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ABSTRACT: We have investigated the interactions between the antimicrobial peptide Novicidin (Nc) and vesicles containing the phospholipid DOPC, with various amounts of DOPG and cholesterol using circular dichroism spectroscopy, calcein release, equilibrium dialysis and isothermal titration calorimetry. Nc adopts a random coil structure in the absence of lipids and in the presence of vesicles containing 100% DOPC. Lipids with 25–40% DOPG induce the highest level of helicity in Nc; higher DOPG levels lead to lower helicity levels and an altered tertiary arrangement of the peptide. However, the ability of Nc to permeabilize vesicles correlates not with helicity but rather with its overall membrane affinity, which is enthalpically favorable but opposed by entropy. Permeabilization declines with increasing mole percentage PG. Changes in helicity correlate with changes in enthalpy, reflecting the enthalpy of helix formation, but not with affinity. There is also a large favorable enthalpic interaction between Nc and lipids in the absence of negative charge and structural changes. Cholesterol slightly reduces membrane permeabilization but has little effect on Nc affinity and secondary structure, and probably protects the membrane by inducing the liquid ordered state. We conclude that helicity is not a prerequisite for activity, and charge–charge interactions are not the only major driving force for AMP interactions with membranes. Our data are compatible with a model in which a superficial binding mode with a large membrane surface binding area per peptide is more efficient than a more intimate embedding within the membrane environment.
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ABSTRACT: We have investigated the interactions between the antimicrobial peptide Novicidin (Nc) and vesicles containing the phospholipid DOPC, with various amounts of DOPG and cholesterol using circular dichroism spectroscopy, calcein release, equilibrium dialysis and isothermal titration calorimetry. Nc adopts a random coil structure in the absence of lipids and in the presence of vesicles containing 100% DOPC. Lipids with 25-40% DOPG induce the highest level of helicity in Nc; higher DOPG levels lead to lower helicity levels and an altered tertiary arrangement of the peptide. However, the ability of Nc to permeabilize vesicles correlates not with helicity but rather with its overall membrane affinity, which is enthalpically favourable but opposed by entropy. Permeabilization declines with increasing mole percentage PG. Changes in helicity correlate with changes in enthalpy, reflecting the enthalpy of helix formation, but not with affinity. There is also a large favorable enthalpic interaction between Nc and lipids in the absence of negative charge and structural changes. Cholesterol slightly reduces membrane permeabilization but has little effect on Nc affinity and secondary structure, and probably protects the membrane by inducing the liquid ordered state. We conclude that helicity is not a prerequisite for activity, and charge-charge interactions are not the only major driving force for AMP interactions with membranes. Our data are compatible with a model in which a superficial binding mode with a large membrane surface binding area per peptide is more efficient than a more intimate embedding within the membrane environment.
Biochimica et Biophysica Acta 04/2013; · 4.66 Impact Factor
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ABSTRACT: The fap operon, encoding functional amyloids in Pseudomonas (Fap), is present in most pseudomonads, but so far the expression and importance for biofilm formation has only been investigated for P. fluorescens strain UK4. In this study, we demonstrate the capacity of P. aeruginosa PAO1, P. fluorescens Pf-5, and P. putida F1 to express Fap fibrils, and investigated the effect of Fap expression on aggregation and biofilm formation. The fap operon in all three Pseudomonas species conferred the ability to express Fap fibrils as shown using a recombinant approach. This Fap overexpression consistently resulted in highly aggregative phenotypes and in increased biofilm formation. Detailed biophysical investigations of purified fibrils confirmed FapC as the main fibril monomer and supported the role of FapB as a minor, nucleating constituent as also indicated by bioinformatic analysis. Bioinformatics analysis suggested FapF and FapD as a potential β-barrel membrane pore and protease, respectively. Manipulation of the fap operon showed that FapA affects monomer composition of the final amyloid fibril, and that FapB is an amyloid protein, probably a nucleator for FapC polymerization. Our study highlights the fap operon as a molecular machine for functional amyloid formation.
