Increase In Alpha-helix Content Research Articles

Analysis of Insulin Allostery in Solution and Solid State With FTIR

The insulin hexamer acts as an allosteric unit mediated by homotropic and heterotropic effects shifting the equilibrium between three distinct conformational states (T(6), R(3)T(3) and R(6)). The homotropic ligand phenol stabilises the R(6) state by binding to hydrophobic pockets only present in the R(6) state and shifts the equilibrium towards the R(6) state. The structural difference between the T(6) and R(6) state is primarily a change in the B1-B8 residues from extended conformation (T(6)) to alpha-helix (R(6)). The aim of this study was to investigate FTIR as an alternative method to monitor the T-R transition in the insulin hexamer upon phenol binding, and in addition to explore the advantage of infrared spectroscopy to measure solid state samples, and support the ability to maintain an allosteric state upon drying. The FTIR spectra of insulin in solution showed an increase in alpha-helix upon phenol binding and correlated well with the transition measured by CD yielding similar dissociation constants. Furthermore it was possible to maintain the increase in alpha-helix upon phenol binding after lyophilisation. The overall structure of the FTIR spectra changed upon lyophilisation but an increase in alpha-helix content was retained. Reconstitution of lyophilised insulin resulted in a change in structure resembling the structure of insulin prior to lyophilisation. Principal component analysis of all spectra was computed resulting in distinct clusters, and most variation in the data set could be explained by PC1 corresponding to a change in alpha-helix.

Single‐Molecule FRET Reveals Structural Heterogeneity of SDS‐Bound α‐Synuclein

SDS-concentration-dependent alpha-synuclein structure: Upon interaction with SDS, alpha Syn folds into a structure with two antiparallel alpha-helices. We show from single-molecule FRET that alpha Synn adopts this conformation in an all-or-none fashion below the SDS critical micelle concentration. Population of the folded species is directly coupled to an increase in alpha-helix content; this suggests that the entire N terminus is involved in the transaction.

Structural characterization of two peptides corresponding to Helix 4 of Apolipoprotein E

Apolipoprotein E (ApoE) is a ligand for the low density lipoprotein (LDL) receptor. The amino-terminal domain of ApoE is a 4-helix bundle and helix 4 is involved in LDL receptor binding. This region associates with lipids and undergoes structural changes which allow a key arginine residue, Arg172 to participate in receptor binding. To characterize this region and to study lipid-induced conformational changes we developed 2 synthetic peptides: one corresponding to residues 128-164 (ApoE-164) and the other corresponding to residues 128-183 (ApoE-183). Secondary structure prediction indicates amphipathic alpha-helical structure for both peptides. Circular dichroism spectroscopy (CD) studies show that ApoE-164 exhibits concentration-dependent self association whereas ApoE-183 does not. To study the effect of lipids on the two peptides we used three detergents: 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), dodecyl phosphocholine(DPC), and octyl-beta-d-glucopyranoside(BetaOG). CD shows that both peptides exhibit an increase in alpha-helix content at the critical micelle concentration (CMC) for all three detergents. The alpha-helix content for both peptides in the absence of detergent is about 30%. Upon the addition of DHPC, DPC or BetaOG there is a 30-40% increase in alpha-helicity at the CMC. This approach is directed towards understanding (i) how lipids induce a conformational change in the 165-183 region, and (ii) how the relative orientation of Arg172 is changed such that it contributes to LDL receptor binding.

The inhibitory effect of various indolyl amino acid derivatives on arginase activity in macrophages

Numerous indolyl amino acids and their derivatives inhibited arginase activity. The inhibition was found to be non-competitive, - at least partly - allosteric, and independent on manganese ions in the active site, and it cannot be explained by the dissociation of arginase homotrimers. Indole alone is weakly inhibitory; however, the presence of three-carbon side chains and their net charges is favorable for the inhibition. The binding of the inhibitory compounds caused only minor changes in the steric structure of arginase: a slight increase in alpha-helix content was detected by circular dichroism together with a decrease in parallel pleated sheet and beta-turn sections. A slight alteration in the tertiary structure was also found using tryptophane fluorescence studies, but buried apolar side chains were not transposed to the protein surface. Computer studies that were performed did not provide additional structural information.

