Effect of calcium (II) and magnesium (II) ions on the 18-23 gamma-carboxyglutamic acid containing cyclic peptide loop of bovine prothrombin. An AMBER molecular mechanics study

The effect of calcium (II) and magnesium (II) ions on the conformation of the 18-23 cyclic peptide loop of bovine prothrombin are investigated by the molecular mechanics program AMBER (Assisted Model Building with Energy Refinement). The work is an extension of an earlier paper (Eastman et al., Int. J. Peptide Protein Res. 27, 1986, 530-553) that employed the program ECEPP (Empirical Conformational Energy Program for Peptides). In the absence of either metal ion, or in the presence of either one Ca(II) or one Mg(II) ion, the lowest-energy forms found by AMBER have the Gla21-Pro22 peptide bond in a trans conformation. In the presence of two Ca(II) or Mg(II) ions, the loop form of lowest energy is decidedly cis. The coordination about the Ca(II) and Mg(II) ions is different in both the single and double metal cases. In addition, the peptide chains that emerge from the loop are oriented parallel to each other in the lowest-energy complex with two Ca(II) ions, but are not parallel in the lowest-energy complex with two Mg(II) ions.     

Stress and strain in staphylococcal nuclease.

Protein molecules generally adopt a tertiary structure in which all backbone and side chain conformations are arranged in local energy minima; however, in several well-refined protein structures examples of locally strained geometries, such as cis peptide bonds, have been observed. Staphylococcal nuclease A contains a single cis peptide bond between residues Lys 116 and Pro 117 within a type VIa beta-turn. Alternative native folded forms of nuclease A have been detected by NMR spectroscopy and attributed to a mixture of cis and trans isomers at the Lys 116-Pro 117 peptide bond. Analyses of nuclease variants K116G and K116A by NMR spectroscopy and X-ray crystallography are reported herein. The structure of K116A is indistinguishable from that of nuclease A, including a cis 116-117 peptide bond (92% populated in solution). The overall fold of K116G is also indistinguishable from nuclease A except in the region of the substitution (residues 112-117), which contains a predominantly trans Gly 116-Pro 117 peptide bond (80% populated in solution). Both Lys and Ala would be prohibited from adopting the backbone conformation of Gly 116 due to steric clashes between the beta-carbon and the surrounding residues. One explanation for these results is that the position of the ends of the residue 112-117 loop only allow trans conformations where the local backbone interactions associated with the phi and psi torsion angles are strained. When the 116-117 peptide bond is cis, less strained backbone conformations are available. Thus the relaxation of the backbone strain intrinsic to the trans conformation compensates for the energetically unfavorable cis X-Pro peptide bond. With the removal of the side chain from residue 116 (K116G), the backbone strain of the trans conformation is reduced to the point that the conformation associated with the cis peptide bond is no longer favorable.     

The importance of anchorage in determining a strained protein loop conformation.

We examine the role of the conformational restriction imposed by constrained ends of a protein loop on the determination of a strained loop conformation. The Lys 116-Pro 117 peptide bond of staphylococcal nuclease A exists in equilibrium between the cis and trans isomers. The folded protein favors the strained cis isomer with an occupancy of 90%. This peptide bond is contained in a solvent-exposed, flexible loop of residues 112-117 whose ends are anchored by Val 111 and Asn 118. Asn 118 is constrained by 2 side-chain hydrogen bonds. We investigate the importance of this constraint by replacing Asn 118 with aspartate, alanine, and glycine. We found that removing 1 or more of the hydrogen bonds observed in Asn 118 stabilizes the trans configuration over the cis configuration. By protonating the Asp 118 side chain of N118D through decreased pH, the hydrogen bonding character of Asp 118 approached that of Asn 118 in nuclease A, and the cis configuration was stabilized relative to the trans configuration. These data suggest that the rigid anchoring of the loop end is important in establishing the strained cis conformation. The segment of residues 112-117 in nuclease A provides a promising model system for study of the basic principles that determine polypeptide conformations. Such studies could be useful in the rational design or redesign of protein molecules.     

