Random Coil Values Research Articles

Impact of pH-mediated in situ enzymatic pretreatment on food waste decomposition and biomethane production: Performance, mechanism, and key metabolic pathways

pH value universally presents the great influence on hydrolysis efficiency of food waste (FW) by in situ enzymatic pretreatment, while the influencing mechanism of pH value on enzymatic decomposition of FW is not clear at present. This study innovatively in situ prepared a microbial-based compound enzyme (MCE) from FW, as well as systematically deciphered the impact and underlying mechanism of pH-mediated MCE pretreatment on FW decomposition and biomethane production. The dissolution, biodegradability and monomer formation of FW were found to be promoted by MCE pretreatment, and the highest hydrolysis efficiency was obtained at pH 5, while the biomethane yield was accordingly increased by 30.89 % compared to the control. The dissection of Fourier transform infrared spectroscopy (FTIR) results unraveled that the reaction order of FW components was protein → lipid → starch → cellulose in the acid-enhanced process, while the order shifted to starch → protein → cellulose → lipid in the alkali-enhanced process. Moreover, the looser protein structure was obtained after MCE pretreatment due to α-helix/(β-sheet + random coil) value was declined from 60.35 % to 57.01 %-49.03 %. These changes induced by pH-mediated MCE pretreatment reshaped the bacterial microbial community in favor of biomethane production, and the metagenomic analysis further demonstrated that MCE pretreatment had the highest abundances of genes related to metabolic functions of level 2 and 3 at pH 5, while the abundances of over 90 % key enzyme-encoding genes associated with hydrolysis, acidogenesis and methanogenesis were also increased during the anaerobic digestion process, therefore harvesting the highest biomethane yield.

Carbamazepine improves hydrogen production from anaerobic fermentation of waste activated sludge

The widespread use of pharmaceuticals sparked concerns about their potential environmental effects. Previous studies demonstrated that most of the pharmaceuticals investigated had detrimental effects on wastewater or sludge treatment. However, it was found in this work that carbamazepine at environmentally relevant levels increased hydrogen production from anaerobic fermentation of waste activated sludge (WAS). Experimental results exhibited that with an increment of carbamazepine from 7 to 51 mg/kg VSS, the maximal hydrogen yield increased from 14.9 ± 0.6 to 23.5 ± 0.3 mL/g VSS. Mechanism analysis showed that carbamazepine accelerated the release of organics from solid to liquid phase, offering more substrates for hydrogen production. Carbamazepine did not affect hydrogen producing processes but inhibited the processes relevant to hydrogen consumption. Molecular docking demonstrated that carbamazepine and sulfite reductase protein were joined by hydrogen bond and ASP-597 and VAL-598 were the active sites. These contributed to the contact between carbamazepine and sulfite reductase, thereby affecting the hydrogen consumption process. The reduced α -helix ratio and α -helix / (β -plate + random coil) values indicated that the presence of carbamazepine led to the destroy of the hydrogen network, leading the collapse of protein structure. Further investigation revealed that carbamazepine reduced the corresponding genes with hydrogen consumption, especially involved in the steps of CO2 → fmdA → Formyl-MFR → 5,10-Methylere-THMPT → mer → 5,10-Methyl-THM(S)PT (CO2-reduction), 5,10-Methyl-THM(S)PT → mtrD/E → Methyl-CoM → mcrA/B → CH4 (CO2-reduction and aceticlastic reaction) and Sulfate → sat → APS → aprB → Sulfite → cysJ → Sulfide (sulfate reduction).

Random coil chemical shifts for serine, threonine and tyrosine phosphorylation over a broad pH range

Phosphorylation is one of the main regulators of cellular signaling typically occurring in flexible parts of folded proteins and in intrinsically disordered regions. It can have distinct effects on the chemical environment as well as on the structural properties near the modification site. Secondary chemical shift analysis is the main NMR method for detection of transiently formed secondary structure in intrinsically disordered proteins (IDPs) and the reliability of the analysis depends on an appropriate choice of random coil model. Random coil chemical shifts and sequence correction factors were previously determined for an Ac-QQXQQ-NH2-peptide series with X being any of the 20 common amino acids. However, a matching dataset on the phosphorylated states has so far only been incompletely determined or determined only at a single pH value. Here we extend the database by the addition of the random coil chemical shifts of the phosphorylated states of serine, threonine and tyrosine measured over a range of pH values covering the pKas of the phosphates and at several temperatures (www.bio.ku.dk/sbinlab/randomcoil). The combined results allow for accurate random coil chemical shift determination of phosphorylated regions at any pH and temperature, minimizing systematic biases of the secondary chemical shifts. Comparison of chemical shifts using random coil sets with and without inclusion of the phosphoryl group, revealed under/over estimations of helicity of up to 33%. The expanded set of random coil values will improve the reliability in detection and quantification of transient secondary structure in phosphorylation-modified IDPs.