Effects of the Core Lipid on the Energetics of Binding of ApoA-I to Model Lipoprotein Particles of Different Sizes

Interaction of apolipoproteins (apo) with lipid surfaces plays crucial roles in lipoprotein metabolism and cholesterol homeostasis. To elucidate the thermodynamics of binding of apoA-I to lipid, we used lipid emulsions composed of triolein (TO) and egg phosphatidylcholine (PC) as lipoprotein models. Determination of the level of binding of wild-type (WT) apoA-I and some deletion mutants to large (120 nm diameter; LEM) and small (35 nm diameter; SEM) emulsions indicated that N-terminal (residues 44-65) and C-terminal (residues 190-243 and 223-243) deletions have large effects on lipid interaction, whereas deletion of the central region (residues 123-166) has little effect. Substitution of amino acids at either L230 or L230, L233, and Y236 with proline residues also decreases the level of binding, indicating that an alpha-helix conformation in this C-terminal region is required for efficient lipid binding. Calorimetry showed that binding of WT apoA-I to SEM generates endothermic heat (DeltaH approximately 30 kcal/mol) in contrast to the exothermic heat (ca. -85 kcal/mol) generated upon binding to LEM and egg PC small unilamellar vesicles (SUV). This exothermic heat arises from an approximately 25% increase in alpha-helix content, and it drives the binding of apoA-I to LEM and SUV. There is a similar increase in alpha-helix content of apoA-I upon binding to either SEM or SUV, but the binding of apoA-I to SEM is an entropy-driven process. These results suggest that the presence of a core triglyceride modifies the highly curved SEM surface packing and thereby the thermodynamics of apoA-I binding in a manner that compensates for the exothermic heat generated by alpha-helix formation.

Structural Characteristics of Purified β-Conglycinin from Soybeans Stored under Four Conditions

Four storage conditions including adverse conditions [84% relative humidity (RH), 30 degrees C], mild conditions (57% RH, 20 degrees C), cold conditions (4 degrees C), and uncontrolled ambient conditions were used for storing soybeans. The storage time was 9 months for the adverse conditions and 18 months for the other three conditions. Beta-conglycinin was purified and characterized with respect to its molecular properties. After storage under the adverse conditions, beta-conglycinin showed no significant changes in total sugar content, surface hydrophobicity, free SH and SS bonds, and amino acid composition within 6 months; however, it showed a significant decrease in surface hydrophobicity and a significant increase in total free SH and total SH including SS content after 6 months. Analysis of the secondary structure showed a significant increase in alpha-helix content, but a significant decrease in beta-sheet content after 3 months. For the other three conditions, no significant changes occurred to the structures of beta-conglycinin when compared to the control. The molecular mass of beta-conglycinin remained in the range of 199-212 kDa for all conditions during the entire storage periods.

α-Helix Formation Is Required for High Affinity Binding of Human Apolipoprotein A-I to Lipids

Apolipoprotein (apo) A-I is thought to undergo a conformational change during lipid association that results in the transition of random coil to alpha-helix. Using a series of deletion mutants lacking different regions along the molecule, we examined the contribution of alpha-helix formation in apoA-I to the binding to egg phosphatidylcholine (PC) small unilamellar vesicles (SUV). Binding isotherms determined by gel filtration showed that apoA-I binds to SUV with high affinity and deletions in the C-terminal region markedly decrease the affinity. Circular dichroism measurements demonstrated that binding to SUV led to an increase in alpha-helix content, but the helix content was somewhat less than in reconstituted discoidal PC.apoA-I complexes for all apoA-I variants, suggesting that the helical structure of apoA-I on SUV is different from that in discs. Isothermal titration calorimetry showed that the binding of apoA-I to SUV is accompanied by a large exothermic heat and deletions in the C-terminal regions greatly decrease the heat. Analysis of the rate of release of heat on binding, as well as the kinetics of quenching of tryptophan fluorescence by brominated PC, indicated that the opening of the N-terminal helix bundle is a rate-limiting step in apoA-I binding to the SUV surface. Significantly, the correlation of thermodynamic parameters of binding with the increase in the number of helical residues revealed that the contribution of alpha-helix formation upon lipid binding to the enthalpy and the free energy of the binding of apoA-I is -1.1 and -0.04 kcal/mol per residue, respectively. These results indicate that alpha-helix formation, especially in the C-terminal regions, provides the energetic source for high affinity binding of apoA-I to lipids.

Membrane charge and curvature determine interaction with acyl-CoA binding protein (ACBP) and fatty acyl-CoA targeting.

