constraints obtained from the nuclear magnetic resonance data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water and dimethyl sulfoxide, eledoisin prefers to be in an extended chain conformation, whereas in the presence of perdeuterated dodecylphosphocholine micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M11) of the peptide. N terminus, though less defined, also displays some degree of order and a possible turn structure. The conformation adopted by eledoisin in the presence of dodecylphosphocholine micelles is similar to the structural motif typical of neurokinin-2 selective agonists and with that reported for kassinin in hydrophobic environment.     

Calculating three-dimensional molecular structure from atom-atom distance information: cyclosporin A

In recent years methods for deriving spatial molecular structure from atom-atom distance information have gained in importance due to the emergence of two-dimensional nuclear magnetic resonance (n.m.r) techniques, which make it possible to obtain such distance information for polypeptides, small proteins, sugars, and DNA fragments in solution. Distance geometry (DG) and restrained molecular dynamics (MD) refinement are applied to a cyclic polypeptide, the immunosuppressive drug cyclosporin A, and the results are compared. Two different procedures, DG followed by restrained MD, and straightforward restrained MD starting from the X-ray structure, both lead to a unique conformation that satisfies the 58 experimentally determined distance constraints. The results nicely show the relative merits of DG and restrained MD techniques for determining spatial molecular structure from distance information.     

Lipid induced conformation of the tachykinin peptide Kassinin

Both the aqueous and lipid-induced structure of Kassinin, a dodecapeptide of amphibian origin, has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H-NMR) spectroscopy and distance geometry calculations. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water Kassinin prefers to be in an extended chain conformation, in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M12) of the peptide. N-terminus though less defined also displays some degree of order and a possible turn structure. The conformation adopted by Kassinin in the presence of DPC micelles is consistent with the structural motif typical of neurokinin-1 selective agonists and with that reported for Eledoisin in hydrophobic environment.     

NMR study of the solution conformation of rat atrial natriuretic factor 7-23 in sodium dodecyl sulfate micelles

The conformation of the cyclic portion (7-23) of naturally occurring rat atrial natriuretic factor, ANF(1-28), has been examined in sodium dodecyl sulfate (SDS) micelles using high-resolution NMR techniques. Evidence is presented which shows that ANF(7-23) has several regions of definable structure in SDS micelles which were not observed in earlier studies in bulk solvents. The 1H NMR resonances of ANF(7-23) in SDS micelles were assigned using sequential assignment techniques, and the conformational properties were analyzed primarily from proton-proton distances obtained from the quantitative analysis of two-dimensional nuclear Overhauser effect spectra. Three-dimensional structures consistent with the NMR data were generated by using distance geometry and constrained minimization/dynamics. Several similar but not identical structures were found which adequately satisfied the NMR constraints. Although none of the structures adopted a standard secondary structure, the conformations of three different sections of the peptide, 8-13, 14-17, and 18-21, were nearly identical in all of the predicted structures when individually superimposed.     

Protein Folding: A Few Random Thoughts

Abstract

Both the aqueous and lipid-induced structure of Kassinin, a dodecapeptide of amphibian origin, has been studied by two-dimensional proton nuclear magnetic resonance (2D ¹H-NMR) spectroscopy and distance geometry calculations. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water Kassinin prefers to be in an extended chain conformation, in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M12) of the peptide. N-terminus though less defined also displays some degree of order and a possible turn structure. The conformation adopted by Kassinin in the presence of DPC micelles is consistent with the structural motif typical of neurokinin-1 selective agonists and with that reported for Eledoisin in hydrophobic environment.     

The solution structure of human transforming growth factor alpha

The solution structure of transforming growth factor alpha has been determined by a combination of high-resolution 1H-nuclear magnetic resonance and distance geometry and restrained molecular dynamics. The 382 restraints derived from the NMR experiments were used to calculate many distance geometry structures, which were then refined by restrained molecular mechanics. Five of these structures were further refined using a variety of methods. Comparison of independently measured parameters, such as calculated hydrogen bonding patterns and experimental amide exchange rates, have been used to evaluate the accuracy of the structures. Also, possible mechanisms to explain the pH-dependent conformational interconversion observed are suggested. Finally comparisons between this work and others on this topic have been made.     