MicrobiologyOpen. 03/2013;
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Shuai Zhang,
Maria Andreasen,
Jakob T Nielsen,
Lei Liu,
Erik H Nielsen,
Jie Song,
Gang Ji,
Fei Sun,
Troels Skrydstrup,
Flemming Besenbacher,
Niels C Nielsen, Daniel E Otzen,
Mingdong Dong
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ABSTRACT: Uncontrolled misfolding of proteins leading to the formation of amyloid deposits is associated with more than 40 types of diseases, such as neurodegenerative diseases and type-2 diabetes. These irreversible amyloid fibrils typically assemble in distinct stages. Transitions among the various intermediate stages are the subject of many studies but are not yet fully elucidated. Here, we combine high-resolution atomic force microscopy and quantitative nanomechanical mapping to determine the self-assembled structures of the decapeptide hIAPP(20-29), which is considered to be the fibrillating core fragment of the human islet amyloid polypeptide (hIAPP) involved in type-2 diabetes. We successfully follow the evolution of hIAPP(20-29) nanostructures over time, calculate the average thickening speed of small ribbon-like structures, and provide evidence of the coexistence of ribbon and helical fibrils, highlighting a key step within the self-assembly model. In addition, the mutations of individual side chains of wide-type hIAPP(20-29) shift this balance and destabilize the helical fibrils sufficiently relative to the twisted ribbons to lead to their complete elimination. We combine atomic force microscopy structures, mechanical properties, and solid-state NMR structural information to build a molecular model containing β sheets in cross-β motifs as the basis of self-assembled amyloids.
Proceedings of the National Academy of Sciences 02/2013; · 9.68 Impact Factor
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ABSTRACT: Ribosomal protein S6 fibrillates readily at slightly elevated temperatures and acidic pH. We find that S6 fibrillation is retarded rather than favored when the protein concentration is increased above a threshold concentration of around 3.5mg/ml. We name this threshold concentration C(FR), the concentration at which fibrillation is retarded. Our data are consistent with a model in which this inhibition is due to the formation of an off-pathway oligomeric species with native-like secondary structure. The oligomeric species dominates at high protein concentrations but exists in dynamic equilibrium with the monomer so that seeding with fibrils can overrule oligomer formation and favors fibrillation under C(FR) conditions. Thus, fibrillation competes with formation of off-pathway oligomers, probably due to a monomeric conversion step that is required to commit the protein to the fibrillation pathway. The S6 oligomer is resistant to pepsin digestion. We also report that S6 forms different types of fibrils dependent on protein concentration. Our observations highlight the multitude of conformational states available to proteins under destabilizing conditions.
Biochimica et Biophysica Acta 01/2013; · 4.66 Impact Factor
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ABSTRACT: Extensive studies on the spontaneous collapse of phospholipid vesicles into supported lipid bilayers (SLBs) have led to procedures which allow SLB formation on a wealth of substrates and lipid compositions. SLBs provide a widely accepted and versatile model system which mimics the natural cell membrane separating the extracellular and intracellular fluids of the living cell. The quartz crystal microbalance with dissipation monitoring (QCM-D) has been central both in the understanding of vesicle collapse into SLBs on various substrates and in probing the kinetics and mechanisms of biomolecular interactions with SLBs in real time. We describe a robust procedure to form SLBs of zwitterionic and charged lipids on SiO(2) sensor crystals which subsequently can be exploited to probe the interaction between proteins and peptides with the SLB.
Methods in molecular biology (Clifton, N.J.) 01/2013; 974:1-21.