Changes of structural and physical properties of semi-gel from Alaska pollock surimi during 4 °C storage

This study compared the semi-gel system of Alaska pollock surimi formed at a low temperature with a heat-induced gel system. The cold semi-gel and heat-induced gel were found to have obvious differences in their degrees of protein unfolding, aggregation, and gel network structures. Microscopic observations and an analysis of their fractal dimensions revealed different degrees of compactness and uniformity in the two network structures. Rheological parameters, including the least permanent deformation, equilibrium stress, and frequency exponent, confirmed the non-total aggregation and lower degree of cross-linking in the cold semi-gel. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that the myosin heavy chain (MHC) value gradually decreased, but the light meromyosin (LMM) value remained stable during cold storage. Furthermore, a decrease in the α-helix and an increase in the β-sheet and random coil values, as revealed by Fourier-transform infrared (FT-IR) spectroscopy, indicated that the proteins unfolded differently in the two gel systems. By comparing the times required for the cold semi-gel and heat-induced gel to form, we concluded that the formation process of heat-induced gel is intertwined while it is temporally separated in cold storage. Overall, myosin was selected as the starting point for establishing a schema chart to characterize the gelation processes of the cold semi-gel and heat-induced gel. These findings are helpful for understanding the gelation process of surimi in refrigerated storage.

The Dynamic Landscape of the Full-Length HIV-1 Transactivator of Transcription.

The type 1 human immunodeficiency virus (HIV-1) transactivator of transcription (Tat) is a small RNA-binding protein essential for viral gene expression and replication. It has also been shown to bind to a large number of human proteins and to modulate many different cellular activities. We have used nuclear magnetic resonance (NMR) spectroscopy and hydrogen exchange chemistry to measure backbone dynamics over the millisecond to picosecond time scales. Sequential backbone assignment was facilitated by several isotope labeling schemes, including uniform labeling, site-specific labeling, and unlabeling. (15)N NMR relaxation parameters were measured and analyzed by reduced spectral density mapping and the Lipari-Szabo Model-Free approach to characterize the backbone dynamics on the picosecond to nanosecond time scale. The results indicate that the protein exists in an extended disordered conformational ensemble. NMR relaxation dispersion profiles show that on the millisecond time scale no conformational exchange is detected for any of the residues, supporting the model of a disordered backbone. NMR chemical shift differences from random coil values suggest that some segments of the protein have a modest propensity to fold; comparison to X-ray diffraction structures of Tat complexes indicates that some segments of the protein function through an induced-fit mechanism whereas other segments likely operate by conformational selection. Surprisingly, measured hydrogen exchange rates are higher than predicted for a disordered polymer, but this is explained as being caused by the high net charge on the protein that enhances base-catalyzed hydrogen exchange. The dynamics results provide a deeper understanding of the protein conformational ensemble and form a foundation for future studies of the conformational changes that accompany the formation of the superelongation complex that activates viral transcription.

Improved validation of IDP ensembles by one-bond Cα-Hα scalar couplings.

Intrinsically disordered proteins (IDPs) are best described by ensembles of conformations and a variety of approaches have been developed to determine IDP ensembles. Because of the large number of conformations, however, cross-validation of the determined ensembles by independent experimental data is crucial. The (1)JCαHα coupling constant is particularly suited for cross-validation, because it has a large magnitude and mostly depends on the often less accessible dihedral angle ψ. Here, we reinvestigated the connection between (1)JCαHα values and protein backbone dihedral angles. We show that accurate amino-acid specific random coil values of the (1)JCαHα coupling constant, in combination with a reparameterized empirical Karplus-type equation, allow for reliable cross-validation of molecular ensembles of IDPs.