Structure of Ca2+ prothrombin fragment 1 including the conformation of the Gla domain

The structure of Ca2+ prothrombin fragment 1 has been solved at 2.8-A resolution by X-ray crystallographic methods. Most of the Gla domain of fragment 1 (residues 1-48), which is high homologous with the N-terminal regions of six other blood proteins, cannot be identified in the electron density map of the apo structure. This is not the case when crystals are grown in the presence of Ca2+ ions where the Gla domain exhibits a well-defined folded structure. The folding of the Gla domain is dominated by secondary structure: (a) 3.0 turns of alpha-helix (25%) and (b) five short beta-strands arranged into two beta-structural units (40%). The Cys18-Cys23 disulfide of the small conserved loop of Gla domains is close to a cluster of conserved aromatic residues. The resulting interaction is probably responsible for the fluorescence quenching event accompanying Ca2+ ion binding. Since the Gla domain approximates a discoid, all the Gla residues are easily accessible to solvent. The arrangement of the paired Gla residues (7-8, 20-21, 26-27) is highly suggestive in that they essentially line one edge of the Gla domain creating a potentially intense electronegative environment. This region might well be that associated with phospholipid binding. The kringle structure of Ca2+ fragment 1 is essentially indistinguishable from that of the apoprotein at this stage.     

Cyclic retro-inverso dipeptides with two aromatic side chains. II. Conformational analysis.

The conformations of cis and trans cyclic retro-inverso dipeptides--2-[(4-hydroxy)benzyl]-5-benzyl-4,6(1H,2H,3H,5H)-pyrimidinedi one (c[mTyr-gPhe]), and 2-benzyl-5-amino-5-[(4-hydroxy)benzyl]-4,6(1H,2H,3H,5H)-pyrimidinedione (c[mTyr-gPhe]), and 2-benzyl-5-amino-5-[(4-hydroxy)benzyl]-4,6(1H,2H,3H,5H)-pyrimidinedione (c[(alpha-amino)mTyr-gPhe])--and the parent cyclic dipeptides--c[tyrosyl-phenylalanine] (cis-c[L-Tyr-L-Phe]) and c[tyrosyl-D-phenylalanine] (trans-c[L-Tyr-D-Phe])--were studied by using 1H-nmr spectroscopy and semiempirical energy calculations. In the cis compounds of all the cyclic retro-inverso and parent dipeptides, the most stable conformer has both aromatic side chains sharing the space over the backbone ring in a "face-to-face" fashion. All the trans compounds predominantly assume a "sandwich" conformation in which the two aromatic rings are folded back over the backbone ring on opposite sides. However, different conformational preferences were observed for the backbones between the retro-inverso and parent cyclic dipeptides. The parent cyclic dipeptide trans-c[L-Tyr-D-Phe] adopts two types of boat structures with different side-chain orientations in almost equal amounts: one with the Tyr side chain in a pseudoaxial position and the Phe side chain in a pseudoequatorial position, the other with the Tyr side chain in a pseudoequatorial position and the Phe side chain in a pseudoaxial position. On the other hand, the cyclic retro-inverso dipeptides trans-c[mPhe-gTyr] and trans c[mTyr-gPhe] assume only one type of boat structure in which the malonyl side chain is in a pseudoequatorial and the gem-diamino side chain is in a pseudoaxial position. In addition to the preferred conformations, the conformational energies of the C alpha--C beta bonds in the malonyl and gem-diamino residues were estimated from the temperature variation of vicinal 1H--1H coupling constants for the H--C alpha--C beta--H groupings observed for the trans isomers of cyclic retro-inverso dipeptides. The energies were evaluated to be 1.1 and 1.8 kcal mol-1 for the malonyl and gem-diamino residues, respectively. Applying these energies to the parent cyclic dipeptide trans-c[L-Tyr-D-Phe], the observed fractions of three side-chain conformations are reasonably reproduced. The conformational energies as well as conformational properties of the molecules estimated in this investigation may be useful to refine force constants for both parent and retro-inverso peptides with aromatic side chains.     