Although acyl-CoA binding protein (ACBP) stimulates utilization of long-chain fatty acyl-CoA by a variety of membrane-bound enzymes, it is not known whether ACBP directly interacts with membranes. To test this hypothesis, mouse recombinant (mr) ACBP was engineered to contain the native mouse ACBP amino acid sequence expressed as a fusion protein at high levels (>150 mg/L) in Escherichia coli. Purification and cleavage of the fusion tag resulted in mrACBP identical to native ACBP as shown by mass (10000.5 Da) and amino acid sequence (peptide mapping after proteolysis) determined by matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectroscopy. The mrACBP was functionally active as shown by binding of cis-parinaroyl-CoA with high affinity, K(d) = 12 +/- 2 nM, at a single binding site, stimulating oleoyl-CoA utilization by microsomal glycerol-3-phosphate acyltransferase 3.2-fold and protecting oleoyl-CoA from microsomal acyl-CoA hydrolase. Direct interaction of mrACBP with membranes was demonstrated by two independent methods: (i) Circular dichroism showed an 8% increase in alpha-helix content of mrACBP in the presence of anionic phospholipid-rich, but not neutral, small unilamellar vesicles (SUV). (ii) Membrane filtration confirmed that mrACBP bound to anionic phospholipid-rich SUV but only weakly interacted with neutral SUV or large unilamellar vesicles (LUV), regardless of charge. (iii) The mrACBP-oleoyl-CoA complex transferred 2-3-fold more oleoyl-CoA to anionic phospholipid-rich SUV than to anionic phospholipid-rich LUV and neutral SUV or LUV. Conversely, mrACBP extracted less oleoyl-CoA from anionic phospholipid-rich SUV. Taken together, these data indicated for the first time that mrACBP interacted preferentially with anionic phospholipid-rich, highly curved membranes to facilitate transfer of ACBP-bound ligands.

Effects of zinc on creatine kinase: activity changes, conformational changes, and aggregation.

The effects of zinc on creatine kinase (CK) are very distinctive compared with other bivalent metal ions. Zinc up to 0.1 mM induced increases in CK activity, accompanied by significant hydrophobic surface exposure and increase in alpha-helix content of CK. Zinc over 0.1 mM denatured and inactived CK. In the presence of 0.1 mM zinc, the CK activity was very close to that of the native CK, but its conformation changed greatly. The kinetic courses of CK inactivation and conformational change in the presence of 1 mM zinc were measured to determine apparent rate constants of inactivation and conformational change. Zinc over 0.05 mM induced CK aggregation at 37 degrees C, and the aggregation was dependent on zinc concentration, CK concentration, and temperature. The inactivation and aggregation can be reversed by EDTA. An explanation for CK aggregation induced by zinc is proposed, as well as a mechanism for CK abnormality in Alzheimer's disease.

Human apolipoprotein C-II forms twisted amyloid ribbons and closed loops.

Human apolipoprotein C-II (apoC-II) self-associates in solution to form aggregates with the characteristics of amyloid including red-green birefringence in the presence of Congo Red under cross-polarized light, increased fluorescence in the presence of thioflavin T, and a fibrous structure when examined by electron microscopy. ApoC-II was expressed and purified from Escherichia coli and rapidly exchanged from 5 M guanidine hydrochloride into 100 mM sodium phosphate, pH 7.4, to a final concentration of 0.3 mg/mL. This apoC-II was initially soluble, eluting as low molecular weight species in gel filtration experiments using Sephadex G-50. Circular dichroism (CD) spectroscopy indicated predominantly unordered structure. Upon incubation for 24 h, apoC-II self-associated into high molecular weight aggregates as indicated by elution in the void volume of a Sephadex G-50 column, by rapid sedimentation in an analytical ultracentrifuge, and by increased light scattering. CD spectroscopy indicated an increase in beta-sheet content, while fluorescence emission spectroscopy of the single tryptophan revealed a blue shift and an increase in maximum intensity, suggesting repositioning of the tryptophan into a less polar environment. Electron microscopy of apoC-II aggregates revealed a novel looped-ribbon morphology (width 12 nm) and several isolated closed loops. Like all of the conserved plasma apolipoproteins, apoC-II contains amphipathic helical regions that account for the increase in alpha-helix content on lipid binding. The increase in beta-structure accompanying apoC-II fibril formation points to an alternative folding pathway and an in vitro system to explore the general tendency of apolipoproteins to form amyloid in vivo.