PH Dependence of Amylin Fibrillization

In type 2 diabetics, the hormone amylin misfolds into amyloid plaques implicated in the destruction of the pancreatic β-cells that make insulin and amylin. The aggregative misfolding of amylin is pH-dependent, and exposure of the hormone to acidic and basic environments could be physiologically important. Amylin has two ionizable residues between pH 3 and 9: the α-amino group and His18. Our approach to measuring the pKa values for these sites has been to look at the pH dependence of fibrillization in amylin variants that have only one of the two groups. The α-amino group at the unstructured N-terminus of amylin has a pKa near 8.0, similar to the value in random coil models. By contrast, His18, which is involved in the intermolecular β-sheet structure of the fibrils, has a pKa that is lowered to 5.0 in the fibrils compared to the random coil value of 6.5. The lowered pKa of His18 is due to the hydrophobic environment of the residue, and electrostatic repulsion between positively charged His18 residues on neighboring amylin molecules in the fibril. His18 acts as an electrostatic switch inhibiting fibrillization in its charged state. The presence of a charged side chain at position 18 also affects fibril morphology and lowers amylin cytotoxicity toward a MIN6 mouse model of pancreatic β-cells. In addition to the two expected pKa values, we detected an apparent pKa of ~4.0 for the amylin-derived peptide NAc-SNNFGAILSS-NH2, which has no titratable groups. This pKa is due to the pH-induced ionization of the dye thioflavin T. By using alternative methods to follow fibrillization such as the dye Nile Red or turbidimetry, we were able to distinguish between the titration of the dye and groups on the peptide. Large differences in reaction kinetics were observed between the different methods at acidic pH, because of charges on the ThT dye, which hinder fibril formation much like the charges on the protein.

13C structuring shifts for the analysis of model β-hairpins and β-sheets in proteins: diagnostic shifts appear only at the cross-strand H-bonded residues

The present studies have shown that (13)C=O, (13)C(α) and (13)C(β) of H-bonded strand residues in β-hairpins provide additional probes for quantitating the extent of folding in β-hairpins and other β-sheet models. Large differences in the structuring shifts (CSDs) of these (13)C sites in H-bonded versus non-H-bonded sites are observed: the differences between H-bonded and non-H-bonded sites are greater than 1.2 ppm for all three (13)C probes. This prompts us to suggest that efforts to determine the extent of hairpin folding from (13)C shifts should be based exclusively on the observation at the cross-strand H-bonded sites. Furthermore, the statistics suggest the (13)C' and (13)C(β) CSDs will provide the best differentiation with 100%-folded CSD values approaching -2.6 and +3 ppm, respectively, for the H-bonded sites. These conclusions can be extended to edge-strands of protein β-sheets. Our survey of reported (13)C shifts in β-proteins indicates that some of the currently employed random coil values need to be adjusted, particularly for ionization-induced effects.

Impact of Hydrostatic Pressure on an Intrinsically Disordered Protein: A High‐Pressure NMR Study of α‐Synuclein

The impact of pressure on the backbone (15) N, (1) H and (13) C chemical shifts in N-terminally acetylated α-synuclein has been evaluated over a pressure range 1-2500 bar. Even while the chemical shifts fall very close to random coil values, as expected for an intrinsically disordered protein, substantial deviations in the pressure dependence of the chemical shifts are seen relative to those in short model peptides. In particular, the nonlinear pressure response of the (1) H(N) chemical shifts, which commonly is associated with the presence of low-lying "excited states", is much larger in α-synuclein than in model peptides. The linear pressure response of (1) H(N) chemical shift, commonly linked to H-bond length change, correlates well with those in short model peptides, and is found to be anticorrelated with its temperature dependence. The pressure dependence of (13) C chemical shifts shows remarkably large variations, even when accounting for residue type, and do not point to a clear shift in population between different regions of the Ramachandran map. However, a nearly universal decrease in (3) JHN-Hα by 0.22 ± 0.05 Hz suggests a slight increase in population of the polyproline II region at 2500 bar. The first six residues of N-terminally acetylated synuclein show a transient of approximately 15% population of α-helix, which slightly diminishes at 2500 bar. The backbone dynamics of the protein is not visibly affected beyond the effect of slight increase in water viscosity at 2500 bar.

