The structure of a mutant insulin uncouples receptor binding from protein allostery. An electrostatic block to the TR transition

The zinc insulin hexamer undergoes allosteric reorganization among three conformational states, designated T(6), T(3)R(3)(f), and R(6). Although the free monomer in solution (the active species) resembles the classical T-state, an R-like conformational change is proposed to occur upon receptor binding. Here, we distinguish between the conformational requirements of receptor binding and the crystallographic TR transition by design of an active variant refractory to such reorganization. Our strategy exploits the contrasting environments of His(B5) in wild-type structures: on the T(6) surface but within an intersubunit crevice in R-containing hexamers. The TR transition is associated with a marked reduction in His(B5) pK(a), in turn predicting that a positive charge at this site would destabilize the R-specific crevice. Remarkably, substitution of His(B5) (conserved among eutherian mammals) by Arg (occasionally observed among other vertebrates) blocks the TR transition, as probed in solution by optical spectroscopy. Similarly, crystallization of Arg(B5)-insulin in the presence of phenol (ordinarily a potent inducer of the TR transition) yields T(6) hexamers rather than R(6) as obtained in control studies of wild-type insulin. The variant structure, determined at a resolution of 1.3A, closely resembles the wild-type T(6) hexamer. Whereas Arg(B5) is exposed on the protein surface, its side chain participates in a solvent-stabilized network of contacts similar to those involving His(B5) in wild-type T-states. The substantial receptor-binding activity of Arg(B5)-insulin (40% relative to wild type) demonstrates that the function of an insulin monomer can be uncoupled from its allosteric reorganization within zinc-stabilized hexamers.     

Insulin at pH 2: structural analysis of the conditions promoting insulin fibre formation

When insulin solutions are subjected to acid, heat and agitation, the normal pattern of insulin assembly (dimers-->tetramers-->hexamers) is disrupted; the molecule undergoes conformational changes allowing it to follow an alternative aggregation pathway (via a monomeric species) leading to the formation of insoluble amyloid fibres. To investigate the effect of acid pH on the conformation and aggregation state of the protein, the crystal structure of human insulin at pH 2.1 has been determined to 1.6 A resolution. The structure reveals that the native fold is maintained at low pH, and that the molecule is still capable of forming dimers similar to those found in hexameric insulin structures at higher pH. Sulphate ions are incorporated into the molecule and the crystal lattice where they neutralise positive charges on the protein, stabilising its structure and facilitating crystallisation. The sulphate interactions are associated with local deformations in the protein, which may indicate that the structure is more plastic at low pH. Transmission electron microscopy analysis of insulin fibres reveals that the appearance of the fibres is greatly influenced by the type of acid employed. Sulphuric acid produces distinctive highly bunched, truncated fibres, suggesting that the sulphate ions have a sophisticated role to play in fibre formation, rather as they do in the crystal structure. Analytical ultracentrifugation studies show that in the absence of heating, insulin is predominantly dimeric in mineral acids, whereas in acetic acid the equilibrium is shifted towards the monomer. Hence, the effect of acid on the aggregation state of insulin is also complex. These results suggest that acid conditions increase the susceptibility of the molecule to conformational change and dissociation, and enhance the rate of fibrillation by providing a charged environment in which the attractive forces between the protein molecules is increased.     

Formation and carbon monoxide‐dependent dissociation of Allochromatium vinosum cytochrome c′ oligomers using domain‐swapped dimers

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c′ from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four‐helix bundle structure containing a covalently bound five‐coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer–monomer transition according to the absence and presence of CO. Herein, domain‐swapped dimeric AVCP was constructed and utilized to form a tetramer and high‐order oligomers. The X‐ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain‐swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain‐swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit–subunit interactions at the interfaces of the domain‐swapped dimer and monomer subunits in the tetramer were also similar to the subunit–subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain‐swapped dimer and two monomers upon CO binding. Without monomers, the domain‐swapped dimers formed tetramers, hexamers, and higher‐order oligomers in the absence of CO, whereas the oligomers dissociated to domain‐swapped dimers in the presence of CO, demonstrating that the domain‐swapped dimer maintains the CO‐induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer–monomer transition protein.     

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.     

