A conserved histidine in insulin is required for the foldability of human proinsulin: structure and function of an ALAB5 analog

The insulins of eutherian mammals contain histidines at positions B5 and B10. The role of His(B10) is well defined: although not required in the mature hormone for receptor binding, in the islet beta cell this side chain functions in targeting proinsulin to glucose-regulated secretory granules and provides axial zincbinding sites in storage hexamers. In contrast, the role of His(B5) is less well understood. Here, we demonstrate that its substitution with Ala markedly impairs insulin chain combination in vitro and blocks the folding and secretion of human proinsulin in a transfected mammalian cell line. The structure and stability of an Ala(B5)-insulin analog were investigated in an engineered monomer (DKP-insulin). Despite its impaired foldability, the structure of the Ala(B5) analog retains a native-like T-state conformation. At the site of substitution, interchain nuclear Overhauser effects are observed between the methyl resonance of Ala(B5) and side chains in the A chain; these nuclear Overhauser effects resemble those characteristic of His(B5) in native insulin. Substantial receptor binding activity is retained (80 +/- 10% relative to the parent monomer). Although the thermodynamic stability of the Ala(B5) analog is decreased (DeltaDeltaG(u) = 1.7 +/- 0.1 kcal/mol), consistent with loss of His(B5)-related interchain packing and hydrogen bonds, control studies suggest that this decrement cannot account for its impaired foldability. We propose that nascent long-range interactions by His(B5) facilitate alignment of Cys(A7) and Cys(B7) in protein-folding intermediates; its conservation thus reflects mechanisms of oxidative folding rather than structure-function relationships in the native state.     

Chiral mutagenesis of insulin. Contribution of the B20-B23 beta-turn to activity and stability

Insulin contains a beta-turn (residues B20-B23) interposed between two receptor-binding elements, the central alpha-helix of the B chain (B9-B19) and its C-terminal beta-strand (B24-B28). The turn contains conserved glycines at B20 and B23. Although insulin exhibits marked conformational variability among crystal forms, these glycines consistently maintain positive phi dihedral angles within a classic type-I beta-turn. Because the Ramachandran conformations of GlyB20 and GlyB23 are ordinarily forbidden to L-amino acids, turn architecture may contribute to structure or function. Here, we employ "chiral mutagenesis," comparison of corresponding D- and L-Ala substitutions, to investigate this turn. Control substitutions are introduced at GluB21, a neighboring residue exhibiting a conventional (negative) phi angle. The D- and L-Ala substitutions at B23 are associated with a marked stereospecific difference in activity. Whereas the D-AlaB23 analog retains native activity, the L analog exhibits a 20-fold decrease in receptor binding. By contrast, D- and L-AlaB20 analogs each exhibit high activity. Stereospecific differences between the thermodynamic stabilities of the analogs are nonetheless more pronounced at B20 (delta deltaG(u) 2.0 kcal/mole) than at B23 (delta deltaG(u) 0.7 kcal/mole). Control substitutions at B21 are well tolerated without significant stereospecificity. Chiral mutagenesis thus defines the complementary contributions of these conserved glycines to protein stability (GlyB20) or receptor recognition (GlyB23).     

Design of an active ultrastable single-chain insulin analog: synthesis, structure, and therapeutic implications

Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (<3 residues) prevent induced fit upon receptor binding and so are essentially without biological activity. Substantial but incomplete activity can be regained with increasing linker length. Here, we describe the design, structure, and function of a single-chain insulin analog (SCI-57) containing a 6-residue linker (GGGPRR). Native receptor-binding affinity (130 +/- 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of SCI-57 is markedly increased (DeltaDeltaG(u) = 0.7 +/- 0.1 kcal/mol relative to the corresponding two-chain analog and 1.9 +/- 0.1 kcal/mol relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects demonstrates that a native-like fold is maintained in solution. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts Val(A3) at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 alpha-helix. Comparison between SCI-57 and its parent two-chain analog reveals striking enhancement of multiple native-like nuclear Overhauser effects within the tethered protein. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultrastable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world.     