Laser Raman spectroscopy of calf bone Gla protein

The Raman spectra of solid calf bone Gla protein in its native state, decarboxylated, with reduced disulfide bond, and as the calcium salt have been obtained. The amide I and III bands are consistent with the presence of alpha-helical, antiparallel beta-sheet, and random-coil regions in all four forms of bone Gla protein. Random coil appears to be the prevailing conformation. The protein conformation in the calcium salt exhibits an increased alpha-helix character compared to the native protein. No significant differences in the backbone conformation are observed among the native, decarboxylated, and reduced forms of bone Gla protein. The Raman band at 504 cm-1, due to the disulfide stretching vibration in native bone Gla protein, is unchanged upon decarboxylation and binding of Ca2+ to the protein, indicating the absence of any changes in the conformation around the disulfide bond in these protein species. The tryptophan and most of the tyrosine residues appear to be 'exposed' rather than 'buried' in the native protein. The environment of at least one of the phenylalanine residues changes when Ca2+ is bound to bone Gla protein. A small change also appears to take place in the environment of at least one of the tyrosine residues upon Ca2+-binding or reduction of the disulfide bond.     

A comparison of the CHARMM, AMBER and ECEPP potentials for peptides. I. Conformational predictions for the tandemly repeated peptide (Asn-Ala-Asn-Pro)9

A search for low-energy helical and near-helical conformations of the tandemly repeated peptide (Asn-Ala-Asn-Pro)9 was undertaken by minimization of the CHARMM potential energy function from eight starting conformations; the latter were obtained from the two low-energy conformations of this repeated peptide found by Gibson & Scheraga, Proc. Natl. Acad. Sci. USA 83, 5649-5653 (1986), and the single conformation found by Brooks et al., Proc. Natl. Acad. Sci. USA 84, 4470-4474 (1987), and from modifications of these three conformations. The same eight starting conformations, as determined by dihedral angles, were used for minimizations of the AMBER and ECEPP potentials. Comparison of the final conformations by least-squares superposition of their C alpha atoms, and by inspection of the parameters of the ideal helix or coiled coil that most closely matched the coordinates of their C alpha atoms in a least-squares sense, showed that: (1) energy minimization, starting from the same conformation but using any two different potentials, could lead to final conformations whose resemblance to each other varied from acceptable to highly unsatisfactory; (2) the ordering of the final energy-minimized conformations, and the energy differences between them, were quite different for all three potentials; (3) the extent of agreement or disagreement between pairs of conformations generated using CHARMM and AMBER, CHARMM and ECEPP, or AMBER and ECEPP, respectively, was not significantly different. The lowest-energy conformation generated using each of the potentials was a left-handed helix, whose pitch and number of residues per turn were similar to those of the left-handed helix found by Gibson & Scheraga. Although the starting conformation which led to the lowest-energy conformation was different for all three potentials, pairwise superposition of the C alpha atoms in the final conformations showed root-mean-square deviations of only 1.0-1.3 A. It is concluded that energy minimizations starting from a large enough sample of initial conformations might on occasion lead to essentially the same conformational prediction whichever potential is used; however, if the sample of starting points is small, predictions based on the three potentials will usually diverge.     

Characterization of secondary amide peptide bond isomerization: thermodynamics and kinetics from 2D NMR spectroscopy

Secondary amide cis peptide bonds are of even lower abundance than the cis tertiary amide bonds of prolines, yet they are of biochemical importance. Using 2D NMR exchange spectroscopy (EXSY) we investigated the formation of cis peptide bonds in several oligopeptides: Ac-G-G-G-NH(2) , Ac-I-G-G-NH(2) , Ac-I-G-G-N-NH(2) and its cyclic form: I-G-G-N in dimethylsulfoxide (DMSO). From the NMR studies, using the amide protons as monitors, an occurrence of 0.13-0.23% of cis bonds was obtained at 296 K. The rate constants for the trans to cis conversion determined from 2D EXSY spectroscopy were 4-9 × 10(-3) s(-1) . Multiple minor conformations were detected for most peptide bonds. From their thermodynamic and kinetic properties the cis isomers are distinguished from minor trans isomers that appear because of an adjacent cis peptide bond. Solvent and sequence effects were investigated utilizing N-methylacetamide (NMA) and various peptides, which revealed a unique enthalpy profile in DMSO. The cyclization of a tetrapeptide resulted in greatly lowered cis populations and slower isomerization rates compared to its linear counterpart, further highlighting the impact of structural constraints.     