Intramolecular dynamics of low molecular weight protein tyrosine phosphatase in monomer-dimer equilibrium studied by NMR: a model for changes in dynamics upon target binding

Low molecular weight protein tyrosine phosphatase (LMW-PTP) dimerizes in the phosphate-bound state in solution with a dissociation constant of K(d)=1.5(+/-0.1)mM and an off-rate on the order of 10(4)s(-1). 1H and 15N NMR chemical shifts identify the dimer interface, which is in excellent agreement with that observed in the crystal structure of the dimeric S19A mutant. Two tyrosine residues of each molecule interact with the active site of the other molecule, implying that the dimer may be taken as a model for a complex between LMW-PTP and a target protein. 15N relaxation rates for the monomeric and dimeric states were extrapolated from relaxation data acquired at four different protein concentrations. Relaxation data of satisfactory precision were extracted for the monomer, enabling model-free analyses of backbone fluctuations on pico- to nanosecond time scales. The dimer relaxation data are of lower quality due to extrapolation errors and the possible presence of higher-order oligomers at higher concentrations. A qualitative comparison of order parameters in the monomeric and apparent dimeric states shows that loops forming the dimer interface become rigidified upon dimerization. Qualitative information on monomer-dimer exchange and intramolecular conformational exchange was obtained from the concentration dependence of auto- and cross-correlated relaxation rates. The loop containing the catalytically important Asp129 fluctuates between different conformations in both the monomeric and dimeric (target bound) states. The exchange rate compares rather well with that of the catalyzed reaction step, supporting existing hypotheses that catalysis and enzyme dynamics may be coupled. The side-chain of Trp49, which is important for substrate specificity, exhibits conformational dynamics in the monomer that are largely quenched upon formation of the dimer, suggesting that binding is associated with the selection of a single side-chain conformer.     

Vibrational spectrum of the unordered polypeptide chain: A Raman study of feather keratin

Raman spectra of native and solubilized feather keratin have been obtained, and the amide I and amide III regions have been analyzed by band resolution techniques. The amide I region of the native form indicates that at least 64% of the protein has an antiparallel chain pleated sheet structure, the remainder being unordered. For the solubilized keratin all of the protein is in an unordered state. The amide III region is not as easily analyzed into component contributions. Normal vibration analyses on N-acetyl-L -alanine-N-methylamide support the conclusion that the amide III region is not as satisfactory as the amide I region in characterizing unordered structures. Even in the latter case caution must be used, since the observed amide I band is an average over the conformational distribution in the particular unordered system.     

SxIP binding disrupts the constitutive homodimer interface of EB1 and stabilizes EB1 monomer

SxIP is a microtubule tip localizing signal found in many +TIP proteins that bind to the hydrophobic cavity of the C-terminal domain of end binding protein 1 (EB1) and then positively regulate the microtubule plus-end tracking of EBs. However, the exact mechanism of microtubule activation of EBs in the presence of SxIP signaling motif is not known. Here, we studied the effect of SxIP peptide on the native conformation of EB1 in solution. Using various NMR experiments, we found that SxIP peptide promoted the dissociation of natively formed EB1 dimer. We also discovered that I224A mutation of EB1 resulted in an unfolded C-terminal domain, which upon binding with the SxIP motif folded to its native structure. Molecular dynamics simulations also confirmed the relative structural stability of EB1 monomer in the SxIP bound state. Residual dipolar couplings and heteronuclear NOE analysis suggested that the binding of SxIP peptide at the C-terminal domain of EB1 decreased the dynamics and conformational flexibility of the N-terminal domain involved in EB1-microtubule interaction. The SxIP-induced disruption of the dimeric interactions in EB1, coupled with the reduction in conformational flexibility of the N-terminal domain of EB1, might facilitate the microtubule association of EB1.     

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.     

Conformational flexibility of aqueous monomeric and dimeric insulin: a molecular dynamics study

A series of molecular dynamics simulations have been used to investigate the nature of monomeric and dimeric insulin in aqueous solution. It is shown that in the absence of crystal contacts both monomeric and dimeric insulin have a high degree of intrinsic flexibility. Neither of the two monomer conformations of 2Zn crystalline insulin appears to be favored in solution nor is the asymmetry of the crystal dimer reduced in the absence of crystal contacts. A shift is observed in the relative positions of molecules 1 and 2 in the dimer compared with that found in the crystal, which may have consequences for the prediction of the effects of mutants in the monomer-monomer interface designed to alter the self-association properties of insulin.     

