Comparative 2D NMR studies of human insulin and des-pentapeptide insulin: sequential resonance assignment and implications for protein dynamics and receptor recognition

The solution structure and dynamics of human insulin are investigated by 2D 1H NMR spectroscopy in reference to a previously analyzed analogue, des-pentapeptide(B26-B30) insulin (DPI; Hua, Q.X., & Weiss, M.A. (1990) Biochemistry 29, 10545-10555). This spectroscopic comparison is of interest since (i) the structure of the C-terminal region of the B-chain has not been determined in the monomeric state and (ii) the role of this region in binding to the insulin receptor has been the subject of long-standing speculation. The present NMR studies are conducted in the presence of an organic cosolvent (20% acetic acid), under which conditions both proteins are monomeric and stably folded. Complete sequential assignment of human insulin is obtained and leads to the following conclusions. (1) The secondary structure of the insulin monomer (three alpha-helices and B-chain beta-turn) is similar to that observed in the 2-Zn crystal state. (2) The folding of DPI is essentially the same as the corresponding portion of intact insulin, in accord with the similarities between their respective crystal structures. However, differences between insulin and DPI are observed in the extent of conformational broadening of amide resonances, indicating that the presence or absence of residues B26-B30 influences the overall dynamics of the protein on the millisecond time scale. (3) Residues B24-B28 adopt an extended configuration in the monomer and pack against the hydrophobic core as in crystallographic dimers; residues B29 and B30 are largely disordered. This configuration differs from that described in a more organic milieu (35% acetonitrile; Kline, A.D., & Justice, R.M., Jr. (1990) Biochemistry 29, 2906-2913), suggesting that the conformation of insulin in the latter study may have been influenced by solvent composition. (4) The insulin fold is shown to provide a model for collective motions in a protein with implications for the mechanism of protein-protein recognition. To our knowledge, this paper describes the first detailed analysis of a protein NMR spectrum under conditions of extensive conformational broadening. Such an analysis is made possible in the present case by comparative study of an analogue (DPI) with more tractable spectroscopic properties.     

Flexibility and bioactivity of insulin: an NMR investigation of the solution structure and folding of an unusually flexible human insulin mutant with increased biological activity

The structure and folding of a novel human insulin mutant, [Thr(B27) --> Pro, Pro(B28) --> Thr]insulin (PT insulin), in aqueous solution and in mixtures of water and 2,2,2-trifluoroethanol (TFE) have been studied by NMR spectroscopy. It was found that PT insulin has a highly flexible structure in pure water and is present in at least two different conformations, although with an overall tertiary structure similar to that of native insulin. Furthermore, the native helical structures are poorly defined. Surprisingly, the mutant has a biological activity about 50% higher than native insulin. In contrast, in TFE/water solution the mutant reveals a propensity of forming a well-defined structure at the secondary structure level, similar to monomeric native insulin. Thus, as shown by a detailed determination of the structure from 208 distance restraints and 52 torsion angle restraints by distance geometry, simulated annealing, and restrained energy minimization, the native insulin helices (A2-A7, A13-A19, and B10-B19) as well as the beta-turn (B20-B23) are formed in 35% TFE. However, the amount of tertiary structure is decreased significantly in TFE/water solution. The obtained results suggest that only an overall tertiary fold, as observed for PT insulin in pure water, is necessary for expressing the biological activity of insulin, as long as the molecule is flexible and retains the propensity to form the secondary structure required for its receptor binding. In contrast, a compact secondary structure, as found for native insulin in solution, is unnecessary for the biological activity. A model for the receptor binding of insulin is suggested that relates the increased bioactivity to the enhanced flexibility of the mutant.     

Three-dimensional solution structure of an insulin dimer. A study of the B9(Asp) mutant of human insulin using nuclear magnetic resonance, distance geometry and restrained molecular dynamics.