Calcium-dependent alpha-helical structure in osteocalcin

Osteocalcin is an abundant Ca2+-binding protein of bone containing three residues of vitamin K dependent gamma-carboxyglutamic acid (Gla) among its 49 (human, monkey, cow) or 50 (chicken) amino acids. Gla side chains participate directly in the binding of Ca2+ ions and the adsorption of osteocalcin to hydroxylapatite (HA) surfaces in vivo and in vitro. Osteocalcin exhibits a major conformational change when Ca2+ is bound. Metal-free chicken osteocalcin is a random coil with only 8% of its residues in the alpha helix as revealed by circular dichroism. In the presence of physiological levels of Ca2+, 38% of the protein adopts the alpha-helical conformation with a transition midpoint at 0.75 mM Ca2+ in a rapid, reversible fashion which (1) requires an intact disulfide bridge, (2) is proportionally diminished when Gla residues are decarboxylated to Glu, (3) is insensitive to 1.5 m NaCl, and (4) can be mimicked by other cations. Tyr fluorescence, UV difference spectra, and Tyr reactivity to tetranitromethane corroborate the conformational change. Homologous monkey osteocalcin also exhibits Ca2+-dependent structure. Integration of predictive calculations from osteocalcin sequence has yielded a structural model for the protein, the dominant features of which include two opposing alpha-helical domains of 9-12 residues each, connected by a bea turn and stabilized by the Cys23-Cys29 disulfide bond. Cation binding permits realization of the full alph a-helical potential by partial neutralization of high anionic charge in the helical domains. Periodic Gla occurrence at positions 17, 21, and 24 has been strongly conserved throughout evolution and places all Gla side chains on the same face of one alpha helix spaced at intervals of approximately 5.4 A, closely paralleling the interatomic separation of Ca2+ in the HA lattice. Helical osteocalcin has greatly increased affinity for HA; thus, the Ca2+-induced structural transition may perform an informational role related to bone metabolism.     

Escherichia coli thioredoxin folds into two compact forms of different stability to urea denaturation

The urea-induced denaturation of Escherichia coli thioredoxin and thioredoxin variants has been examined by electrophoresis on urea gradient slab gels by the method of Creighton [Creighton, T. (1986) Methods Enzymol. 131, 156-172]. Thioredoxin has only two cysteine residues, and these form a redox-active disulfide at the active site. Oxidized thioredoxin-S2 and reduced thioredoxin-(SH)2 each show two folded isomers with a large difference in stability to urea denaturation. The difference in stability is greater for the isomers of oxidized than for the isomers of reduced thioredoxin. At 2 degrees C, the urea concentrations at the denaturation midpoint are approximately 8 and 4.3 M for the oxidized isomers and 4.8 and 3.7 M for the reduced isomers. The difference between the gel patterns of samples applied in native versus denaturing buffer, and at 2 and 25 degrees C, is characteristic for the involvement of a cis-proline-trans-proline isomerization. The data very strongly suggest that the two folded forms of different stabilities correspond to the cis and trans isomers of the highly conserved Pro 76 peptide bond, which is cis in the crystal structure of oxidized thioredoxin. Urea gel experiments with the mutant thioredoxin P76A, with alanine substituted for proline at position 76, corroborate this interpretation. The electrophoretic banding pattern diagnostic for an involvement of proline isomerization in urea denaturation is not observed for oxidized P76A. In broad estimates of delta G degree for the native-denatured transition, the difference in delta G degree (no urea) between the putative cis and trans isomers of the Ile 75-Pro 76 peptide bond is approximately 3 kcal/mol for oxidized thioredoxin and approximately 1.5 kcal/mol for reduced thioredoxin. Since cis oxidized thioredoxin is much more stable than trans, folded oxidized thioredoxin is essentially all cis. In folded reduced thioredoxin, cis and trans interconvert slowly, on the minute time scale at 2 and 25 degrees C. In the absence of urea, the folded reduced thioredoxin is less than a few percent trans. Three additional mutants with additions or substitutions at the active site also show electrophoresis banding patterns consistent with a difference in stability between cis and trans isomers.     

The utility of side-chain cyclization in determining the receptor-bound conformation of peptides: cyclic tripeptides and angiotensin II.