The conformational stability and flexibility of insulin with an additional intramolecular cross-link

The conformational stability and flexibility of insulin containing a cross-link between the alpha-amino group of the A-chain to the epsilon-amino group of Lys29 of the B-chain was examined. The cross-link varied in length from 2 to 12 carbon atoms. The conformational stability was determined by guanidine hydrochloride-induced equilibrium denaturation and flexibility was assessed by H2O/D2O amide exchange. The cross-link has substantial effects on both conformational stability and flexibility which depend on its length. In general, the addition of a cross-link enhances conformational stability and decreases flexibility. The optimal length for enhanced stability and decreased flexibility was the 6-carbon link. For the 6-carbon link the Gibbs free energy of unfolding was 8.0 kcal/mol compared to 4.5 kcal/mol for insulin, and the amide exchange rate decreased by at least 3-fold. A very short cross-link (i.e. the 2-carbon link) caused conformational strain that was detectable by a lack of stabilization in the Gibbs free energy of unfolding and enhancement in the amide exchange rate compared to insulin. The effect of the cross-link length on insulin hydrodynamic properties is discussed relative to previously obtained receptor binding results.     

Activation of the insulin receptor's kinase domain changes the rate-determining step of substrate phosphorylation

The insulin receptor and many other protein kinases are activated by relief of intrasteric inhibition that is regulated by reversible phosphorylation. The changes accompanying activation of the insulin receptor's kinase domain were analyzed using steady-state kinetics, viscometric analysis, and equilibrium binding measurements. Peptide phosphorylation catalyzed by the unphosphorylated basal-state kinase is limited by a slow rate of the chemical step, and the activated enzyme is limited by product release rates. Underlying these changes were a 36-fold increase in the rate constant for the chemical step of the enzyme-catalyzed reaction, a 5-fold increase in the affinity for MgATP, and an 8-fold increase in the affinity for peptide substrate. This results in binding of substrates that is 2.2 kcal/mol more favorable and a free energy barrier for transition state formation that is lowered by 2.1 kcal/mol in the activated enzyme. Therefore, the change in conformational free energy inherent in the protein after autophosphorylation [Bishop, S. M., Ross, J. B. A., and Kohanski, R. A. (1999) Biochemistry 38, 3079-3089] is equally distributed between formation of the substrate ternary complex and formation of the transition state complex.     

Folding thermodynamics and kinetics of YNMG RNA hairpins: specific incorporation of 8-bromoguanosine leads to stabilization by enhancement of the folding rate

Modified nucleotides allow fundamental energetic and kinetic properties of nucleic acids to be probed. Here, we demonstrate that an RNA hairpin containing the nucleotide analogue 8-bromoguanosine (8BrG or G), gcUUCGgc, has enhanced stability relative to the unmodified hairpin, with DeltaDeltaG(37)(degrees)= -0.69 +/- 0.15 kcal mol(-1) and DeltaT(M) = +6.8 +/- 1.4 degrees C. NMR spectroscopic data suggest that the enhanced stability of gcUUCGgc does not arise from the native state; laser temperature-jump experiments support this notion, as gcUUCGgc and gcUUCGgc have similar unfolding rate constants, but the folding rate constant of gcUUCGgc is 4.1-fold faster at 37.5 degrees C and 2.8-fold faster under isoenergetic conditions. On the basis of these findings, we propose that 8BrG reduces the conformational entropy of the denatured state, resulting in an accelerated conformational search for the native state and enhanced stability.     