The solution structure of the B9(Asp) mutant of human insulin has been determined by two-dimensional 1H nuclear magnetic resonance spectroscopy. Thirty structures were calculated by distance geometry from 451 interproton distance restraints based on intra-residue, sequential and long-range nuclear Overhauser enhancement data, 17 restraints on phi torsional angles obtained from 3JH alpha HN coupling constants, and the restraints from 17 hydrogen bonds, and the three disulphide bridges. The distance geometry structures were optimized using restrained molecular dynamics (RMD) and energy minimization. The average root-mean-square deviation for the best 20 RMD refined structures is 2.26 A for the backbone and 3.14 A for all atoms if the less well-defined N and C-terminal residues are excluded. The helical regions are better defined, with root-mean-square deviation values of 1.11 A for the backbone and 2.03 A for all atoms. The data analysis and the calculations show that B9(Asp) insulin, in water solution at the applied pH (1.8 to 1.9), is a well-defined dimer with no detectable difference between the two monomers. The association of the two monomers in the solution dimer is relatively loose as compared with the crystal dimer. The overall secondary and tertiary structures of the monomers in the 2Zn crystal hexamer is found to be preserved. The conformation-averaged NMR structures obtained for the monomer is close to the structure of molecule 1 in the hexamer of the 2Zn insulin crystal. However, minor, but significant deviations from this structure, as well as from the structure of monomeric insulin in solution, exist and are ascribed to the absence of the hexamer and crystal packing forces, and to the presence of monomer-monomer interactions, respectively. Thus, the monomer in the solution dimer shows a conformation similar to that of the crystal monomer in molecular regions close to the monomer-monomer interface, whereas it assumes a conformation similar to that of the solution structure of monomeric insulin in other regions, suggesting that B9(Asp) insulin adopts a monomer-like conformation when this is not inconsistent with the monomer-monomer arrangement in the dimer.     

Mutations at the dimer, hexamer, and receptor-binding surfaces of insulin independently affect insulin-insulin and insulin-receptor interactions.

Mutagenesis of the dimer- and hexamer-forming surfaces of insulin yields analogues with reduced tendencies to aggregate and dramatically altered pharmacokinetic properties. We recently showed that one such analogue, HisB10----Asp, ProB28----Lys, LysB29----Pro human insulin (DKP-insulin), has enhanced affinity for the insulin receptor and is useful for studying the structure of the insulin monomer under physiologic solvent conditions [Weiss, M. A., Hua, Q. X., Lynch, C. S., Frank, B. H., & Shoelson, S. E. (1991) Biochemistry 30, 7373-7389]. DKP-insulin retains native secondary and tertiary structure in solution and may therefore provide an appropriate baseline for further studies of related analogues containing additional substitutions within the receptor-binding surface of insulin. To test this, we prepared a family of DKP analogues having potency-altering substitutions at the B24 and B25 positions using a streamlined approach to enzymatic semisynthesis which negates the need for amino-group protection. For comparison, similar analogues of native human insulin were prepared by standard semisynthetic methods. The DKP analogues show a reduced tendency to self-associate, as indicated by 1H-NMR resonance line widths. In addition, CD spectra indicate that (with one exception) the native insulin fold is retained in each analogue; the exception, PheB24----Gly, induces similar perturbations in both native insulin and DKP-insulin backgrounds. Notably, analogous substitutions exhibit parallel trends in receptor-binding potency over a wide range of affinities: D-PheB24 greater than unsubstituted greater than GlyB24 greater than SerB24 greater than AlaB25 greater than LeuB25 greater than SerB25, whether the substitution was in a native human or DKP-insulin background. Such "template independence" reflects an absence of functional interactions between the B24 and B25 sites and additional substitutions in DKP-insulin and demonstrates that mutations in discrete surfaces of insulin have independent effects on protein structure and function. In particular, the respective receptor-recognition (PheB24, PheB25), hexamer-forming (HisB10), and dimer-forming (ProB28, LysB29) surfaces of insulin may be regarded as independent targets for protein design. DKP-insulin provides an appropriate biophysical model for defining structure-function relationships in a monomeric template.     