The effect of side-chain cyclization on accessible backbone conformations of tripeptides, X-Ala-Y (X and/or Y = Cys, Hcy (Hcy: homocysteine), cis 4-mercaptoproline (MPc), and trans 4-mercaptoproline (MPt)), was elucidated using two variants of systematic conformational search. In addition to cyclization through a disulfide bond, the thioether (-S-CH2-) and amide (-CO-NH-) side-chain analogues of Cys-Ala-Cys and Hcy-Ala-Hcy were evaluated. The number of valid backbone conformations and the allowed phi, psi space were evaluated for each compound, and the ability of the cyclic tripeptides to accommodate beta-turn conformations was examined in order to assess the value of cyclization in limiting conformational freedom. Based on the number of conformations, cyclization was highly effective in reducing the backbone degree of freedom: in order of decreasing number of conformations, Ala-Ala-Ala 1 > Hcy-Ala-Hcy 2 > Cys-Ala-Hcy 3 approximately equal to Hcy-Ala-Cys 4 > MPc-Ala-Hcy 5, 7 > Cys-Ala-Cys 6 > MPc-Ala-Cys 8 > Hcy-Ala-MPt 9 > Cys-Ala-MPt 10 approximately equal to MPc-Ala-MPt 11. Although Hcy-Ala-Hcy 2 had the greatest number of conformations of the cyclic peptides studied, it was still greatly constrained relative to its linear analogue 1. The bicyclic ring system introduced by MP was even more effective in constraining the cycle, having greater impact at position 3 than at position 1. Under the conditions of the study, cyclization of MP-containing analogues could be effected only with the cis isomer (MPc) at position 1 and/or the trans isomer (MPt) at position 3. Sterically allowed conformations of Ala2 for the cyclic tripeptides 2-4 were generally similar to those of the linear tripeptide 1, while those of Cys-Ala-Cys 6 and MPc-Ala-Hcy 7 were restricted to a smaller region of phi 2, psi 2 space: the right- and left-handed alpha-helical conformation and the beta-conformation. This trend was even more pronounced for Hcy-Ala-MPt 9, Cys-Ala-MPt 10, and MPc-Ala-MPt 11, in which Ala2 was severely restricted to a very small region of phi, psi space: the left-handed alpha-helical conformation for 9-11, plus the beta conformation for 9. This suggests that MP at the 3-position is incompatible with a right-handed alpha-helical conformation at position 2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystal structure of Mg2+- and Ca2+-bound Gla domain of factor IX complexed with binding protein

Factor IX is an indispensable protein required in the blood coagulation cascade. It binds to the surface of phospholipid membrane by means of a gamma-carboxyglutamic acid (Gla) domain situated at the N terminus. Recently, we showed that physiological concentrations of Mg2+ ions affect the native conformation of the Gla domain and in doing so augment the biological activity of factor IXa and binding affinity with its binding protein even in the presence of Ca2+ ions. Here we report on the crystal structures of the Mg2+/Ca2+-bound and Ca2+-bound (Mg2+-free) factor IX Gla domain (IXGD1-46) in complex with its binding protein (IX-bp) at 1.55 and 1.80 A resolutions, respectively. Three Mg2+ and five Ca2+ ions were bound in the Mg2+/Ca2+-bound IXGD1-46, and the Mg2+ ions were replaced by Ca2+ ions in Mg2+-free IXGD1-46. Comparison of Mg2+/Ca2+-bound with Ca2+-bound structures of the complexes showed that Mg2+ ion, which formed a bridge between IXGD1-46 and IX-bp, forced IXGD1-46 to rotate 4 degrees relative to IX-bp and hence might be the cause of a more tight interaction between the molecules than in the case of the Mg2+-free structure. The results clearly suggest that Mg2+ ions are required to maintain native conformation and in vivo function of factor IX Gla domain during blood coagulation.     

Graphical representation of the salient conformational features of protein residues.

A composite plot for depicting in two dimensions the conformation and the secondary structural features of protein residues has been developed. Instead of presenting the exact values of the main- and side-chain torsion angles (φ, psi and chi(1)), it indicates the region in the three-dimensional conformational space to which a residue belongs. Other structural aspects, like the presence of a cis peptide bond and disulfide linkages, are also displayed. The plot may be used to recognize patterns in the backbone and side-chain conformation along a polypeptide chain and to compare protein structures derived from X-ray crystallography, NMR spectroscopy or molecular modelling studies and also to highlight the effect of mutation on structure.     