Effects of Zn(II) binding and apoprotein structural stability on the conformation change of designed antennafinger proteins

Ligand-induced conformation change is a general strategy for controlling protein function. In this work, we demonstrate the relationships between ligand binding and conformational stability using a previously designed protein, Ant-F, which undergoes a conformation change upon Zn(II) binding. To investigate the effect of stabilization of the apo structure on the conformation change, we also created a novel protein, Ant-F-H1, into which mutations are introduced to increase its stability over that of Ant-F. The chemical denaturation experiments clarified that apo-Ant-F-H1 is more stable than apo-Ant-F (DeltaDeltaG = -1.28 kcal/mol) and that the stability of holo-Ant-F-H1 is almost the same as that of holo-Ant-F. The Zn(II) binding assay shows that the affinity of Zn(II) for Ant-F-H1 is weaker than that for Ant-F (DeltaDeltaG = 1.40 kcal/mol). A large part of the increased value of free energy in stability corresponds to the decreased value of free energy in Zn(II) binding, indicating that the stability of the apo structure directly affects the conformation change. The denaturation experiments also reveal that Zn(II) destabilizes the conformation of both proteins. From the thermodynamic linkage, Zn(II) is thought to bind to the unfolded state with high affinity. These results suggest that the binding of Zn(II) to the unfolded state is an important factor in the conformational change as well as the stability of the apo and holo structures.     

Role of the minor energetic determinants of chicken egg white lysozyme (HEWL) to the stability of the HEWL.antibody scFv-10 complex

Seven of the 13 non-glycine contact amino acids in the hen (chicken) egg white lysozyme (HEWL) epitope for antibody Fab-10 each contribute < or =0.3 kcal/mol to the change in free energy (DeltaDeltaG(D)) from wild type (WT) when replaced by alanine (nullspots), and three others each give (0.7 < DeltaDeltaG(D) < or = 1. 0) kcal/mol (warm spots) (Rajpal et al. Protein Sci 1998;7:1868-1874). The low DeltaDeltaG(D) values introduced by alanine mutations present an opportunity to explore accurately their cumulative effects, as the sum of the combined DeltaDeltaG(D) values is not so large as to destabilize the complex beyond the range of accurate measurement. Substitution of six of the seven null spot residues by alanine leads to a cumulative DeltaDeltaG(D) = 2.25 +/- 0.04 kcal/mol, whereas the sum of the six individual changes is only -0.36 +/- 0.32 kcal/mol. The triple warm spot mutation generates a DeltaDeltaG(D) = 5.11 +/- 0.06 kcal/mol versus DeltaDeltaG(D) = 2.52 +/- 0.22 kcal/mol for the sum of the three individuals. The non-additivity in the individual DeltaDeltaG(D) values for the alanine mutations may indicate that these residues provide a conformationally stabilizing effect on the hot spot residues, each of which exhibits DeltaDeltaG(D) > 4.0 kcal/mol on alanine substitution.     

DeltaG-based prediction and experimental confirmation of SYCRP1-binding sites on the Synechocystis genome

DNA-binding sites for SYCRP1, which is a regulatory protein of the cyanobacterium Synechocystissp. PCC6803, were predicted for the whole genome sequence by estimating changes in the binding free energy () for SYCRP1 for those sites. The values were calculated by summing DeltaDeltaG values derived from systematic single base-pair substitution experiments (symmetrical and cooperative binding model). Of the calculated binding sites, 23 sites with a value <3.9kcal.mol(-1) located upstream or between the ORFs were selected as putative binding sites for SYCRP1. In order to confirm whether SYCRP1 actually binds to these binding sites or not, 11 sites with the lowest values were tested experimentally, and we confirmed that SYCRP1 binds to ten of the 11 sites with a DeltaDeltaG(total) value <3.9kcal.mol(-1). The best correlation coefficient between and the observed DeltaDeltaG(total) for binding of SYCRP1 to those sites was 0.78. These results suggest that the DeltaDeltaG values derived from systematic single base-pair experiments may be used to screen for potential binding sites of a regulatory protein in the genome sequence.     