Changes in secondary structure follow the dissociation of human insulin hexamers: a circular dichroism study

Vacuum UV circular dichroism spectra measured down to 178 nm for hexameric 2-zinc human insulin, zinc-free human insulin, and the two engineered and biologically active monomeric mutants, [B/S9D] and [B/S9D,T27E] human insulin, show significant differences. The secondary structure analysis of the 2-zinc human insulin (T6) in neutral solution was determined: 57% helix, 1% beta-strand, 18% turn, and 24% random coil. This is very close to the corresponding crystal structure showing that the solution and solid structures are similar. The secondary structure of the monomer shows a 10-15% increase in antiparallel beta-structure and a corresponding reduction in random coil structure. These structural changes are consistent with an independent analysis of the corresponding difference spectra. The advantage of secondary structure analyses of difference spectra is that the contribution of odd spectral features stemming mainly from side chain chromophores is minimized and the sensitivity of the analyses improved. Analysis of the CD spectra of T6 2-zinc, zinc-free human insulin and monomeric mutant insulin by singular value decomposition indicates that the secondary structure changes following the dissociation of hexamers into dimers and monomers are two-state processes.     

Toward the solution structure of human insulin: sequential 2D 1H NMR assignment of a des-pentapeptide analogue and comparison with crystal structure

2D 1H NMR studies are presented of des-pentapeptide-insulin, an analogue of human insulin lacking the C-terminal five residues of the B chain. Removal of these residues, which are not required for function, is shown to reduce conformational broadening previously described in the spectrum of intact insulin [Weiss et al. (1989) Biochemistry 28, 9855-9873]. This difference presumably reflects more rapid internal motions in the fragment, which lead to more complete averaging of chemical shifts on the NMR time scale. Sequential 1H NMR assignment and preliminary structural analysis demonstrate retention in solution of the three alpha-helices observed in the crystal state and the relative orientation of the receptor-binding surfaces. These studies provide a foundation for determining the solution structure of insulin.     

Two-dimensional NMR and photo-CIDNP studies of the insulin monomer: assignment of aromatic resonances with application to protein folding, structure, and dynamics

The aromatic 1H NMR resonances of the insulin monomer are assigned at 500 MHz by comparative studies of chemically modified and genetically altered variants, including a mutant insulin (PheB25----Leu) associated with diabetes mellitus. The two histidines, three phenylalanines, and four tyrosines are observed to be in distinct local environments; their assignment provides sensitive markers for studies of tertiary structure, protein dynamics, and protein folding. The environments of the tyrosine residues have also been investigated by photochemically induced dynamic nuclear polarization (photo-CIDNP) and analyzed in relation to packing constraints in the crystal structures of insulin. Dimerization involving specific B-chain interactions is observed with increasing protein concentration and is shown to depend on temperature, pH, and solvent composition. In the monomer large variations are observed in the line widths of amide resonances, suggesting intermediate exchange among conformational substates; such substates may relate to conformational changes observed in different crystal states and proposed to occur in the hormone-receptor complex. Additional evidence for multiple conformations in solution is provided by comparative studies of an insulin analogue containing a peptide bond between residues B29 and A1 (mini-proinsulin). This analogue forms dimers and higher-order oligomers under conditions in which native insulin is monomeric, suggesting that the B29-A1 peptide bond stabilizes a conformational substate favorable for dimerization. Such stabilization is not observed in corresponding studies of native proinsulin, in which a 35-residue connecting peptide joins residues B30 and A1; this extended tether is presumably too flexible to constrain the conformation of the B-chain. The differences between proinsulin and mini-proinsulin suggest a structural mechanism for the observation that the fully reduced B29-A1 analogue folds more efficiently than proinsulin to form the correct pattern of disulfide bonds. These results are discussed in relation to molecular mechanics calculations of insulin based on the available crystal structures.     

Crystal structure of allo-Ile(A2)-insulin, an inactive chiral analogue: implications for the mechanism of receptor binding