Role of the hexapeptide disulfide loop in the gamma-carboxyglutamic acid domain of protein C in Ca2+-mediated structural and functional properties

The anticoagulant and immunomodulatory effects of protein C (PC) rely on the presence of the N-terminal gamma-carboxyglutamic acid (Gla) domain. This domain is strongly conserved among vitamin K-dependent blood proteins and, in addition to a high relative content of Gla, contains a hexapeptide disulfide loop between Cys residues 17 and 22. In the present study, the contribution of the hexapeptide loop toward Gla domain structure and function was evaluated using wild-type and Cys17/Cys22-alkylated synthetic peptide analogues of the 47-residue Gla domain/helical stack of PC. Circular dichroism and intrinsic fluorescence measurements revealed significant differences in the metal ion-dependent conformations of the two peptides. Disruption of the disulfide loop slightly altered the capacity of the peptide to interact with acidic phospholipid (PL) vesicles. The affinity of the alkylated peptide for soluble endothelial protein C receptor (EPCR), as demonstrated by surface plasmon resonance studies, was increased compared with the wild-type species, although total binding was compromised. These results suggest that the disulfide loop of PC contributes to the overall Ca(2+)-dependent conformation but is not strictly required for PL membrane binding or EPCR recognition.     

A further insight into the binding of blood clotting factors to membranes

The active site of factor Xa, labelled with dansylglutamylglycylarginine (DnsEGR) is sensitive to association with Ca2+, factor Va and phospholipids. When bound to factor Va, DnsEGR-factor-Xa does not change the composition of the binding site of factor Va, as shown by fluorescence energy-transfer experiments between the Trp residues of factor Va and pyrene-labelled phospholipids. Prothrombin was cleaved by alpha-chymotrypsin into two parts: N-terminal residues 1-41 (peptide 1-41) containing the gamma-carboxyglutamic acid residues (Gla), and des-(1-41)-prothrombin; their membrane association was investigated. Peptide 1-41 contains the aromatic residues Tyr and Trp in positions 24 and 41, respectively, and is suitable for fluorescence spectroscopy. The absence of fluorescence energy transfer between these residues suggests that they are more than 2.8 nm apart. Binding of Ca2+ and of phospholipids involves essentially the Tyr residue, while the C-terminal characteristics of the Trp residue remain unchanged. The conformational change which takes place on binding does not shorten the distance between Tyr and Trp beyond 2.8 nm. Our conclusion is that peptide 1-41 has an extended conformation. This result is compatible with the disordered character of the Gla region found in the crystalline structure of fragment 1 of prothrombin. Ca2+ induces a greater fluorescence energy transfer between prothrombin and membranes labelled with pyrene but has no influence on the binding of des-(1-41)-prothrombin. Moreover, the binding curves of des(1-41)-prothrombin are similar to those of prothrombin in the absence of Ca2+. It is concluded that the Ca2+-independent association of prothrombin with membranes involves essentially that part of the prothrombin molecule deleted in the Gla region.     

Crystal and molecular structure of L-valyl-L-lysine hydrochloride.

L-Valyl-L-lysine hydrochloride, C11N3O3H23 HCl, crystallizes in the monoclinic space group P2(1) with a = 5.438(5), b = 14.188(5), c = 9.521(5) A, beta = 95.38(2) degrees and Z = 2. The crystal structure, solved by direct methods, refined to R = 0.036, using full matrix least-squares method. The peptide exists in a zwitterionic form, with the N atom of the lysine side-chain protonated. The two gamma-carbons of the valine side-chain have positional disorder, giving rise to two conformations, chi 1(11) = -67.3 and 65.9 degrees, one of which (65.9 degrees) is sterically less favourable and has been found to be less popular amongst residues branching at beta-C. The lysine side-chain has the geometry of g- tgt, not seen in crystal structures of the dipeptides reported so far. Interestingly, chi 2(3) (63.6 degrees) of lysine side-chain has a gauche+ conformation unlike in most of the other structures, where it is trans. The neighbouring peptide molecules are hydrogen bonded in a head-to-tail fashion, a rather uncommon interaction in lysine peptide structures. The structure shows considerable similarity with that of L-Lys-L-Val HCl in conformational angles and H-bond interactions [4].     