Increasing protein conformational stability by optimizing beta-turn sequence

Protein conformational stability is an important concern in many fields. Here, we describe a strategy for significantly increasing conformational stability by optimizing beta-turn sequence. Proline and glycine residues are statistically preferred at several beta-turn positions, presumably because their unique side-chains contribute favorably to conformational stability in certain beta-turn positions. However, beta-turn sequences often deviate from preferred proline or preferred glycine. Therefore, our strategy involves replacing non-proline and non-glycine beta-turn residues with preferred proline or preferred glycine residues. Here, we develop guidelines for selecting appropriate mutations, and present results for five mutations (S31P, S42G, S48P, T76P, and Q77G) that significantly increase the conformational stability of RNase Sa. The increases in stability ranged from 0.7 kcal/mol to 1.3 kcal/mol. The strategy was successful in overlapping or isolated beta-turns, at buried (up to 50%) or completely exposed sites, and at relatively flexible or inflexible sites. Considering the significant number of beta-turn residues in every globular protein and the frequent deviation of beta-turn sequences from preferred proline and preferred glycine residues, this simple, efficient strategy will be useful for increasing the conformational stability of proteins.     

A cavity-forming mutation in insulin induces segmental unfolding of a surrounding α-helix

To investigate the cooperativity of insulin's structure, a cavity-forming substitution was introduced within the hydrophobic core of an engineered monomer. The substitution, Ile(A2)-->Ala in the A1-A8 alpha-helix, does not impair disulfide pairing between chains. In accord with past studies of cavity-forming mutations in globular proteins, a decrement was observed in thermodynamic stability (DeltaDeltaG(u) 0.4-1.2 kcal/mole). Unexpectedly, CD studies indicate an attenuated alpha-helix content, which is assigned by NMR spectroscopy to selective destabilization of the A1-A8 segment. The analog's solution structure is otherwise similar to that of native insulin, including the B chain's supersecondary structure and a major portion of the hydrophobic core. Our results show that (1) a cavity-forming mutation in a globular protein can lead to segmental unfolding, (2) tertiary packing of Ile(A2), a residue of low helical propensity, stabilizes the A1-A8 alpha-helix, and (3) folding of this segment is not required for native disulfide pairing or overall structure. We discuss these results in relation to a hierarchical pathway of protein folding and misfolding. The Ala(A2) analog's low biological activity (0.5% relative to the parent monomer) highlights the importance of the A1-A8 alpha-helix in receptor recognition.     

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.     

Calorimetric examination of high-affinity Src SH2 domain-tyrosyl phosphopeptide binding: dissection of the phosphopeptide sequence specificity and coupling energetics

SH2 domains are protein modules which interact with specific tyrosine phosphorylated sequences in target proteins. The SH2 domain of the Src kinase binds with high affinity to a tyrosine phosphorylated peptide containing the amino acids Glu, Glu, and Ile (EEI) at the positions +1, +2, and +3 C-terminal to the phosphotyrosine, respectively. To investigate the degree of selectivity of the Src SH2 domain for each amino acid of the EEI motif, the binding thermodynamics of a panel of substitutions at the +1 (Gln, Asp, Ala, Gly), +2 (Gln, Asp, Ala, Gly), and +3 (Leu, Val, Ala, Gly) positions were examined using titration microcalorimetry. It was revealed that the Src SH2 domain is insensitive (DeltaDeltaG degrees 相似文献   