The crystal structure of an inactive chiral analogue of insulin containing nonstandard substitution allo-Ile(A2) is described at 2.0 A resolution. In native insulin, the invariant Ile(A2) side chain anchors the N-terminal alpha-helix of the A-chain to the hydrophobic core. The structure of the variant protein was determined by molecular replacement as a T(3)R(3) zinc hexamer. Whereas respective T- and R-state main-chain structures are similar to those of native insulin (main-chain root-mean-square deviations (RMSD) of 0.45 and 0.54 A, respectively), differences in core packing are observed near the variant side chain. The R-state core resembles that of the native R-state with a local inversion of A2 orientation (core side chain RMSD 0.75 A excluding A2); in the T-state, allo-Ile(A2) exhibits an altered conformation in association with the reorganization of the surrounding side chains (RMSD 0.98 A). Surprisingly, the core of the R-state is similar to that observed in solution nuclear magnetic resonance (NMR) studies of an engineered T-like monomer containing the same chiral substitution (allo-Ile(A2)-DKP-insulin; Xu, B., Hua, Q. X., Nakagawa, S. H., Jia, W., Chu, Y. C., Katsoyannis, P. G., and Weiss, M. A. (2002) J. Mol. Biol. 316, 435-441). Simulation of NOESY spectra based on crystallographic protomers enables the analysis of similarities and differences in solution. The different responses of the T- and R-state cores to chiral perturbation illustrates both their intrinsic plasticity and constraints imposed by hexamer assembly. Although variant T- and R-protomers retain nativelike protein surfaces, the receptor-binding activity of allo-Ile(A2)-insulin is low (2% relative to native insulin). This seeming paradox suggests that insulin undergoes a change in conformation to expose Ile(A2) at the hormone-receptor interface.     

Two-dimensional NMR studies of Des-(B26-B30)-insulin: sequence-specific resonance assignments and effects of solvent composition.

Des-pentapeptide-insulin (DPI), a monomeric analogue which lacks the C-terminal five residues of the B-chain, provides a tractable model for 2D-NMR studies of insulin under a variety of solvent conditions. In this paper we present the sequential assignment of DPI at pH 1.8 and 25 degrees C in 10% deuterated DMSO/90% H2O; the chemical shifts are in general similar to those recently described in the absence of an organic cosolvent [1], in 20% acetic acid [2] and (for intact insulin) in 35% acetonitrile [3]. Under each of these solvent conditions qualitative analysis of the 2D-NMR data indicates that the major elements of secondary structure observed in the crystal state (three alpha-helices and B-chain beta-turn) are retained in solution. However, there is disagreement in the literature regarding the stability of the insulin fold, as monitored by amide-proton exchange rates and long-range nuclear Overhauser enhancements [1-3]. In contrast to a previous study [1], we observe slowly exchanging amide resonances (in freshly prepared D2O solutions) and nonlocal NOEs under each of the solvent conditions described, implying the existence of a stably folded secondary structure and hydrophobic core. The slowly-exchanging resonances are assigned to the central alpha-helix of the B-chain, the ends of the adjoining beta-turn, and the two A-chain alpha-helices. Qualitative analysis of long-range NOEs indicates that the major features of the crystal state are retained under these solvent conditions.     

Proton nuclear magnetic resonance study of the B9(Asp) mutant of human insulin. Sequential assignment and secondary structure

The sequence-specific 1H nuclear magnetic resonance (n.m.r.) assignment of 49 of the 51 amino acid residues of human B9(Asp) insulin in water at low pH is reported. Spin systems were identified using a series of two-dimensional n.m.r. techniques. For the majority of the amino acid residues with unique spin systems, particularly Ala, Thr, Val, Leu, Ile and Lys, the complete spin systems were identified. Sequence-specific assignments were obtained from sequential nuclear Overhauser enhancement (NOE) connectivities. The results indicate that the solution structure of the mutant closely resembles the crystal structure of native insulin. Thus, the NOE data reveal three helical domains all consistent with the secondary structure of the native human 2Zn insulin in the crystal phase. Numerous slowly exchanging amide protons support these structural elements, and indicate a relatively stable structure of the protein. A corresponding resemblance of the tertiary structures in the two phases is also suggested by slowly exchanging amide protons, and by the extreme chemical shift values observed for the beta-protons of B15(Leu) that agree with a close contact between this residue and the aromatic rings of B24(Phe) and B26(Tyr), as found in the crystal structure of the 2Zn insulin. Finally, there are clear indications that the B9(Asp) insulin mutant exists primarily as a dimer under the given conditions.     