Proteins can convert to beta-sheet in single crystals

Raman microscopy was used to follow conformational changes in single protein crystals. Crystals of native insulin and of the 5S and 12S subunits of the enzyme transcarboxylase showed a mixture of Raman marker bands signifying alpha-helix, beta-sheet and nonordered secondary structure. However, by reducing the S-S bonds in the insulin crystal, or by lowering the pH for the 5S crystal, or by soaking substrates into the 12S crystal, the secondary structure in each crystal became predominantly beta-sheet. The beta-form crystals could be dissolved only with difficulty and yielded high-molecular weight protein aggregates, indicating that the beta-sheet formation involves intermolecular contacts. Although their morphology appeared unchanged, the crystals no longer diffracted X-rays. Using crystals that had not been exposed to laser light, the dye thioflavin T formed highly fluorescent complexes with the "beta-transformed" crystals but not with the native crystals.     

Ultraviolet resonance Raman spectroscopy of distamycin complexes with poly(dA)-poly(dT) and poly(dA-dT): role of H-bonding

Raman spectra are reported for distamycin, excited at 320 nm, in resonance with the first strong absorption band of the chromophore. Qualitative band assignments to pyrrole ring and amide modes are made on the basis of frequency shifts observed in D2O. When distamycin is dissolved in dimethyl sulfoxide or dimethylformamide, large (30 cm-1) upshifts are seen for the band assigned to amide I, while amides II and III shift down appreciably. Similar but smaller shifts are seen when distamycin is bound to poly(dA-dT) and poly(dA)-poly(dT). Examination of literature data for N-methylacetamide in various solvents shows that the amide I frequencies correlate well with solvent acceptor number but poorly with solvent donor number. This behavior implies that acceptor interactions with the C = O group are more important than donor interactions with the N-H group in polarizing the amide bond and stabilizing the zwitterionic resonance form. The resonance Raman spectra therefore imply that the distamycin C = O groups, despite being exposed to solvent, are less strongly H-bonded in the polynucleotide complexes than in aqueous distamycin, perhaps because of orienting influences of the nearby backbone phosphate groups. In this respect, the poly(dA-dT) and poly(dA)-poly(dT) complexes are the same, showing the same RR frequencies. Resonance Raman spectra were also obtained at 200-nm excitation, where modes of the DNA residues are enhanced. The spectra were essentially the same with and without distamycin, except for a perceptable narrowing of the adenine modes of poly(dA-dT), suggesting a reduction in conformational flexibility of the polymer upon drug binding.     

Conformational states of the bacteriophage P22 capsid subunit in relation to self-assembly

The formation of closed icosahedral capsids from a single species of coat protein subunit requires that the subunits assume different conformations at different lattice positions. In the double-stranded DNA bacteriophage P22, formation of correctly dimensioned capsids is mediated by interaction between coat protein subunits and scaffolding protein. Raman spectroscopy has been employed to compare the conformations of coat protein subunits which have been polymerized to form capsids in the presence and absence of the of scaffolding protein display a Raman spectrum characterized by a broad amide I band centered at 1665 cm-1 with a discernible shoulder near 1653 cm-1, and a broad amide III profile centered at 1238 cm-1 but asymmetrically skewed to higher frequency. These spectral features indicate that the protein conformation in procapsid shells is rich in beta-sheet secondary structure but contains also a significant distribution of alpha-helix. When biologically active, purified subunits assemble in the absence of scaffolding protein, they form polydisperse multimers lacking the proper dimensions of procapsid closed shells. We designate these multimers as "associated subunits" (AS). The Raman spectrum of associated subunits indicates a narrower distribution of secondary structure. The associated subunits are characterized by a sharper and more intense Raman amide I band at 1666 cm-1, with no prominent amide I shoulder of lower frequency. An analogous narrowing of the Raman amide III profile is also observed for AS particles, with an accompanying shift of the amide III band center to 1235 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secondary structure of alpha-synuclein oligomers: characterization by raman and atomic force microscopy

