Solution structure of the recombinant human oncoprotein p13MTCP1

The human oncoprotein p13MTCP1 is coded by the MTCP1 gene, a gene involved in chromosomal translocations associated with T-cell prolymphocytic leukemia, a rare form of human leukemia with a mature T-cell phenotype. The primary sequence of p13MTCP1 is highly and only homologous to that of p14TCL1, a product coded by the gene TCL1 which is also involved in T-cell prolymphocytic leukemia. These two proteins probably represent the first members of a new family of oncogenic proteins. We present the three-dimensional solution structure of the recombinant p13MTCP1 determined by homonuclear proton two-dimensional NMR methods at 600 MHz. After proton resonance assignments, a total of 1253 distance restraints and 64 dihedral restraints were collected. The solution structure of p13MTCP1 is presented as a set of 20 DYANA structures. The rmsd values with respect to the mean structure for the backbone and all heavy atoms for the conformer family are 1.07 +/- 0.19 and 1.71 +/- 0.17 A, when the structured core of the protein (residues 11-103) is considered. The solution structure of p13MTCP1 consists of an orthogonal beta-barrel, composed of eight antiparallel beta-strands which present an original arrangement. The two beta-pleated loops which emerge from this barrel might constitute the interaction surface with a potential molecular partner.     

Determination of the three-dimensional solution structure of Raphanus sativus antifungal protein 1 by 1H NMR

Raphanus sativus Antifungal Protein 1 (Rs-AFP1) is a 51 amino acid residue plant defensin isolated from radish (Raphanus sativus L.) seeds. The three-dimensional structure in aqueous solution has been determined from two-dimensional 1H NMR data recorded at 500 MHz using the DIANA/REDAC calculation protocols. Experimental constraints consisted of 787 interproton distances extracted from NOE cross-peaks, 89 torsional constraints from 106 vicinal interproton coupling constants and 32 stereospecific assignments of prochiral protons. Further refinement by simulated annealing resulted in a set of 20 structures having pairwise root-mean-square differences of 1.35(+/- 0.35) A over the backbone heavy atoms and 2.11(+/- 0.46) A over all heavy atoms. The molecule adopts a compact globular fold comprising an alpha-helix from Asn18 till Leu28 and a triple-stranded beta-sheet (beta 1 = Lys2-Arg6, beta 2 = His33-Tyr38 and beta 3 = His43-Pro50). The central strand of this beta-sheet is connected by two disulfide bridges (Cys21-Cys45 and Cys25-Cys47) to the alpha-helix. The connection between beta-strand 2 and 3 is formed by a type VIa beta-turn. Even the loop (Pro7 to Asn17) between beta-strand 1 and the alpha-helix is relatively well defined. The structure of Raphanus sativus Antifungal Protein 1 features all the characteristics of the "cysteine stabilized alpha beta motif". A comparison of the complete structure and of the regions important for interaction with the fungal receptor according to a mutational study, is made with the structure of gamma-thionin, a plant defensin that has no antifungal activity. It is concluded that this interaction is both electrostatic and specific, and some possible scenarios for the mode of action are given.     

Structure of hen lysozyme in solution

The structure of the 129-residue protein hen lysozyme has been determined in solution by two-dimensional 1H nuclear magnetic resonance methods. 1158 NOE distance restraints, and 68 phi and 24 chi 1 dihedral angle restraints were employed in conjunction with distance geometry and simulated annealing procedures. The overall C alpha root-mean-square deviation from the average for 16 calculated structures is 1.8(+/- 0.2) A, but excluding 14 residues in exposed disordered regions, this value reduces to 1.3(+/- 0.2) A. Regions of secondary structure, and the four alpha-helices in particular, are well defined (C alpha root-mean-square deviation 0.8(+/- 0.3) A for helices). The main-chain fold is closely similar to structures of the protein in the crystalline state. Furthermore, many of the internal side-chains are found in well-defined conformational states in the solution structures, and these correspond well with the conformational states found in the crystal. The general high level of definition of mainchain and many internal side-chains in the solution structures is reinforced by the results of an analysis of coupling constants and ring current shifts. Many side-chains on the surface, however, are highly disordered amongst the set of solution structures. In certain cases this disorder has been shown to be dynamic in origin by the examination of 3J alpha beta coupling constants.     