Spectroscopic characterization of the conformational adaptability of Bombyx mori apolipophorin III

Apolipophorin III (apoLp-III) from the silkmoth, Bombyx mori, has been over-expressed in Escherichia coli, purified and characterized. Far-UV CD spectroscopic analysis revealed 65% alpha-helix secondary structure. Near-UV CD spectra obtained in buffer or complexed with dimyristoylglycerophosphocholine (DMPC), provided evidence that apoLp-III alpha-helices reorient upon interaction with lipid, indicative of a protein conformational change. In guanidine hydrochloride (GdnHCl) denaturation studies, a transition midpoint of 0.33 M was observed, corresponding to a DeltaGDH2O = 2.46 kcal. mol-1. Fluorescence studies of the sole tryptophan residue (Trp40) in apoLp-III revealed an emission lambdamax = 327 nm. Compared to free tryptophan, Stern-Volmer constants (KSV) for acrylamide and KI quenching of Trp40 fluorescence were decreased by 20-fold and sevenfold, respectively. In studies of apoLp-III-DMPC disc complexes, far-UV CD spectroscopy revealed an increase in alpha-helix content to approximately 85% and a ninefold increase in the GdnHCl-induced denaturation transition midpoint to 3 M. In studies of lipid interaction, apoLp-III was shown to disrupt both negatively charged and zwitterionic phospholipid bilayer vesicles, transforming them into discoidal complexes. Characterization of apoLp-III-DMPC discs, using 5-doxyl or 12-doxyl stearic acid as lipid-based quenching agents, revealed that Trp40 localizes near the phospholipid polar head groups. KSV values for acrylamide and KI quenching of intrinsic fluorescence of apoLp-III-DMPC discs indicate that Trp40 is embedded in the lipid milieu, with little or no accessibility to the aqueous quenchers. Given the large amount of alpha-helix in apoLp-III, the data presented support a model in which amphipathic alpha-helical segments are stabilized by helix-helix interactions and lipid association induces a protein conformational change which results in substitution of helix-helix interactions for helix-lipid contacts.

DNA-induced conformational change of Gaf1, a novel GATA factor in Schizosaccharomyces pombe.

A novel GATA factor in Schizosaccharomyces pombe, Gaf1, containing one zinc-finger motif was studied for conformational change that was induced by DNA-binding. Gaf1 was shown to bind to the upstream activation sequence of a gene in Saccharomyces cerevisiae containing GATA element by gel mobility shift assay. Circular dichroism spectra of Gaf1 indicated an increase of alpha-helix content of Gaf1 occurred upon binding to the upstream activation sequence. These results suggest that the binding of Gaf1 to the GATA element is required for the conformational change that may precede transactivation of the target gene(s).

Conformational studies on a peptide fragment representing the RNA‐binding N‐terminus of a viral coat protein using circular dichroism and NMR spectroscopy

Conformational studies were performed on a synthetic pentacosapeptide representing the RNA-binding N-terminal region of the coat protein of cowpea chlorotic mottle virus. The effects of ionic strength, addition of (oligo)phosphates and temperature on the conformation of this highly positively charged peptide containing six arginines and three lysines were studied. CD experiments show that the peptide has 15-18% alpha-helical conformation and about 80% random-coil conformation in the absence of inorganic salt at 25 degrees C, and 20-21% alpha-helical conformation under the same conditions at 10 degrees C. Addition of inorganic salts results in an increase of alpha-helix content, up to 42% in the presence of oligophosphate with an average chain length of 18 phosphates, which was used as an RNA analog. NMR experiments show that the alpha-helix formation starts in the region between Thr9 and Gln12, and is extended in the direction of the C terminus. Relaxation measurements show that binding to oligophosphates of increasing length results in reduced internal mobilities of the positively charged side chains of the arginyl and lysyl residues and of the side chain of Thr9 in the alpha-helical region. The alpha-helix formation in the N-terminal part of this viral coat protein upon binding of phosphate groups to the positively charged side chains is suggested to play an essential role in RNA binding.

Glycation of calmodulin: chemistry and structural and functional consequences

In the presence of Ca2+ and glucose, calmodulin incorporates 2.5 mol of glucose/mol of protein. In the absence of Ca2+, only 1.5 mol of glucose is incorporated per mole of calmodulin. Glycation of calmodulin is associated with variable reductions in its capacity to activate three Ca2+/calmodulin-dependent brain target enzyme systems, including adenylyl cyclase, phosphodiesterase, and protein kinase. In addition, glycated calmodulin exhibits a 54% reduction in its Ca2+ binding capacity. Isolated CNBr cleavage fragments of glycated calmodulin suggest that glycation follows a nonspecific pattern in that each of seven available lysines is susceptible to modification. A limit observed on the extent of glycation appears related to the accompanying increase in negative charge on the protein. Glycation results in minimal structural rearrangements in calmodulin, and the Ca2+-induced increase in alpha-helix content and radius of gyration is the same for glycated and unmodified calmodulin. Since glycated calmodulin's Ca2+ binding capacity is reduced, this implies that the Ca2+-induced conformational changes in calmodulin do not require all four Ca2+ binding sites to be occupied. Examination of the lysine positions in calmodulin suggests that Ca2+ binding to domains II and IV is sufficient to induce these changes. The functional consequences of calmodulin glycation therefore cannot be attributed to inhibition of these conformational changes. An alternative explanation is that the inhibition arises from interference at the target enzyme binding site by bound glucose. While glycation shows minimal structural effects, a large pH dependence is observed for the alpha-helix content of unmodified calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular dichroism and nucleotide and phosphate-induced conformational changes of mitochondrial adenosinetriphosphatase.