Formation of alpha-synuclein aggregates is proposed to be a crucial event in the pathogenesis of Parkinson's disease. Large soluble oligomeric species are observed as probable intermediates during fibril formation and these, or related aggregates, may constitute the toxic element that triggers neurodegeneration. Unfortunately, there is a paucity of information regarding the structure and composition of these oligomers. Here, the morphology and the conformational characteristics of the oligomers and filaments are investigated by a combined atomic force microscopy (AFM) and Raman microscopic approach on a common mica surface. AFM showed that in vitro early stage oligomers were globular with variable heights, while prolonged incubation caused the oligomers to become elongated as protofilaments. The height of the subsequently formed alpha-synuclein filaments was similar to that of the protofilaments. Analysis of the Raman amide I band profiles of the different alpha-synuclein oligomers establishes that the spheroidal oligomers contain a significant amount of alpha-helical secondary structure (47%), which decreases to about 37% in protofilaments. At the same time, when protofilaments form, beta-sheet structure increases to about 54% from the approximately 29% observed in spheroidal oligomers. Upon filament formation, the major conformation is beta-sheet (66%), confirmed by narrowing of the amide I band and the profile maximum shifting to 1667 cm(-1). The accumulation of spheroidal oligomers of increasing size but unchanged vibrational spectra during the fibrillization process suggests that a cooperative conformational change may contribute to the kinetic control of fibrillization.     

Toward the active conformation of insulin: stereospecific modulation of a structural switch in the B chain

How insulin binds to the insulin receptor has long been a subject of speculation. Although the structure of the free hormone has been extensively characterized, a variety of evidence suggests that a conformational change occurs upon receptor binding. Here, we employ chiral mutagenesis, comparison of corresponding d and l amino acid substitutions, to investigate a possible switch in the B-chain. To investigate the interrelation of structure, function, and stability, isomeric analogs have been synthesized in which an invariant glycine in a beta-turn (Gly(B8)) is replaced by d- or l-Ser. The d substitution enhances stability (DeltaDeltaG(u) 0.9 kcal/mol) but impairs receptor binding by 100-fold; by contrast, the l substitution markedly impairs stability (DeltaDeltaG(u) -3.0 kcal/mol) with only 2-fold reduction in receptor binding. Although the isomeric structures each retain a native-like overall fold, the l-Ser(B8) analog exhibits fewer helix-related and long range nuclear Overhauser effects than does the d-Ser(B8) analog or native monomer. Evidence for enhanced conformational fluctuations in the unstable analog is provided by its attenuated CD spectrum. The inverse relationship between stereospecific stabilization and receptor binding strongly suggests that the B7-B10 beta-turn changes conformation on receptor binding.     

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.     

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 relation of conformation and association of insulin to receptor binding; x-ray and circular-dichroism studies on bovine and hystricomorph insulins.

Crystal and solution structure studies on insulins of different sequences and of widely different receptor binding affinities are reported. Bovine insulin, studied as a control, has a circular dichroism spectrum which is dependent both on protein concentration and zinc concentration. The spectrum appears to be related to the level of association of the insulin molecules. This implies that when using circular dichroism to compare solution structures of insulin derivatives or species variants one must make the comparison at equivalent levels of association and not merely at the same concentration. Changes in circular dichroism are related to the known crystal structure of zinc insulin hexamers. The chinchilla insulin spectrum shows a reduced zinc dependence in low-salt conditions which correlates with the inability to form crystals in similar conditions. This is attributed to an amino acid substitution at position B4. Crystals are obtained in high-salt conditions and X-ray diffraction patterns show these to be isomorphous with bovine 4Zn insulin crystals. Guinea pig insulin failed to crystallise under conditions which are normally conducive to the formation of crystals of zinc insulin hexamers and the circular dichroism showed no zinc dependence. This is consistent with a monomeric structure. The significance of the association behaviour of chinchilla and guinea pig insulins may be in the storage of the hormone in vivo. Whereas the monomeric form of chinchilla insulin has a structure closely related to bovine insulin, the circular dichroism indicates a gross structural difference for guinea pig insulin. This may be similar to that in des-A21, des-B30-insulin, as both lack the Arg-B22--Asn-A21 carboxylate ion pair. The similarity of structure of chinchilla and bovine insulins is reflected in their receptor binding whereas the low receptor binding of guinea pig insulin probably results from the changes in its conformation rather than an alteration in residues of a receptor binding region.     