Three-dimensional organization of a human water channel

Aquaporins (AQP) are members of the major intrinsic protein (MIP) superfamily of integral membrane proteins and facilitate water transport in various eukaryotes and prokaryotes. The archetypal aquaporin AQP1 is a partly glycosylated water-selective channel that is widely expressed in the plasma membranes of several water-permeable epithelial and endothelial cells. Here we report the three-dimensional structure of deglycosylated, human erythrocyte AQP1, determined at 7 A resolution in the membrane plane by electron crystallography of frozen-hydrated two-dimensional crystals. The structure has an inplane, intramolecular 2-fold axis of symmetry located in the hydrophobic core of the bilayer. The AQP1 monomer is composed of six membrane-spanning, tilted alpha-helices. These helices form a barrel that encloses a vestibular region leading to the water-selective channel, which is outlined by densities attributed to the functionally important NPA boxes and their bridges to the surrounding helices. The intramolecular symmetry within the AQP1 molecule represents a new motif for the topology and design of membrane protein channels, and is a simple and elegant solution to the problem of bidirectional transport across the bilayer.     

A novel RNA-binding motif in influenza A virus non-structural protein 1

The solution NMR structure of the RNA-binding domain from influenza virus non-structural protein 1 exhibits a novel dimeric six-helical protein fold. Distributions of basic residues and conserved salt bridges of dimeric NS1(1-73) suggest that the face containing antiparallel helices 2 and 2' forms a novel arginine-rich nucleic acid binding motif.     

Solution structure by NMR of circulin A: a macrocyclic knotted peptide having anti-HIV activity

The three-dimensional solution structure of circulin A, a 30 residue polypeptide from the African plant Chassalia parvifolia, has been determined using two-dimensional 1H-NMR spectroscopy. Circulin A was originally identified based upon its inhibition of the cytopathic effects and replication of the human immunodeficiency virus. Structural restraints consisting of 369 interproton distances inferred from nuclear Overhauser effects, and 21 backbone dihedral and nine chi1 angle restraints from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimisation in the program X-PLOR. The final set of 12 structures had mean pairwise rms differences over the whole molecule of 0.91 A for the backbone atom, and 1.68 A for all heavy atoms. For the well-defined region encompassing residues 2-12 and 18-27, the corresponding values were 0.71 and 1.66 A, respectively. Circulin A adopts a compact structure consisting of beta-turns and a distorted segment of triple-stranded beta-sheet. Fluorescence spectroscopy provided additional evidence for a solvent-exposed Trp residue. The molecule is stabilised by three disulfide bonds, two of which form an embedded loop completed by the backbone fragments connecting the cysteine residues. A third disulfide bond threads through the centre of this loop to form a "cystine-knot" motif. This motif is present in a range of other biologically active proteins, including omega-contoxin GVIA and Cucurbita maxima trypsin inhibitor. Circulin A belongs to a novel class of macrocyclic peptides which have been isolated from plants in the Rubiaceae family. The global fold of circulin A is similar to kalata B1, the only member of this class for which a structure has previously been determined.     