Hierarchical protein "un-design": insulin's intrachain disulfide bridge tethers a recognition alpha-helix

A hierarchical pathway of protein folding can enable segmental unfolding by design. A monomeric insulin analogue containing pairwise substitution of internal A6-A11 cystine with serine [[Ser(A6),Ser(A11),Asp(B10),Lys(B28),Pro(B29)]insulin (DKP[A6-A11](Ser))] was previously investigated as a model of an oxidative protein-folding intermediate [Hua, Q. X., et al. (1996) J. Mol. Biol. 264, 390-403]. Its structure exhibits local unfolding of an adjoining amphipathic alpha-helix (residues A1-A8), leading to a 2000-fold reduction in activity. Such severe loss of function, unusual among mutant insulins, is proposed to reflect the cost of induced fit: receptor-directed restoration of the alpha-helix and its engagement in the hormone's hydrophobic core. To test this hypothesis, we have synthesized and characterized the corresponding alanine analogue [[Ala(A6),Ala(A11),Asp(B10),Lys(B28), Pro(B29)]insulin (DKP[A6-A11](Ala))]. Untethering the A6-A11 disulfide bridge by either amino acid causes similar perturbations in structure and dynamics as probed by circular dichroism and (1)H NMR spectroscopy. The analogues also exhibit similar decrements in thermodynamic stability relative to that of the parent monomer as probed by equilibrium denaturation studies (Delta Delta G(u) = 3.0 +/- 0.5 kcal/mol). Despite such similarities, the alanine analogue is 50 times more active than the serine analogue. Enhanced receptor binding (Delta Delta G = 2.2 kcal/mol) is in accord with alanine's greater helical propensity and more favorable hydrophobic-transfer free energy. The success of an induced-fit model highlights the applicability of general folding principles to a complex binding process. Comparison of DKP[A6-A11](Ser) and DKP[A6-A11](Ala) supports the hypothesis that the native A1-A8 alpha-helix functions as a preformed recognition element tethered by insulin's intrachain disulfide bridge. Segmental unfolding by design provides a novel approach to dissecting structure-activity relationships.     

Heteronuclear 2D NMR studies of an engineered insulin monomer: assignment and characterization of the receptor-binding surface by selective 2H and 13C labeling with application to protein design

Insulin provides an important model for the application of genetic engineering to rational protein design and has been well characterized in the crystal state. However, self-association of insulin in solution has precluded complementary 2D NMR study under physiological conditions. We demonstrate here that such limitations may be circumvented by the use of a monomeric analogue that contains three amino acid substitutions on the protein surface (HisB10----Asp, ProB28----Lys, and LysB29----Pro); this analogue (designated DKP-insulin) retains native receptor-binding potency. Comparative 1H NMR studies of native human insulin and a series of three related analogues--(i) the singly substituted analogue [HisB10----Asp], (ii) the doubly substituted analogue [ProB28----Lys; LysB29----Pro], and (iii) DKP-insulin--demonstrate progressive reduction in concentration-dependent line-broadening in accord with the results of analytical ultracentrifugation. Extensive nonlocal interactions are observed in the NOESY spectrum of DKP-insulin, indicating that this analogue adopts a compact and stably folded structure as a monomer in overall accord with crystal models. Site-specific 2H and 13C isotopic labels are introduced by semisynthesis as probes for the structure and dynamics of the receptor-binding surface. These studies confirm and extend under physiological conditions the results of a previous 2D NMR analysis of native insulin in 20% acetic acid [Hua, Q. X., & Weiss, M. A. (1991) Biochemistry 30, 5505-5515]. Implications for the role of protein flexibility in receptor recognition are discussed with application to the design of novel insulin analogues.     

Nonlocal structural perturbations in a mutant human insulin: sequential resonance assignment and 13C-isotope-aided 2D-NMR studies of [PheB24-->Gly]insulin with implications for receptor recognition.

Insulin's mechanism of receptor binding is not well understood despite extensive study by mutagenesis and X-ray crystallography. Of particular interest are "anomalous" analogues whose bioactivities are not readily rationalized by crystal structures. Here the structure and dynamics of one such analogue (GlyB24-insulin) are investigated by circular dichroism (CD) and isotope-aided 2D-NMR spectroscopy. The mutant insulin retains near-native receptor-binding affinity despite a nonconservative substitution (PheB24-->Gly) in the receptor-binding surface. Relative to native insulin, GlyB24-insulin exhibits reduced dimerization; the monomer (the active species) exhibits partial loss of ordered structure, as indicated by CD studies and motional narrowing of selected 1H-NMR resonance. 2D-NMR studies demonstrate that the B-chain beta-turn (residues B20-23) and beta-strand (residues B24-B28) are destabilized; essentially native alpha-helical secondary structure (residues A3-A8, A13-A18, and B9-B19) is otherwise maintained. 13C-Isotope-edited NOESY studies demonstrate that long-range contacts observed between the B-chain beta-strand and the alpha-helical core in native insulin are absent in the mutant. Implications for the mechanism of insulin's interaction with its receptor are discussed.     