Physicochemical basis for the rapid time-action of LysB28ProB29-insulin: dissociation of a protein-ligand complex.

The rate-limiting step for the absorption of insulin solutions after subcutaneous injection is considered to be the dissociation of self-associated hexamers to monomers. To accelerate this absorption process, insulin analogues have been designed that possess full biological activity and yet have greatly diminished tendencies to self-associate. Sedimentation velocity and static light scattering results show that the presence of zinc and phenolic ligands (m-cresol and/or phenol) cause one such insulin analogue, LysB28ProB29-human insulin (LysPro), to associate into a hexameric complex. Most importantly, this ligand-bound hexamer retains its rapid-acting pharmacokinetics and pharmacodynamics. The dissociation of the stabilized hexameric analogue has been studied in vitro using static light scattering as well as in vivo using a female pig pharmacodynamic model. Retention of rapid time-action is hypothesized to be due to altered subunit packing within the hexamer. Evidence for modified monomer-monomer interactions has been observed in the X-ray crystal structure of a zinc LysPro hexamer (Ciszak E et al., 1995, Structure 3:615-622). The solution state behavior of LysPro, reported here, has been interpreted with respect to the crystal structure results. In addition, the phenolic ligand binding differences between LysPro and insulin have been compared using isothermal titrating calorimetry and visible absorption spectroscopy of cobalt-containing hexamers. These studies establish that rapid-acting insulin analogues of this type can be stabilized in solution via the formation of hexamer complexes with altered dissociation properties.     

Proinsulin is refractory to protein fibrillation: topological protection of a precursor protein from cross-beta assembly

Insulin is susceptible to fibrillation, a misfolding process leading to well ordered cross-beta assembly. Protection from fibrillation in beta cells is provided by sequestration of the susceptible monomer within zinc hexamers. We demonstrate that proinsulin is refractory to fibrillation under conditions that promote the rapid fibrillation of zinc-free insulin. Proinsulin fibrils, as probed by Raman microscopy, are nonetheless similar in structure to insulin fibrils. The connecting peptide, although not well ordered in native proinsulin, participates in a fibril-specific beta-sheet. Native insulin and proinsulin exhibit similar free energies of unfolding as inferred from guanidine denaturation studies: relative amyloidogenicities are thus not correlated with global stability. Strikingly, the susceptibility of proinsulin to fibrillation is increased by scission of the connecting peptide at single sites. We thus propose that the connecting peptide constrains a large scale conformational change in the misfolded protein. A tethering mechanism is proposed based on a model of an insulin protofilament derived from electron-microscopic image reconstruction. The proposed relationship between cross-beta assembly and protein topology is supported by studies of single-chain analogs (mini-proinsulin and insulin-like growth factor I) in which foreshortened connecting peptides further retard fibrillation. In addition to its classic function to facilitate disulfide pairing, the connecting peptide may protect beta cells from toxic protein misfolding in the endoplasmic reticulum.     

The solution conformations of gramicidin A and its analogs

The conformational states in dioxane and ethanol of gramicidin A and of analogs varying in chain length and amino acid sequence have been studied. Infrared, CD, and polarization of fluorescence spectra of the peptides were measured, from which dimerization constants were determined and spectral characteristics of the monomeric and dimeric states obtained. Resonance splitting of the amide I ir band has been calculated for all gramicidin A models proposed earlier. Detailed comparison of the experimental and computed spectra showed that the four dimeric gramicidin species present in solution are predominantly antiparallel double ?ππ_ld helices in equilibrium with smaller amounts of head-to-head associated π_LD helices. The gramicidin A monomer was found to be a π_LD^4.4 helix in dioxane. For each conformational form the number of residues per turn and the helical sense were determined. The relationship between the amino acid sequence and the structure and stability of the dimer in the series of gramicidin A and its analogs is discussed. The above findings are rationalized in terms of the membrane channel properties of gramicidin A, in particular the conformational rearrangements occurring during the passage of metal ions through the channel and also the differences in conformation of the antibiotic in nonpolar solutions and in the membrane.