Backbone folding of the polypeptide cardiac stimulant anthopleurin-A determined by nuclear magnetic resonance, distance geometry and molecular dynamics

The solution conformation of the cardiac stimulatory sea anemone polypeptide anthopleurin-A has been characterised using distance geometry and restrained molecular dynamics calculations. A set of 253 approximate interproton distance restraints and 14 peptide backbone torsion angle restraints derived from two-dimensional 1H-NMR spectra at 500 MHz were used as input for these calculations. 13 structures generated by either metric matrix or variable target function distance geometry calculations were refined using energy minimisation and restrained molecular dynamics. The resulting structures contain a region of twisted antiparellel beta-sheet to which two separate regions of unordered chain are linked by three disulphide bonds. Two loops, one including Pro-41 and the other encompassing residues 10-18, are poorly defined by the NOE data.     

Refinement of the solution structure of the RNA-DNA hybrid 5'-[r(GCA)d(TGC)]2. Combined use of nuclear magnetic resonance and restrained molecular dynamics

The solution conformation of the self-complementary RNA-DNA hybrid hexamer 5'-[r(GCA)d(TGC)]2 is investigated by NMR spectroscopy and restrained molecular dynamics. The 1H-NMR spectrum is assigned in a sequential manner using two-dimensional homonuclear Hartmann-Hahn and nuclear Overhauser enhancement spectroscopy. From the latter a set of 178 approximate interproton distance restraints are determined and used as the basis of a structure refinement by restrained molecular dynamics. Eight independent calculations are carried out, four from a classical A-type geometry and four from a classical B-type one. Convergence is achieved to very similar A-type structures with an average atomic root mean square difference between them of 1.0 +/- 0.2 A. The converged structures exhibit variations in helical parameters similar to those found previously for the analogue RNA hexamer 5'-r(GCAUGC)2 [(1988) Biochemistry 27, 1735-1743].     

PFG-NMR investigations of the binding of cationic neuropeptides to anionic and zwitterionic micelles

The mechanism by which peptides bind to micelles is believed to be a two-phase process, involving (i). initial electrostatic interactions between the peptide and micelle surface, followed by (ii). hydrophobic interactions between peptide side chains and the micelle core. To better characterize the electrostatic portion of this process, a series of pulse field gradient nuclear magnetic resonance (PFG-NMR) spectroscopic experiments were conducted on a group of neuropeptides with varying net cationic charges (+1 to +3) and charge location to determine both their diffusion coefficients and partition coefficients when in the presence of detergent micelles. Two types of micelles were chosen for the study, namely anionic sodium dodecylsulfate (SDS) and zwitterionic dodecylphosphocholine (DPC) micelles. Results obtained from this investigation indicate that in the case of the anionic SDS micelles, peptides with a larger net positive charge bind to a greater extent than those with a lesser net positive charge (bradykinin > substance P > neurokinin A > Met-enkephalin). In contrast, when in the presence of zwitterionic DPC micelles, the degree of mixed-charge nature of the peptide affects binding (neurokinin A > substance P > Met-enkephalin > bradykinin). Partition coefficients between the peptides and the micelles follow similar trends for both micelle types. Diffusion coefficients for the peptides in SDS micelles, when ranked from largest to smallest, follow a trend where increasing net positive charge results in the smallest diffusion coefficient: Met-enkephalin > neurokinin A > bradykinin > substance P. Diffusion coefficients when in the presence of DPC micelles, when ranked from largest to smallest, follow a trend where the presence of negatively-charged side chains results in the smallest diffusion coefficient: bradykinin > Met-enkephalin > substance P > neurokinin A.     