Replacement of His23 by Cys in a zinc finger of HIV-1 NCp7 led to a change in 1H NMR-derived 3D structure and to a loss of biological activity

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1), which is necessary for the formation of infectious virions, contains two zinc fingers of the Cys-X2-Cys-X4-His-X4-Cys form. To elucidate the importance of this particular motif, well conserved in retroviruses and retroelements, we substituted the histidine residue by a cysteine in the first zinc binding domain 13VKCFNCGKEGHTARNCRA30. The structures of the mutated and native zinc complexed peptides were studied by two-dimensional 600 MHz 1H nuclear magnetic resonance (NMR) in aqueous solution. The nuclear Overhauser effects were used as constraints to determine the solution structures using DIANA software followed by AMBER energy refinement. The results show that native and mutant peptides fold into non-identical three-dimensional structures, probably accounting for the loss of retrovirus infectivity following the His-Cys point mutation.     

NMR solution structure of the receptor binding domain of Pseudomonas aeruginosa pilin strain P1. Identification of a beta-turn

The solution structure of the peptide antigen from the receptor binding domain of Pseudomonas aeruginosa strain P1 has been determined using two-dimensional 1H NMR techniques. Ensembles of solution conformations for the trans form of this 23-residue disulfide bridged peptide have been generated using a simulated annealing procedure in conjunction with distance and torsion angle restraints derived from NMR data. Comparison of the NMR-derived solution structures of the P1 peptide with those previously determined for the 17-residue PAK, PAO and KB7 strain peptides [McInnes, C., et al. (1993) Biochemistry 32, 13432-13440; Campbell, A.P., et al. (1995) Biochemistry 34, 16255-16268] reveals the common structural motif of a beta-turn, which may be the necessary structural requirement for recognition of a common cell surface receptor and a common cross-reactive antibody to which all four strains bind. The importance of this conserved beta-turn in the PAK, PAO, KB7 and P1 peptides is discussed with regard to the design of a synthetic peptide vaccine effective against multiple strains of Pseudomonas aeruginosa infections.     

Solution structure, rotational diffusion anisotropy and local backbone dynamics of Rhodobacter capsulatus cytochrome c2

The solution structure, backbone dynamics and rotational diffusion of the Rhodobacter capsulatus cytochrome c2 have been determined using heteronuclear NMR spectroscopy. In all, 1204 NOE-derived distances were used in the structure calculation to give a final ensemble with 0.59(+/-0.08) A rms deviation for the backbone atoms (C, Calpha and N) with respect to the mean coordinates. There is no major difference between the solution structure and the previously solved X-ray crystal structure (1.07(+/-0.07) A rms difference for the backbone atoms), although certain significant local structural differences have been identified. This protein contains five helical regions and a histidine-heme binding domain, connected by a series of structured loops. The orientation of the helices provides an excellent sampling of angular space and thus allows a precise characterization of the anisotropic diffusion tensor. Analysis of the hydrodynamics of the protein has been performed by interpretation of the 15N relaxation data using isotropic, axially asymmetric and fully anisotropic diffusion tensors. The protein can be shown to exhibit significant anisotropic reorientation with a diffusion tensor with principal axes values of 1.405(+/-0.031)x10(7) s-1, 1.566(+/-0.051)x10(7) s-1 and 1.829(+/-0.054)x10(7) s-1. Hydrodynamic calculations performed on the solution structure predict values of 1.399x10(7) s-1, 1.500x10(7) s-1 and 1.863x10(7) s-1 when a solvent shell of 3.5 A is included in the calculation. The optimal orientation of the diffusion tensor has been incorporated into a hybrid Lipari-Szabo type local motion-anisotropic rotational diffusion model to characterize the local mobility in the molecule. The mobility parameters thus extracted show a quantitative improvement with respect to the model-free analysis assuming isotropic reorientation; helical regions exhibit similar dynamic properties and fewer residues require more complex models of internal motion. While the molecule is essentially rigid, a tripeptide loop region (residues 101 to 103) exhibits flexibility in the range of 20 to 30 ps, which appears to be correlated with the order in the NMR solution structure.     