The conformational changes induced by the binding of different effectors on F1-ATPase are investigated by using circular dichroism and are related to enzyme activity. The hydrophilic part of the terminal enzyme of oxidative phosphorylation, F1-ATPase, solubilized from the pig heart mitochondrial membrane contains both regulatory and catalytic sites which can bind nucleotides and phosphate. The circular dichroic spectra of F1-ATPase in the absence or in the presence of ADP, Mg2+, phosphate, and the substrate analogue guanosine 5'-(beta, gamma-imidotriphosphate) [GMP-P-(NH)P] were recorded and analyzed in terms of secondary structure. The most significant result is a sizable increase from 35% to 42% of the alpha-helix content when the enzyme is incubated with all the effectors. Since the kinetic study showed that GMP-P(NH)P is a competitive inhibitor of MgATP with or without preincubation of the enzyme with ADP and phosphate, it was concluded that the catalytic and regulatory sites can be simultaneously occupied by ADP and GMP-P-(NH)P. The increase of alpha-helix content is then interpreted by a conformational change that occurs only after occupation of both types of sites.

Quantitative analysis of calcium binding to porcine intestinal calcium-binding protein.

An equilibrium dialysis study has revealed that porcine intestinal calcium-binding protein (CaBP) has two binding sites for Ca2+ whose dissociation constants (Kd) are the same, 0.56 microM. The intrinsic fluorescence spectrum of the CaBP shows a peak (at 303 nm) in tyrosine band. Ca2+ binding to the CaBP induces a monophasic increase in the intensity of intrinsic fluorescence without any shift to either excitation or emission maximum. The change in the fluorescence intensity induced by Ca2+ binding is complete at a bound Ca2+/CaBP molar ratio of about 2, and the apparent Kd value is 0.51 microM. The same bound Ca2+/CaBP molar ratio has been obtained from the maximal changes in UV absorption and CD spectrum of the CaBP upon addition of Ca2+. A CD study has shown an about 5% increase in alpha-helix content in the CaBP at the maximal binding of Ca2+. All these results indicate that the porcine intestinal CaBP has two high-affinity binding sites with equal affinity for Ca2+ and suggest that it undergoes a quantitative conformation change accompanying Ca2+ binding in the vicinity of single tyrosine and phenylalanine residues of the protein molecule.

Micellar complexes of human apolipoprotein A-I with phosphatidylcholines and cholesterol prepared from cholate-lipid dispersions.

Micellar complexes of human apolipoprotein A-I and phosphatidylcholine, with or without cholesterol, were prepared by adding apolipoprotein A-I (apo A-I) to sodium cholate-lipid mixtures. Cholate was removed by dialysis and the apo A-I.lipid complexes were isolated by gel filtration chromatography or by density gradient ultracentrifugation. The lipid mixtures consisted of dipalmitoylphosphatidylcholine or egg yolk phosphatidylcholine in the presence of various molar ratios of cholesterol. The formation of complexes was examined at different phosphatidylcholine (PC)-to-apo A-I ratios, PC-to-cholate ratios, and cholate concentrations. Yields of complexes were maximal when incubation and dialysis were performed near the transition temperature of the PC. Upon lipid binding and complex formation, apo A-I experienced a significant increase in alpha-helix content, and a blue shift in the intrinsic tryptophan fluorescence. In all lipid-protein incubation mixtures, from 600:1 to 75:1, PC/apo A-I (molar ratios), relatively small, stable complexes were present which gave maximum yields at incubation ratios similar to their isolated stoichiometries of 75:1 to 140:1, PC/apo A-I (molar ratios). For the isolated complexes, molecular weights were determined by sedimentation equilibrium to be in the range from 220,000 to 260,000; fluorescence polarization using the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene showed a broadened and shifted gel to liquid-crystalline phase transition, characteristic of micellar complexes of apo A-I with PC. Complexes prepared using apo A-I, covalently labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride, had an overall particle rotational relaxation time of 530 ns. On electron micrographs, the complexes, negatively stained with phosphotungstate, appeared as lamellar, discoidal particles.