Crystal structure of destripeptide (B28-B30) insulin: implications for insulin dissociation

Destripeptide (B28-B30) insulin (DTRI) is an insulin analogue that has much weaker association ability than native insulin but keeps most of its biological activity. It can be crystallized from a solution containing zinc ions at near-neutral pH. Its crystal structure has been determined by molecular replacement and refined at 1.9 A resolution. DTRI in the crystal exists as a loose hexamer compared with 2Zn insulin. The hexamer only contains one zinc ion that coordinates to the B10 His residues of three monomers. Although residues B28-B30 are located in the monomer-monomer interface within a dimer, the removal of them can simultaneously weaken both the interactions between monomers within the dimer and the interactions between dimers. Because the B-chain C-terminus of insulin is very flexible, we take the DTRI hexamer as a transition state in the native insulin dissociation process and suggest a possible dissociation process of the insulin hexamer based on the DTRI structure.     

Secondary structure of histones in solution

The secondary structure of histones H2B and H3 from calf thymus has been quantitatively studied in heavy water solutions in a wide range of histone concentrations, pD, and concentrations of sodium chloride by an infrared spectroscopy method. Also, the interactions between molecules of different histones in equimolar mixtures H2A-H2B, H2A-H3, H2A-H4, H2B-H3, H2B-H4, H3-H4, and H2A-H2B-H3-H4 have been investigated using the same method. For H2B and H3 conditions favourable for aggregation have been shown to induce the formation of pleated sheet structure. When the pD and concentration of NaCl are in a physiological range, the secondary structure of H2B and H3 contains about 15% of alpha-helix, 4% of parallel pleated sheet structure, 14% of antipatallel pleated sheet structure in H2B and 18% in H3. For mixtures in all cases, except H2A-H4, there is an interaction between molecules of different histones followed by a reduction of the antiparallel pleated sheet structure content. The data on the secondary structure of histones in different states (under self-association, in mixtures, in nucleosomes, and in chromatin) have been discussed and it is suggested that: 1) the secondary structure of histones in chromatin is essentially similar to that in the state of self-association; 2) in the core nucleosome particle the quantity of DNA (in nucleotide pairs), and the quantities of alpha-helix and antiparallel pleated sheet structure (in peptide groups) satisfy the relation 1 : 1 : 1.     

Multinuclear magnetic resonance studies of Escherichia coli adenylate kinase in free and bound forms. Resonance assignment, secondary structure and ligand binding.

The crystal structure of Escherichia coli adenylate kinase (AKe) revealed three main components: a CORE domain, composed of a five-stranded parallel beta-sheet surrounded by alpha-helices, and two peripheral domains involved in covering the ATP in the active site (LID) and binding of the AMP (NMPbind). We initiated a long-term NMR study aiming to characterize the solution structure, binding mechanism and internal dynamics of the various domains. Using single (15N) and double-labeled (13C and 15N) samples and double- and triple-resonance NMR experiments we assigned 97% of the 1H, 13C and 15N backbone resonances, and proton and 13Cbeta resonances for more than 40% of the side chains in the free protein. Analysis of a 15N-labeled enzyme in complex with the bi-substrate analogue [P1,P5-bis(5'-adenosine)-pentaphosphate] (Ap5A) resulted in the assignment of 90% of the backbone 1H and 15N resonances and 42% of the side chain resonances. Based on short-range NOEs and 1H and 13C secondary chemical shifts, we identified the elements of secondary structure and the topology of the beta-strands in the unliganded form. The alpha-helices and the beta-strands of the parallel beta-sheet in solution have the same limits (+/- 1 residue) as those observed in the crystal. The first helix (alpha1) appears to have a frayed N-terminal side. Significant differences relative to the crystal were noticed in the LID domain, which in solution exhibits four antiparallel beta-strands. The secondary structure of the nucleoside-bound form, as deduced from intramolecular NOEs and the 1Halpha chemical shifts, is similar to that of the free enzyme. The largest chemical shift differences allowed us to map the regions of protein-ligand contacts. 1H/2H exchange experiments performed on free and Ap5A-bound enzymes showed a general decrease of the structural flexibility in the complex which is accompanied by a local increased flexibility on the N-side of the parallel beta-sheet.     