Solution structure of drosomycin, the first inducible antifungal protein from insects

Drosomycin is the first antifungal protein characterized recently among the broad family of inducible peptides and proteins produced by insects to respond to bacterial or septic injuries. It is a small protein of 44 amino acid residues extracted from Drosophila melanogaster that exhibits a potent activity against filamentous fungi. Its three-dimensional structure in aqueous solution was determined using 1H 2D NMR. This structure, involving an alpha-helix and a twisted three-stranded beta-sheet, is stabilized by three disulfide bridges. The corresponding Cysteine Stabilized alpha beta (CS alpha beta) motif, which was found in other defense proteins such as the antibacterial insect defensin A, short- and long-chain scorpion toxins, as well as in plant thionins and potent antifungal plant defensins, appears as remarkably persistent along evolution.     

Solution structure and dynamics of linked cell attachment modules of mouse fibronectin containing the RGD and synergy regions: comparison with the human fibronectin crystal structure

We report the three-dimensional solution structure of the mouse fibronectin cell attachment domain consisting of the linked ninth and tenth type III modules, mFnFn3(9,10). Because the tenth module contains the RGD cell attachment sequence while the ninth contains the synergy region, mFnFn3(9,10) has the cell attachment activity of intact fibronectin. Essentially complete signal assignments and approximately 1800 distance and angle restraints were derived from multidimensional heteronuclear NMR spectra. These restraints were used with a hybrid distance geometry/simulated annealing protocol to generate an ensemble of 20 NMR structures having no distance or angle violations greater than 0.3 A or 3 degrees. Although the beta-sheet core domains of the individual modules are well-ordered structures, having backbone atom rmsd values from the mean structure of 0.51(+/-0.12) and 0.40(+/-0.07) A, respectively, the rmsd of the core atom coordinates increases to 3.63(+/-1.41) A when the core domains of both modules are used to align the coordinates. The latter result is a consequence of the fact that the relative orientation of the two modules is not highly constrained by the NMR restraints. Hence, while structures of the beta-sheet core domains of the NMR structures are very similar to the core domains of the crystal structure of hFnFn3(9,10), the ensemble of NMR structures suggests that the two modules form a less extended and more flexible structure than the fully extended rod-like crystal structure. The radius of gyration, Rg, of mFnFn3(9,10) derived from small-angle neutron scattering measurements, 20.5(+/-0.5) A, agrees with the average Rg calculated for the NMR structures, 20.4 A, and is ca 1 A less than the value of Rg calculated for the X-ray structure. The values of the rotational anisotropy, D ||/D perpendicular, derived from an analysis of 15N relaxation data, range from 1.7 to 2.1, and are significantly less than the anisotropy of 2.67 predicted by hydrodynamic modeling of the crystal coordinates. In contrast, hydrodynamic modeling of the NMR coordinates yields anisotropies in the range of 1.9 to 2.7 (average 2.4(+/-0.2)), with NMR structures bent by more than 20 degrees relative the crystal structure having calculated anisotropies in best agreement with experiment. In addition, the relaxation parameters indicate that several loops in mFnFn3(9,10), including the RGD loop, are flexible on the nanosecond to picosecond time-scale. Taken together, our results suggest that, in solution, the limited set of interactions between the mFnFn3(9,10) modules position the RGD and synergy regions to interact specifically with cell surface integrins, and at the same time permit sufficient flexibility that allows mFnFn3(9,10) to adjust for some variation in integrin structure or environment.     

Solution structure of the superactive monomeric des-[Phe(B25)] human insulin mutant: elucidation of the structural basis for the monomerization of des-[Phe(B25)] insulin and the dimerization of native insulin