Single proline residues can dictate the oxidative folding pathways of cysteine-rich peptides

The cysteine-rich N and C-terminal domains of minicollagen-1 from Hydra nematocysts fold with excesses of oxidized/reduced glutathione (10:1) into globular structures with distinct cystine frameworks despite their identical cysteine sequence pattern. An additional main difference is the cis conformation of a conserved proline residue in the N-terminal and the trans conformation of this residue in the C-terminal domain. Comparative analysis of the oxidative folding revealed for the C-terminal domain a fast and highly cooperative formation of a single disulfide isomer. Conversely, oxidation of the N-terminal domain proceeds mainly via an intermediate that results from the fast quasi-stochastic disulfide formation according to the proximity rule. The rate of conversion of the bead-like isomer into the globular end-product is largely dominated by the trans-to-cis isomerization of the critical proline residue as well assessed by its replacement with (4R)- and (4S)-fluoroproline known to exhibit distinct propensities for the trans and cis conformation, respectively. Independently, whether the trans or cis conformation is favored by these substitutions, both analogues retain sufficient sequence-encoded information to fold almost quantitatively into the identical cystine framework and thus spatial structure of the parent peptide with the critical proline residue as cis isomer, but at rates significantly lower for the (4R) than for the (4S)-fluoroproline analogue. Correspondingly, other sequence-encoded structural elements have to act as a driving force for these unidirectional folding pathways despite the rather simple sequence composition consisting only of aliphatic residues, some proline and only one aromatic residue (tyrosine) in the core parts of the C and N-terminal domains. The two cysteine-rich domains of minicollagen-1 may well represent ideal targets for ab initio structure calculations in order to learn more about the elementary information encoded in such primordial molecules.     

Two-dimensional NMR and structure determination of salmon calcitonin in methanol

The structure of the 32-residue peptide salmon calcitonin (sCT) in 90% MeOH-10% H2O has been investigated by two-dimensional NMR techniques and molecular modeling. Sequential assignments for nearly all of the 32 spin systems have been obtained, and results indicate that the heptaresidue loop formed by the disulfide bond between Cys-1 and Cys-7 is followed by an alpha-helical segment from Val-8 through Tyr-22. A region of conformational heterogeneity is observed for residues 20-25, resulting from the slow isomerism of the cis and trans forms of Pro-23. The C-terminal segment is found to exist in an extended conformation.     

Structural mechanism governing cis and trans isomeric states and an intramolecular switch for cis/trans isomerization of a non-proline peptide bond observed in crystal structures of scorpion toxins

Non-proline cis peptide bonds have been observed in numerous protein crystal structures even though the energetic barrier to this conformation is significant and no non-prolyl-cis/trans-isomerase has been identified to date. While some external factors, such as metal binding or co-factor interaction, have been identified that appear to induce cis/trans isomerization of non-proline peptide bonds, the intrinsic structural basis for their existence and the mechanism governing cis/trans isomerization in proteins remains poorly understood. Here, we report the crystal structure of a newly isolated neurotoxin, the scorpion alpha-like toxin Buthus martensii Karsch (BmK) M7, at 1.4A resolution. BmK M7 crystallizes as a dimer in which the identical non-proline peptide bond between residues 9 and 10 exists either in the cis conformation or as a mixture of cis and trans conformations in either monomer. We also determined the crystal structures of several mutants of BmK M1, a representative scorpion alpha-like toxin that contains an identical non-proline cis peptide bond as that observed in BmK M7, in which residues within or neighboring the cis peptide bond were altered. Substitution of an aspartic acid residue for lysine at residue 8 in the BmK M1 (K8D) mutant converted the cis form of the non-proline peptide bond 9-10 into the trans form, revealing an intramolecular switch for cis-to-trans isomerization. Cis/trans interconversion of the switch residue at position 8 appears to be sequence-dependent as the peptide bond between residues 9 and 10 retains its wild-type cis conformation in the BmK M1 (K8Q) mutant structure. The structural interconversion of the isomeric states of the BmK M1 non-proline cis peptide bond may relate to the conversion of the scorpion alpha-toxins subgroups.