Prediction of protein mutant stability using classification and regression tool

Prediction of protein stability upon amino acid substitutions is an important problem in molecular biology and the solving of which would help for designing stable mutants. In this work, we have analyzed the stability of protein mutants using two different datasets of 1396 and 2204 mutants obtained from ProTherm database, respectively for free energy change due to thermal (DeltaDeltaG) and denaturant denaturations (DeltaDeltaG(H(2)O)). We have used a set of 48 physical, chemical energetic and conformational properties of amino acid residues and computed the difference of amino acid properties for each mutant in both sets of data. These differences in amino acid properties have been related to protein stability (DeltaDeltaG and DeltaDeltaG(H(2)O)) and are used to train with classification and regression tool for predicting the stability of protein mutants. Further, we have tested the method with 4 fold, 5 fold and 10 fold cross validation procedures. We found that the physical properties, shape and flexibility are important determinants of protein stability. The classification of mutants based on secondary structure (helix, strand, turn and coil) and solvent accessibility (buried, partially buried, partially exposed and exposed) distinguished the stabilizing/destabilizing mutants at an average accuracy of 81% and 80%, respectively for DeltaDeltaG and DeltaDeltaG(H(2)O). The correlation between the experimental and predicted stability change is 0.61 for DeltaDeltaG and 0.44 for DeltaDeltaG(H(2)O). Further, the free energy change due to the replacement of amino acid residue has been predicted within an average error of 1.08 kcal/mol and 1.37 kcal/mol for thermal and chemical denaturation, respectively. The relative importance of secondary structure and solvent accessibility, and the influence of the dataset on prediction of protein mutant stability have been discussed.     

D614G substitution at the hinge region enhances the stability of trimeric SARS-CoV-2 spike protein

Mutations in the spike protein of SARS-CoV-2 are the major causes for the modulation of ongoing COVID-19 infection. Currently, the D614G substitution in the spike protein has become dominant worldwide. It is associated with higher infectivity than the ancestral (D614)variant. We demonstrate using Gaussian network model-based normal mode analysis that the D614G substitution occurs at the hinge region that facilitates domain-domain motions between receptor binding domain and S2 region of the spike protein. Computer-aided mutagenesis and inter-residue energy calculations reveal that contacts involving D614 are energetically frustrated. However, contacts involving G614 are energetically favourable, implying the substitution strengthens residue contacts that are formed within as well as between protomers. We also find that the free energy difference (ΔΔG) between two variants is -2.6 kcal/mol for closed and -2.0 kcal/mol for 1-RBD up conformation. Thus, the thermodynamic stability has increased upon D614G substitution. Whereas the reverse mutation in spike protein structures having G614 substitution has resulted in the free energy differences of 6.6 kcal/mol and 6.3 kcal/mol for closed and 1-RBD up conformations, respectively, indicating that the overall thermodynamic stability has decreased. These results suggest that the D614G substitution modulates the flexibility of spike protein and confers enhanced thermodynamic stability irrespective of conformational states. This data concurs with the known information demonstrating increased availability of the functional form of spikeprotein trimer upon D614G substitution.     

Fluorous effect in proteins: de novo design and characterization of a four-alpha-helix bundle protein containing hexafluoroleucine

Several studies have demonstrated that proteins incorporating fluorinated analogues of hydrophobic amino acids such as leucine and valine into their hydrophobic cores exhibit increased stability toward thermal denaturation and unfolding by guanidinium chloride. However, estimates for the increase in the thermodynamic stability of a protein (DeltaDeltaG(unfold)) afforded by the substitution of a hydrophobic amino acid with its fluorinated analogue vary quite significantly. To address this, we have designed a peptide that adopts an antiparallel four-helix bundle structure in which the hydrophobic core is packed with leucine, and investigated the effects of substituting the central two layers of the core with L-5,5,5,5',5',5'-hexafluoroleucine (hFLeu). We find that DeltaDeltaG(unfold) is increased by 0.3 kcal/mol per hFLeu residue. This is in good agreement with the predicted increase in DeltaDeltaG(unfold) of 0.4 kcal/mol per residue arising from the increased hydrophobicity of the hFLeu side chain, which we determined experimentally from partitioning measurements on hFLeu and leucine. The increased stability of this fluorinated protein may therefore be ascribed to simple hydrophobic effects, rather than specific "fluorous" interactions between the hFLeu residues.     