An insulin analogue with gamma-amino butyric acid substitution for A13Leu-A14Tyr.

An insulin A chain analogue, [A13-14 GABA, A21 Ala]A chain, for which the dipeptide Leu-Try at A13-A14 was substituted by a non-coded amino acid, gamma-amino butyric acid (GABA) and A21 Asn by Ala, was prepared by stepwise Fmoc solid-phase manual synthesis and then combined with the natural B chain of porcine insulin to yield an insulin analogue, [A13-14 GABA, A21Ala] porcine insulin (GABA substituted insulin). This insulin analogue still retains 50% in vivo biological activity and 59% in receptor binding capacity. It can also be crystallized. These results indicate that its overall conformation is similar to the native form and that the side chains of A13Leu and A14Tyr are not essential for insulin activity. In addition, the replacement of a normal C-N peptide bond by an unnatural C-C bond may have general meaning in structure and function studies of other proteins.     

The solution structure of a monomeric insulin. A two-dimensional 1H-NMR study of des-(B26-B30)-insulin in combination with distance geometry and restrained molecular dynamics.

The solution conformation of des-(B26-B30)-insulin (DPI) has been investigated by 1H-NMR spectroscopy. A set of 250 approximate interproton distance restraints, derived from two-dimensional nuclear Overhauser enhancement spectra, were used as the basis of a structure determination using distance geometry (DG) and distance-bound driven dynamics (DDD). Sixteen DG structures were optimized using energy minimization (EM) and submitted to short 5-ps restrained molecular dynamics (RMD) simulations. A further refinement of the DDD structure with the lowest distance errors was done by energy minimization, a prolonged RMD simulation in vacuo and a time-averaged RMD simulation. An average structure was obtained from a trajectory generated during 20-ps RMD. The final structure was compared with the des-(B26-B30)-insulin crystal structure refined by molecular dynamics and the 2-Zn crystal structure of porcine insulin. This comparison shows that the overall structure of des-(B26-B30)-insulin is retained in solution with respect to the crystal structures with a high flexibility at the N-terminal part of the A chain and at the N-terminal and C-terminal parts of the B chain. In the RMD run a high mobility of Gly A1, Asn A21 and of the side chain of Phe B25 is noticed. One of the conformations adopted by des-(B26-B30)-insulin in solution is similar to that of molecule 1 (Chinese nomenclature) in the crystal structure of porcine insulin.     

Prediction of protein secondary structure based on physical theory. Histones

Secondary structures of histones H1, H2A, H2B, H3, H4 and H5 have been calculated by the computer program ALB based on a molecular theory of protein secondary structure. The predicted secondary structures of all histones are predominantly alpha-helical. The calculated secondary structure of linker histones H1 and H5 is close to that previously obtained from two-dimensional NMR data. For each of the core histones (H2A, H2B, H3, H4) one long alpha-helix and several short ones have been predicted. These long helices can be identified with rods in the low-resolution electron density map.     

Rat relaxin: insulin-like fold predicts a likely receptor binding region

The amino acid sequences for the ovarian hormone relaxin, now determined for pig, rat and shark, indicate that the molecule may have an internal structure similar to that of insulin. The combined results from six secondary structure prediction methods applied to the sequences of both relaxin and insulin support the concept of a similar folding for the B chain between the disulphide bridges. Model building with a computer graphics system has shown that the rat relaxin sequence cannot be superimposed on the 2Zn insulin structure without close contacts occurring between the residues in the central core. However, the residues can be accommodated in the more open framework assumed by 4Zn insulin (molecule I). With the relaxin models built according to the insulin fold, surface residues shared by the three relaxin sequences (B9(Arg), B13(Arg), A13 and A14 (Lys or Arg)) all lie in a localized area on the molecule. This group of residues focuses attention on a larger area on the molecule's surface which may well be the receptor binding site.