The three-dimensional solution structure of des-[Phe(B25)] human insulin has been determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Thirty-five structures were calculated by distance geometry from 581 nuclear Overhauser enhancement-derived distance constraints, ten phi torsional angle restraints, the restraints from 16 helical hydrogen bonds, and three disulfide bridges. The distance geometry structures were optimized using simulated annealing and restrained energy minimization. The average root-mean-square (r.m.s.) deviation for the best 20 refined structures is 1.07 angstroms for the backbone and 1.92 angstroms for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are more well defined, with r.m.s. deviations of 0.64 angstroms for the backbone and 1.51 angstroms for all atoms. It is found that the des-[Phe(B25)] insulin is a monomer under the applied conditions (4.6 to 4.7 mM, pH 3.0, 310 K), that the overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer of native insulin are preserved, and that the conformation-averaged NMR solution structure is close to the structure of molecule 1 in the hexamer. The structure reveals that the lost ability of des-[Phe(B25)] insulin to self-associate is caused by a conformational change of the C-terminal region of the B-chain, which results in an intra-molecular hydrophobic interaction between Pro(B28) and the hydrophobic region Leu(B11)-Leu(B15) of the B-chain alpha-helix. This interaction interferes with the inter-molecular hydrophobic interactions responsible for the dimerization of native insulin, depriving the mutant of the ability to dimerize. Further, the structure displays a series of features that may explain the high potency of the mutant on the basis of the current model for the insulin-receptor interaction. These features are: a change in conformation of the C-terminal region of the B-chain, the absence of strong hydrogen bonds between this region and the rest of the molecule, and a relatively easy accessibility to the Val(A3) residue.     

Conformation of desmopressin, an analogue of the peptide hormone vasopressin, in aqueous solution as determined by NMR spectroscopy

Desmopressin (1-desamino-[DArg8]vasopressin, is a synthetic analogue of the neurohypophyseal peptide hormone vasopressin which has high antidiuretic and antibleeding potency. The structure of desmopressin has been determined in aqueous solution by two-dimensional NMR techniques and molecular dynamics simulations. Both standard and time-averaged distance restraints were used in structure calculations because of the inherent flexibility in small peptides. 21 models calculated with standard restraints were compared with structures refined with time-averaged distance restraints and were found to be good representatives of the conformational ensemble of desmopressin. The macrocyclic ring forms an inverse gamma-turn centered around Gln4. Residues 1 and 2, the disulphide bridge and the three-residue acyclic tail were found to be flexible in solution. Residues 4-6 in the ensemble of calculated structures contain essentially the same backbone conformation as in the crystal structure of pressinoic acid, the cyclic moiety of vasopressin, whereas residues 2-6 superimpose on the NMR-derived conformation of oxytocin bound to neurophysin. The results presented in this work suggest that, in addition to the differences in sequence between desmopressin and vasopressin, differences in conformational and dynamic properties between the two compounds explain their pharmacological differences.     

Dynamical studies of peptide motifs in the Plasmodium falciparum circumsporozoite surface protein by restrained and unrestrained MD simulations

The immunodominant region on the circumsporozoite surface (CS) protein of the malaria parasite Plasmodium falciparum contains 37 repeated copies of a asparagine-alanine-asparagine-proline (NANP) motif NMR studies of linear synthetic peptides containing one, two or three repeat units provided evidence for nascent type I beta-turns within the NPNA cadence in aqueous solution. The beta-turns could be stabilised upon substituting proline for alpha-methylproline (p(Me)) in the dodecamer (NP(Me)NA)3, without loss of the ability to elicit antibodies cross-reactive with P. falciparum sporozoites. In this work, four 4 ns MD simulations of the dodecapeptide Acetyl-(NP(Me)NA)3, in water, using NOE distance restraints, using 3J-coupling constant restraints, using both these restraints and without restraints, were carried out to determine the conformations of this peptide in aqueous solution. An unrestrained MD simulation of the unmethylated Ac-(NPNA)3 peptide in water was also carried out to investigate the effect of the additional methyl groups on the structure and dynamics of the peptide. The application of NOE distance restraints and 3J-coupling constant restraints leads to contradictory results, probably due to different averaging time scales inherent to the measurement of these data, which exceed the 100 ps averaging applied in the simulations. The additional methyl groups lead to more compact structures, which display enhanced local fluctuations. The central tetrapeptide adopts a type I beta-turn, while the outer motifs display more conformational variability. The three motifs in the methylated dodecamer peptide, however, adopt frequently in the distance restrained MD simulation a compact structure such that the outer motifs appear to form a hydrophobic core by stacking of their two proline rings. This arrangement also suggests how a peptide containing multiple tandemly linked copies of a stable beta-turn NPNA motif might adopt a folded stem-like structure, which conceivably may be of biological relevance in the native CS protein.     