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ABSTRACT: Thermomyces lanuginosus Lipase (TlL) is a kinetically stable protein, resistant towards both denaturation and refolding in presence of the ionic surfactant sodium dodecyl sulfate (SDS) and the nonionic surfactant decyl maltoside (DecM). We investigate the pH-dependence of this kinetic stability. At pH 8, TlL remains folded and enzymatically active at multi-mM surfactant concentrations but fails to refold from the acid-urea denatured state at sub-mM concentrations of SDS and DecM, indicating a broad concentration range of kinetic trapping or hysteresis. At pH 8, very few SDS molecules bind to TlL. The hysteresis SDS concentration range shrinks when moving to pH 4 and 6; in this pH range SDS binds as micelle-like clusters. Although hysteresis can be removed by reducing disulfide bonds, destabilizing the native state and lowering the unfolding activation barrier, SDS-sensitivity is not directly linked to intrinsic kinetic stability (its resistance to the general chemical denaturant guanidinium chloride (GdmCl)), since TlL unfolds more slowly in GdmCl at pH 6.0 than at pH 8.0. However, the estimated net charge drops from ~-12 to ~-5 between pH 8 and 6. SDS denatures TlL at pH 6.0 by nucleating via a critical number of bound SDS molecules on the surface of native TlL to form clusters. These results imply that SDS-sensitivity is connected to the availability of appropriately charged regions on the protein. We suggest that conformational rigidity is a necessary but not sufficient feature of SDS-resistance, since this has to be combined with sufficient negative electrostatic potential to avoid extensive SDS binding.
Biochemistry 12/2012; · 3.42 Impact Factor
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ABSTRACT: Outer membrane proteins (OMPs) represent a large group of β-barrel proteins found both in the membranes of both bacteria and eukaryotes. Their general ease of expression and refolding and straightforward methods to monitor their degree of folding conspire to make OMPs excellent model systems to investigate how the membrane environment and other biological factors modulating the membrane insertion and folding of OMPs influence the folding pathway. This review attempts to provide an overview of how these proteins are studied in vitro and what kind of information can reliably be extracted. Numerous choices have to be made in setting the conditions for successful folding/unfolding, and here a major challenge remains to identify conditions that lead to completely reversible folding without any hysteresis. Recent progress indicates that this is possible through rigorous optimization, such as the use of relatively extreme pH and phospholipids with short chain lengths. OMPs are generally kinetically very stable, which means that they both fold and unfold very slowly. Many OMPs cannot even unfold when embedded in lipid vesicles, but recent work has demonstrated that surfactants can provide a useful alternative which can lead to a complete description of the kinetics of folding and unfolding of an OMP. The recent report of the first protein engineering study of an OMP has demonstrated that it may soon be possible to have almost atomic-level resolution of an OMP folding mechanism. Combining this insight with the biological complexity of the membrane environment constitutes an exciting new frontier in membrane protein science. Abbrevations: Cmc, critical micelle concentration; LPS, lipopolysaccharide; LUV, large unilamellar vesicles; OMP, outer membrane protein; PL, phospholipids; SUV, small unilamellar vesicles.
Archives of Biochemistry and Biophysics 11/2012; · 2.93 Impact Factor
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ABSTRACT: The outer membrane protein OmpA from Escherichia coli can fold into lipid vesicles and surfactant micelles from the urea-denatured state. However, a complete kinetic description of the folding and unfolding of OmpA, which can provide the basis for subsequent protein engineering studies of the protein's folding pathway, is lacking. Here we use two different denaturants to probe the unfolding mechanism of OmpA in the presence of the surfactant octyl maltoside (OM). Unfolding of OmpA in the presence of micelles, achieved with the potent denaturant guanidinium chloride (GdmCl), leads to single-phase unfolding. In contrast, OmpA unfolds in urea only below OM's critical micelle concentration, and this occurs in different phases, which we attribute to the existence of states that have bound different amounts of surfactant, from completely "naked" to partly covered by surfactant. Multiple parallel refolding phases are attributed to different levels of collapse prior to folding. Kinetic results used to derive the stability of OmpA in surfactant, using either urea or GdmCl as the denaturing agent, give comparable results and indicate a minimalist three-state folding scheme involving denatured state D, folding intermediate I, and native state N. N and I are stabilized by 15.6 and 2.6 kcal/mol, respectively, relative to D. The periplasmic domain of OmpA does not contribute to stability in surfactant micelles. However, BBP, a minimalist transmembrane β-barrel version of OmpA with shortened loops, is destabilized by ∼10 kcal/mol compared to OmpA, highlighting loop contributions to OmpA stability.