Contribution of Residue B5 to the Folding and Function of Insulin and IGF-I: CONSTRAINTS AND FINE-TUNING IN THE EVOLUTION OF A PROTEIN FAMILY*

Proinsulin exhibits a single structure, whereas insulin-like growth factors refold as two disulfide isomers in equilibrium. Native insulin-related growth factor (IGF)-I has canonical cystines (A6—A11, A7–B7, and A20—B19) maintained by IGF-binding proteins; IGF-swap has alternative pairing (A7–A11, A6—B7, and A20—B19) and impaired activity. Studies of mini-domain models suggest that residue B5 (His in insulin and Thr in IGFs) governs the ambiguity or uniqueness of disulfide pairing. Residue B5, a site of mutation in proinsulin causing neonatal diabetes, is thus of broad biophysical interest. Here, we characterize reciprocal B5 substitutions in the two proteins. In insulin, His^B5 → Thr markedly destabilizes the hormone (ΔΔG_u 2.0 ± 0.2 kcal/mol), impairs chain combination, and blocks cellular secretion of proinsulin. The reciprocal IGF-I substitution Thr^B5 → His (residue 4) specifies a unique structure with native ¹H NMR signature. Chemical shifts and nuclear Overhauser effects are similar to those of native IGF-I. Whereas wild-type IGF-I undergoes thiol-catalyzed disulfide exchange to yield IGF-swap, His^B5-IGF-I retains canonical pairing. Chemical denaturation studies indicate that His^B5 does not significantly enhance thermodynamic stability (ΔΔG_u 0.2 ± 0.2 kcal/mol), implying that the substitution favors canonical pairing by destabilizing competing folds. Whereas the activity of Thr^B5-insulin is decreased 5-fold, His^B5-IGF-I exhibits 2-fold increased affinity for the IGF receptor and augmented post-receptor signaling. We propose that conservation of Thr^B5 in IGF-I, rescued from structural ambiguity by IGF-binding proteins, reflects fine-tuning of signal transduction. In contrast, the conservation of His^B5 in insulin highlights its critical role in insulin biosynthesis.     

Impact of proline residues on parvalbumin stability

Despite its higher net charge and reduced opportunities for favorable tertiary interactions, Ca(2+)-free rat beta-parvalbumin is more stable than rat alpha-parvalbumin. Under conditions wherein alpha denatures at 45.8 degrees C, beta denatures at 53.6 degrees. The homologous chicken beta isoform known as CPV3 also exhibits heightened stability-prompting an inquiry into the stabilizing influence of Pro-21 and Pro-26. Individual P21A and P26A mutations lower the T(m) of rat beta by 3.2 degrees, decreasing conformational stability by 0.74 kcal/mol. Simultaneous replacement of Pro-21 and Pro-26 essentially abolishes the excess stability (DeltaT(m) = -7.6 degrees; DeltaDeltaG(conf) = -1.77 kcal/mol). Significantly, the P21A/P26A variant displays Ca(2+) affinity virtually indistinguishable from wild-type beta, implying that structural alterations in the AB domain do not necessarily influence the divalent ion affinity of the CD-EF domain. The consequences of introducing proline at positions 21 and 26 in rat alpha were also examined. Whereas the H26P mutation raises the T(m) by 5.6 degrees (DeltaDeltaG(conf) = 1.25 kcal/mol), A21P lowers the T(m) by 8.5 degrees (DeltaDeltaG(conf) = -1.9 kcal/mol). Replacement of Ala-21 by proline in an alpha AB/beta CD-EF chimera increases the T(m) by 5.8 degrees (DeltaDeltaG(conf) = 0.95 kcal/mol), implying that the destabilization of alpha by Pro-21 results from steric conflict with a residue in the CD-EF domain. Consistent with that hypothesis, the K80S mutation markedly stabilizes alpha A21P, yielding a protein with a T(m) 2.0 degrees higher than wild-type alpha. The observed differences in stability resulting from proline addition/removal are largely consistent with alterations in main-chain and side-chain conformational entropy.     