Nuclear magnetic resonance solution structure of the fushi tarazu homeodomain from Drosophila and comparison with the Antennapedia homeodomain

The three-dimensional structure of a recombinant 70-residue polypeptide containing the complete fushi tarazu (ftz) homeodomain from Drosophila melanogaster has been determined by nuclear magnetic resonance (NMR) spectroscopy in solution. On the basis of 915 upper distance constraints derived from nuclear Overhauser effects and 178 dihedral angle constraints, a group of 20 conformers representing the solution structure of the ftz homeodomain was computed with the program DIANA and energy-minimized with the program OPAL. The average of the pairwise root-mean-square deviations of the individual NMR conformers relative to the mean coordinates is 0.50 A for the backbone atoms N, C alpha and C' of residues 8 to 53. The molecular architecture includes three helices comprising the residues 10 to 21, 28 to 38, and 42 to 52, a loop of residues 22 to 27 between the helices I and II, and a turn of residues 39 to 41 linking the helices II and III. Comparisons with the structure of the mutant Antennapedia homeodomain with Cys39 replaced by Ser, Antp (C39S), shows that the two proteins contain the same molecular fold for residues 8 to 53, whereas the more flexible fourth helix comprising residues 53 to 59 in the Antp (C39S) homeodomain has no counterpart in the ftz homeodomain. Considering that important intermolecular interactions in the DNA complexes with the Antp, engrailed and Mat alpha 2 homeodomains involve the fourth helix, it was rather unexpected that the stability of the complex of ftz with the BS2 operator site was found to be comparable to or even somewhat higher than that of the Antp complex with BS2. Another difference is that the Antp homeodomain is more stable with respect to thermal denaturation, with denaturation temperatures at pH 4.8 of 27 degrees C and 48 degrees C, respectively, for ftz and Antp.     

Solution structure of Lqh-8/6, a toxin-like peptide from a scorpion venom--structural heterogeneity induced by proline cis/trans isomerization

Lqh-8/6 is a minor fraction isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus. Here we describe the purification, amino acid sequencing and solution structure determination by NMR and molecular modeling of this peptide. Lqh-8/6 is a small polypeptide (38 residues) which contains 8 half-cystines and is highly similar to another venom component, chlorotoxin. Standard homonuclear methods were used to sequentially assign the proton NMR spectra and to collect spatial restraints for structure determination. Two populations, identified early in the assignment step, are in slow interconversion on the NMR timescale. The two conformers were shown to originate from a cis/trans peptidyl-prolyl isomerization. Using a distance geometry program and simulated annealing protocol under the NMR restraints we obtained 10 final structures for the major conformation (trans isomer). None of the structures showed NOE violations larger than 0.05 nm, and the rmsd value relative to the mean structure (considering the main chain atoms in well-defined secondary structure) is 0.07 nm. The three-dimensional structure contains a short alpha-helix strapped on a small antiparallel beta-strand and an N-terminal extended fragment. The sequence/structure and structure/function relationships of the new scorpion toxin-like peptide are discussed in the context of the present structure determination. This toxin shows a stable, highly populated cis conformer of a peptidyl-prolyl peptide bond.     