Biochemistry 09/2012; 51(42):8371-83. · 3.42 Impact Factor
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ABSTRACT: Mutations in the transforming growth factor β-induced protein (TGFBIp) are linked to the development of corneal dystrophies in which abnormal protein deposition in the cornea leads to a loss of corneal transparency and ultimately blindness. Different mutations give rise to phenotypically distinct corneal dystrophies. Most mutations are located in the fourth fasciclin-1 domain (FAS1-4). The amino acid substitution A546T in the FAS1-4 domain is linked to the development of lattice corneal dystrophy with amyloid deposits in the superficial and deep stroma, classifying it as an amyloid disease. Here we provide a detailed description of the fibrillation of the isolated FAS1-4 domain carrying the A546T substitution. The A546T substitution leads to a significant destabilization of FAS1-4 and induces a partially folded structure with increased surface exposure of hydrophobic patches. The mutation also leads to two distinct fibril morphologies. Long straight fibrils composed of pure β-sheet structure are formed at lower concentrations, whereas short and curly fibrils containing a mixture of α-helical and β-sheet structures are formed at higher concentrations. The formation of short and curly fibrils is preceded by the formation of a small number of oligomeric species with high membrane permeabilization potential and rapid fibril formation. The long straight fibrils are formed more slowly and through progressively bigger oligomers that lose their membrane permeabilization potential as fibrillation proceeds beyond the lag phase. These different fibril classes and associated biochemical differences may lead to different clinical symptoms associated with the mutation.
Journal of Biological Chemistry 08/2012; 287(41):34730-42. · 4.77 Impact Factor
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ABSTRACT: Many severe diseases are associated with amyloid fibril deposits in the body caused by protein misfolding. Structural information on amyloid fibrils is accumulating rapidly, but little is known about the assembly of peptides into fibrils at the level of individual molecules. Here we investigate self-assembly of the fibril-forming tetrapeptides KFFE and KVVE on a gold surface under ultraclean vacuum conditions using scanning tunneling microscopy. Combined with restrained molecular dynamics modeling, we identify peptide arrangements with interesting similarities to fibril structures. By resolving individual peptide residues and revealing conformational heterogeneities and dynamics, we demonstrate how conformational correlations may be involved in cooperative fibril growth. Most interestingly, intermolecular interactions prevail over intramolecular interactions, and assembly of the phenyl-rich KFFE peptide appears not to be dominated by π-π interactions. This study offers interesting perspectives for obtaining fundamental single-molecule insights into fibril formation using a surface science approach to study idealized model systems.
ACS Nano 07/2012; 6(8):6882-9. · 10.77 Impact Factor
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ABSTRACT: Bacterial resistance to classical antibiotics is a serious medical problem, which continues to grow. Small antimicrobial peptides represent a potential solution and are increasingly being developed as novel therapeutic agents. Many of these peptides owe their antibacterial activity to the formation of trans-membrane ion-channels resulting in cell lysis. However, to further develop the field of peptide antibiotics, a thorough understanding of their mechanism of action is needed. Alamethicin belongs to a class of peptides called peptaibols and represents one of these antimicrobial peptides. To examine the dynamics of assembly and to facilitate a thorough structural evaluation of the alamethicin ion-channels, we have applied click chemistry for the synthesis of templated alamethicin multimers covalently attached to cyclodextrin-scaffolds. Using oriented circular dichroism, calcein release assays, and single-channel current measurements, the α-helices of the templated multimers were demonstrated to insert into lipid bilayers forming highly efficient and remarkably stable ion-channels.