Three-dimensional structure of potato carboxypeptidase inhibitor in solution. A study using nuclear magnetic resonance, distance geometry, and restrained molecular dynamics

The solution conformation of potato carboxypeptidase inhibitor (CPI) has been investigated by 1H NMR spectroscopy. The spectrum is assigned in a sequential manner by using two-dimensional NMR techniques to identify through-bond and through-space (less than 5 A) connectivities. A set of 309 approximate interproton distance restraints is derived from the two-dimensional nuclear Overhauser enhancement spectra and used as the basis of a three-dimensional structure determination by a combination of metric matrix distance geometry and restrained molecular dynamics calculations. A total of 11 converged distance geometry structures were computed and refined by using restrained molecular dynamics. The average atomic root mean square (rms) difference between the final 11 structures and the mean structure obtained by averaging their coordinates is 1.4 +/- 0.3 A for residues 2-39 and 0.9 +/- 0.2 A for residues 5-37. The corresponding values for all atoms are 1.9 +/- 0.3 and 1.4 +/- 0.2 A, respectively. The larger values for residues 2-38 relative to those for residues 5-37 arise from the fact that the positions of the N- (residues 1-4) and C- (residues 38-39) terminal tails are rather poorly determined, whereas those of the core of the protein (residues 5-37) are well determined by the experimental interproton distance data. The computed structures are very close to the X-ray structure of CPI in its complex with carboxypeptidase, and the backbone atomic rms difference between the mean of the computed structures and the X-ray structure is only 1.2 A. Nevertheless, there are some real differences present which are evidenced by significant deviations between the experimental upper interproton distance limits and the corresponding interproton distances derived from the X-ray structure. These principally occur in two regions, residues 18-20 and residues 28-30, the latter comprising part of the region of secondary contacts between CPI and carboxypeptidase in the X-ray structure.     

The conformations of hirudin in solution: a study using nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The solution conformations of the protein hirudin have been investigated by the combined use of distance geometry and restained molecular dynamics calculations. The basis for the structure determination comprised 359 approximate inter-proton distance restrains and 10 phi backbone torsion angle restrains derived from n.m.r. measurements. It is shown that hirudin is composed of three domains: a central core made up of residues 3-30, 37-46 and 56-57; a protruding 'finger' (residues 31-36) consisting of the tip of an antiparallel beta sheet, and an exposed loop (residues 47-55). The structure of each individual domain is relatively well defined with average backbone atomic r.m.s. differences of <2 A between the final seven converged restrained dynamic structures and the mean structure obtained by averaging their coordinates. The orientation of the two minor domains relative to the central core, however, could not be determined as no long-range (i-h >5) interdomain proton-proton contacts could be observed in the two-dimensional nuclear Overhauser enhancement spectra. From the restrained molecular dynamics calculations it appears that the two minor domains exhibit large rigid-body motions relative to the central core.     

Interaction of a two-transmembrane-helix peptide with lipid bilayers and dodecyl sulfate micelles

To probe structural changes that occur when a membrane protein is transferred from lipid bilayers to SDS micelles, a fragment of bacteriorhodopsin containing transmembrane helical segments A and B was studied by fluorescence spectroscopy, molecular dynamics (MD) simulation, and stopped flow kinetics. In lipid bilayers, F?rster resonance energy transfer (FRET) was observed between tyrosine 57 on helix B and tryptophans 10 and 12 on helix A. FRET efficiency decreased substantially when the peptide was transferred to SDS. MD simulation showed no evidence for significant disruption of helix-helix interactions in SDS micelles. However, a cluster of water molecules was observed to form a hydrogen-bonded network with the phenolic hydroxyl group of tyrosine 57, which probably causes the disappearance of tyrosine-to-tryptophan FRET in SDS. The tryptophan quantum yield decreased in SDS, and the change occurred at nearly the same rate as membrane solubilization. The results provide a clear example of the importance of corroborating distance changes inferred from FRET by using complementary methods.     

A comparison of the restrained molecular dynamics and distance geometry methods for determining three-dimensional structures of proteins on the basis of interproton distances

A direct comparison of the metric matrix distance geometry and restrained molecular dynamics methods for determining three-dimensional structures of proteins on the basis of interproton distances is presented using crambin as a model system. It is shown that both methods reproduce the overall features of the secondary and tertiary structure (shape and polypeptide fold). The region of conformational space sampled by the converged structures generated by the two methods is similar in size, and in both cases the converged structures are distributed about mean structures which are closer to the X-ray structure than any of the individual structures. The restrained molecular dynamics structures are superior to those obtained from distance geometry as regards local backbone conformation, side chain positions and non-bonding energies.     

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.