Graded compression ultrasonography in the assessment of the "tough decision" acute abdomen in childhood

The three-dimensional structure of omega-conotoxin MVIID has been determined in aqueous solution by two-dimensional 1H NMR techniques. A total of 267 relevant upper-bound distance restraints were used to obtain a family of convergent structures using molecular dynamics methods. A standard simulated annealing protocol using the XPLOR program included in ARIA provided a total of 18 final structures. The averaged RMSD between these structures and the mean atomic coordinates was 0.8 +/- 0.3 A for the backbone atoms. The highest mobility was observed in the segments between residues 10 to 13, comprising Tyr 13, one of the residues shown to be important for binding of omega-conotoxin GVIA and MVIIA to N-type calcium channels. The three-dimensional structure is stabilised by the three disulfide bonds and includes a short antiparallel beta-strand between residues 5-8, 23-25 and 19-21. The folding for this non-N-type calcium channel blocker is similar to that previously calculated for omega-conotoxins GVIA, MVIIA and MVIIC. This suggests the disulfide bond pattern fixes the structure. The reported three-dimensional information can be used to advantage in order to highlight the structural parameters involved in discrimination among calcium channel subtypes.     

The effect of point mutations on the free energy of transmembrane alpha-helix dimerization

Glycophorin A forms homodimers through interaction of the single, helical transmembrane domains of the monomers. The dimers are stable in sodium dodecylsulfate (SDS), permitting a number of studies that have identified a critical motif of residues that mediates dimer formation. We have used analytical ultracentrifugation to measure the energy of dimerization in a non-denaturing detergent solution and have observed the changes in energy arising from two of the mutants previously studied. Use of the detergent pentaoxyethylene octyl ether (C8E5) is a great advantage, since its micelles are neutrally buoyant and the detergent allows a reversible association to occur between monomer and dimer states of the glycophorin A transmembrane helices during the time-scale of sedimentation equilibrium. Use of this detergent in analytical ultracentrifugation may enable a wide range of studies of molecular association events in membrane proteins. We find that the glycophorin A transmembrane helix dimerizes with a dissociation constant of 240(+/-50) nM, corresponding to a free energy of dissociation of 9.0(+/-0.1) kcal mol-1. Point mutants that were found to be disruptive in SDS (L75A, I76A) reduced the dimer affinity in the C8E5 detergent environment (Kd=1.7(+/-0.2) microM and 4.2(+/-0.9) microM, respectively). Thus, the earlier findings are placed on a quantitative, relative energy scale of association by our measurements. Molecular modeling and simulations suggest that the energy differences can be accounted for as changes in van der Waals interactions between helices.     

Investigation of the solution structure of chymotrypsin inhibitor 2 using molecular dynamics: comparison to x-ray crystallographic and NMR data

The native solution structure and dynamics of chymotrypsin inhibitor 2 (CI2) have been studied using a long (5.3 ns) molecular dynamics (MD) simulation without any imposed restraints. The majority of the experimentally observed spin-spin coupling constants, short- and long-range nuclear Overhauser effect (NOE) cross peaks and the amide hydrogen exchange behavior were reproduced by the MD simulation. This good correspondence suggests that the major structural features of the protein during the simulation are representative of the true protein structure in solution. Two water molecules formed hydrogen bond bridges between beta2 and beta3, in agreement with X-ray crystallographic data and a recent reassessment of the solution structure using time-averaged NMR restraints during MD refinement. The active-site loop of the protein displayed the greatest structural changes and the highest mobility. When this loop region was excluded, the average Calpha r.m.s. deviation of the simulated solution structures from the crystal structure was approximately 1.5 Angstrom from 0.5 to 5.3 ns. There is structural heterogeneity in particular regions of the NMR-derived solution structures, which could be a result of imprecision or true internal motion. A study of the distribution of mobility through the protein allows us to distinguish between these two alternatives. In particular, deviations in the active-site loop appear to be a result of heightened mobility, which is also supported by good correspondence between calculated and experimental S2 N-H order parameters. On the other hand, other ill-defined regions of the NMR-derived structures are well defined in the simulation and are probably the result of a lack of structural restraints (i.e. NOEs), as opposed to reflecting the true mobility.