The Journal of Physical Chemistry B 06/2012; 116(26):7652-9. · 3.70 Impact Factor
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ABSTRACT: Mixed micelle formation of anionic surfactants sodium dodecyl sulfate (SDS) and sodium lauroyl sarcosine (SLAS) have been studied in water and in 5, 10, and 15mM concentrations of α-cyclodextrin (α-CD) over mole fraction range of α
SDS from 0 to 1. From the conductivity curves, the critical micellar concentration (CMC) for the pure and binary mixtures were evaluated. The degree of counterion association (χ) or counterion dissociation (δ), the equivalent ionic conductivities of the monomeric species (Λ
m), the associated species (Λ
assc), and the micelle (Λ
mic) were evaluated from the slope of the conductivity vs concentration plots. The CMC values have been used to calculate the thermodynamic parameters such as the standard free energy of micelle formation DG0mic\Delta G^{{\rm{0}}}_{{{\rm{mic}}}} and a transfer of standard free energy of micelle
( DG\text0M,tr ){\left( {\Delta G^{{\text{0}}}_{{{\rm{M,tr}}}} } \right)} from the aqueous medium to additive medium computed. The apparent CMC of the surfactants varies linearly with α-CD concentrations. From the dependence of CMC of the surfactants on α-CD concentration, we are able to determine the association constant (K) of surfactant-α-CD inclusion complexes assuming 1:1 stoichiometry. Mixed micelle behaves ideally in the pure water as well as at the different concentrations of α-CD, which was evaluated by using the Clint equation, the regular solution approximation, and Motomura’s formulation. Self-diffusion coefficients of the micelle increased upon the induction of SDS into the micelle. 2D-rotating frame Overhauser effect spectroscopy spectra of SDS and SLAS were recorded in the presence of α-CD to investigate the interaction between H-atoms of the alkyl chain of the surfactants and H-atoms of the hydrophobic cavity of α-CD indicating multiple complexation. The fluorescence anisotropy of rhodamine B has been measured to observe the structural behavior of mixed micelle.
Colloid and Polymer Science 04/2012; 284(8):916-926. · 2.33 Impact Factor
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ABSTRACT: The interactions of α-cyclodextrin (α-CD) with the nonionic surfactant decanoyl-N-methyl-glucamide (Mega-10) and the zwitterionic surfactant dimethyldodecylammoniopropanesulfonate (DPS) in their mixed system
have been studied using interfacial tension, fluorescence, and nuclear magnetic resonance measurements. From the plots of
interfacial tension vs. log of total surfactant concentration, we have obtained values of the surface excess of surfactant,
the critical micellar concentration (cmc), the standard free energy of micelle formation, and association constant of surfactant/α-CD
inclusion complexes (assuming a 1:1 stoichiometry). A comparison of the K
a values obtained for the interaction between α-CD and DPS and Mega-10, respectively, shows that DPS interacts stronger with
α-CD than Mega-10. The experimental mixed cmc was analyzed by the pseudophase separation model and regular solution theory
for the evaluation of ideality or nonideality of the mixed micelle formation. The interaction parameters in the mixed micelle
and the micelle composition at different mole fractions of DPS were also computed. The fluorescence anisotropy (r) values of rhodamine B decreases with the increase of α-CD concentrations.
Colloid and Polymer Science 04/2012; 287(11):1243-1252. · 2.33 Impact Factor
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ABSTRACT: Membrane proteins are vital for biological function, and their action is governed by structural properties critically depending on their interactions with the membranes. This has motivated considerable interest in studies of membrane protein folding and unfolding. Here the structural changes induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl β-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows addressing detergent properties and protein conformations at the same time. The mapping of the states reveals that KcsA undergoes a series of rearrangements which include expansion of the tetramer in several steps followed by dissociation into monomers at 29% TFE. Supplementary studies of DDM and TFE in the absence of KcsA suggest that the disintegration of the tetramer at 29% TFE is caused by TFE dissolving the surrounding DDM rim. Above 34% TFE, KcsA collapses to a new structure that is fully formed at 44% TFE.