Chiral mutagenesis of insulin. Foldability and function are inversely regulated by a stereospecific switch in the B chain

How insulin binds to its receptor is unknown despite decades of investigation. Here, we employ chiral mutagenesis-comparison of corresponding d and l amino acid substitutions in the hormone-to define a structural switch between folding-competent and active conformations. Our strategy is motivated by the T --> R transition, an allosteric feature of zinc-hexamer assembly in which an invariant glycine in the B chain changes conformations. In the classical T state, Gly(B8) lies within a beta-turn and exhibits a positive phi angle (like a d amino acid); in the alternative R state, Gly(B8) is part of an alpha-helix and exhibits a negative phi angle (like an l amino acid). Respective B chain libraries containing mixtures of d or l substitutions at B8 exhibit a stereospecific perturbation of insulin chain combination: l amino acids impede native disulfide pairing, whereas diverse d substitutions are well-tolerated. Strikingly, d substitutions at B8 enhance both synthetic yield and thermodynamic stability but markedly impair biological activity. The NMR structure of such an inactive analogue (as an engineered T-like monomer) is essentially identical to that of native insulin. By contrast, l analogues exhibit impaired folding and stability. Although synthetic yields are very low, such analogues can be highly active. Despite the profound differences between the foldabilities of d and l analogues, crystallization trials suggest that on protein assembly substitutions of either class can be accommodated within classical T or R states. Comparison between such diastereomeric analogues thus implies that the T state represents an inactive but folding-competent conformation. We propose that within folding intermediates the sign of the B8 phi angle exerts kinetic control in a rugged landscape to distinguish between trajectories associated with productive disulfide pairing (positive T-like values) or off-pathway events (negative R-like values). We further propose that the crystallographic T -->R transition in part recapitulates how the conformation of an insulin monomer changes on receptor binding. At the very least the ostensibly unrelated processes of disulfide pairing, allosteric assembly, and receptor binding appear to utilize the same residue as a structural switch; an "ambidextrous" glycine unhindered by the chiral restrictions of the Ramachandran plane. We speculate that this switch operates to protect insulin-and the beta-cell-from protein misfolding.     

Analysis of tachykinin-binding site interactions using NMR and energy calculation data of potent cyclic analogues of substance P

The three-dimensional structures of [Cys3,6,Tyr8]-, [Gly2,Cys3,6,Tyr8]- and [DCys3,Cys6]substance P, designed as conformational analogues of substance P, have been studied by 1H-NMR (500 MHz) in different solvents and by energy calculations. As previously observed for substance P and physalaemin, two tachykinins acting via the NK-1 receptor, [Cys3,6,Tyr8]substance P presents an alpha-helical structure of the 4----8 sequence in methanol. This structure is stabilized by a beta-turn III via the formation of three hydrogen bonds involving the Cys-6, Phe-7 and Tyr-8 NH groups. In contrast to substance P, two of these hydrogen bonds are still present in dimethyl sulfoxide and in water the Cys-6 NH hydrogen bond is the only one remaining, such that a beta-turn structure inside the ring can be envisaged. In close agreement with the NMR data, the energy calculations lead to three types of folding for the core of [Cys3,6,Tyr8]substance P: a beta-turn III, a less stable beta-turn I (delta E = 3 kcal), and a beta-turn II (delta E = 4.6 kcal). The structure of Gly-Leu-Met-NH2 is strongly affected by changing the hydrophobicity of the medium. The most stable calculated conformation is the helix; however, numerous unrelated structures are destabilized by about 2-3 kcal/mol. These data are analyzed and discussed in connection with the high potency of [Cys3,6,Tyr8]substance P for both the NK-1 and NK-3 binding sites; that is the internal region of tachykinins (non-homologous amino acids) might present a similar three-dimensional structure when bound to the receptors (which may be at the origin of some lack of selectivity), whereas paradoxically the selectivity may be due to the common C-terminal sequence.