Biochimica et Biophysica Acta 04/2012; 1818(9):2290-301. · 4.66 Impact Factor
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ABSTRACT: The 219-residue protein p25α stimulates the fibrillation of α-synuclein (αSN) in vitro and colocalizes with it in several α-synucleinopathies. Although p25α does not fibrillate by itself under native conditions in vitro, αSN-free p25α aggregates have also been observed in vivo in, for example, multiple system atrophy. To investigate which environmental conditions might trigger this aggregation, we investigated the effect of polyanionic biomolecules on p25α aggregation. Heparin, polyglutamate, arachidonic acid micelles, and RNA all induce p25α aggregation. More detailed studies using heparin as template for aggregation reveal that a minimum of 10-14 heparin monosaccharide units per heparin polymer are required. Bona fide fibrils are only formed at intermediate heparin concentrations, possibly because an excess of heparin binding sites blocks the inter-p25α contacts required for amyloid formation. Other polyanions also show an optimum for amyloid formation. Aggregation involves only modest structural changes according to both spectroscopic and proteolytic experiments. The aggregates do not seed aggregation of heparin-free p25α, suggesting that heparin is required in stoichiometric amounts to form organized structures. We are able to reproduce these observations in a model involving two levels of binding of p25α to heparin. We conclude that the modest structural changes that p25α undergoes can promote weak intermolecular contacts and that polyanions such as heparin play a central role in stabilizing these aggregates but in multiple ways, leading to different types of aggregates. This highlights the role of non-protein components in promoting protein aggregation in vivo.
Journal of Molecular Biology 02/2012; 421(4-5):601-15. · 4.00 Impact Factor
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ABSTRACT: Osmolytes are small molecules that are exploited by cells as a protective system against stress conditions. They favour compact protein states which makes them stabilize globular proteins in vitro and promote folding. Conversely, this preference for compact states promotes aggregation of unstructured proteins. Here we combine a brief review of the effect of osmolytes on protein fibrillation with a report of the effect of osmolytes on the unstructured peptide hormone glucagon. Our results show that osmolytes either accelerate the fibrillation kinetics or leave them unaffected, with the exception of the osmolyte taurine. Furthermore, the osmolytes that affected the shape of the fibrillation time profile led to fibrils with different structure as revealed by CD. The structural changes induced by Pro, Ser and choline-O-sulfate could be due to specific osmolytes binding to the peptides, stabilizing an otherwise labile fibrillation intermediate.
International Journal of Molecular Sciences 01/2012; 13(3):3801-19. · 2.60 Impact Factor
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Maria Andreasen,
Søren B Nielsen,
Tina Mittag,
Morten Bjerring,
Jakob T Nielsen,
Shuai Zhang,
Erik H Nielsen,
Martin Jeppesen,
Gunna Christiansen,
Flemming Besenbacher,
Mingdong Dong,
Niels Chr Nielsen,
Troels Skrydstrup, Daniel E Otzen
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ABSTRACT: The well-ordered cross β-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form β-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. β-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6-10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe-Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the -NH- groups has been replaced by -CH(2)-) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone.
Biochimica et Biophysica Acta 10/2011; 1824(2):274-85. · 4.66 Impact Factor
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Daniel E Otzen
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ABSTRACT: Taylor et al. report the crystal structure of CsgC, a redox-active member of E. coli's curli-producing Csg operon. The outer membrane protein CsgG is a potential redox-substrate and might exist in an octameric structure with a periplasmatic CsgC binding site, highlighting a potential role for disulfide bonds in curli production.
Structure 09/2011; 19(9):1207-9. · 6.35 Impact Factor