Peptide–Column Interactions and Their Influence on Back Exchange Rates in Hydrogen/Deuterium Exchange-MS

Hydrogen/deuterium exchange (HDX) methods generate useful information on protein structure and dynamics, ideally at the individual residue level. Most MS-based HDX methods involve a rapid proteolytic digestion followed by LC/MS analysis, with exchange kinetics monitored at the peptide level. Localizing specific sites of HDX is usually restricted to a resolution the size of the host peptide because gas-phase processes can scramble deuterium throughout the peptide. Subtractive methods may improve resolution, where deuterium levels of overlapping and nested peptides are used in a subtractive manner to localize exchange to smaller segments. In this study, we explore the underlying assumption of the subtractive method, namely, that the measured back exchange kinetics of a given residue is independent of its host peptide. Using a series of deuterated peptides, we show that secondary structure can be partially retained under quenched conditions, and that interactions between peptides and reversed-phase LC columns may both accelerate and decelerate residue HDX, depending upon peptide sequence and length. Secondary structure is induced through column interactions in peptides with a solution-phase propensity for structure, which has the effect of slowing HDX rates relative to predicted random coil values. Conversely, column interactions can orient random-coil peptide conformers to accelerate HDX, the degree to which correlates with peptide charge in solution, and which can be reversed by using stronger ion pairing reagents. The dependency of these effects on sequence and length suggest that subtractive methods for improving structural resolution in HDX-MS will not offer a straightforward solution for increasing exchange site resolution.

Alpha-Synuclein, an Intrinsically Unstructured Protein. How Interesting Can It Be?

N-terminal acetylation of alpha-synuclein (aS), a 140-residue protein implicated in the etiology of Parkinson's disease, is common in mammals. The impact of this modification on the protein's structure and dynamics in free solution and on its membrane binding properties has been evaluated by both NMR and CD spectroscopy. While in contrast to literature reports, no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in ca 15% transient population of alpha-helical structure for its first six residues. The 1H, 15N, and 13C chemical shifts for residues 13-140 remain unaffected by acetylation. Nevertheless, a substantial increase in affinity of aS for negatively charged lipid membranes is observed, likely to be of strong functional significance. A new method for residue-specific NMR probing of lipid binding is demonstrated for aS and assigns a new putative function to this enigmatic protein. Although free aS in the absence of lipids has backbone chemical shifts that are exceptionally close to random coil values, considerable positional variation for the distribution of its backbone torsion angles and the time scale of local reorientation can be deduced from its 3JHH and NOE data.

The pH-Dependence of Amylin Fibrillization

In type 2 diabetics, the hormone amylin misfolds into amyloid plaques implicated in the destruction of the pancreatic β-cells that make insulin and amylin. The pH-dependence of fibril formation affects fibrillization kinetics, fibril morphology, and possibly toxicity. Amylin is stored in β-cell secretory granules at pH 5.5 and is released into the extracellular matrix at pH 7.4. There are two ionizable residues in amylin: the α-amino group and His 18. Our approach to measuring the pKa values for these sites has been to look at the pH dependence of fibrillization in amylin variants that have only one of the groups: NAc-amylin (for His18) and H18R (for the N-terminus). The fibril-specific dye thioflavin-T (ThT) was used to monitor kinetics. The pKa of His18 is lowered to 5.1 in the fibrils, compared to the random coil value of 6.5. The lowered pKa is due to the unfavorable burial of a charge, and electrostatic repulsion between positively charged His18 residues on neighboring amylin molecules in the fibril. For H18R, which can only be protonated at the N-terminus, we see a pKa ∼8 due to the N-terminus but a second pKa of ∼4.5 due to protonation of ThT. We confirmed this with the amylin-derived peptide NAc-SNNFGAILSS-NH2 which has no titratable groups but still shows a pKa of 4.5 due to ThT. A pKa of 1.9 for free ThT was determined by NMR but with fluorescence we are monitoring the excited-state of the amyloid-bound dye which has a perturbed pKa. By using alternative methods to follow fibrillization such as the dye Nile Red or turbidimetry we were able to distinguish between the effects. Large differences in reaction kinetics were observed between the different methods, due to charges on the dyes, which affect fibril formation much like charges on the protein.

Deuterium isotope shifts for backbone 1H, 15N and 13C nuclei in intrinsically disordered protein α-synuclein

Intrinsically disordered proteins (IDPs) are abundant in nature and characterization of their potential structural propensities remains a widely pursued but challenging task. Analysis of NMR secondary chemical shifts plays an important role in such studies, but the output of such analyses depends on the accuracy of reference random coil chemical shifts. Although uniform perdeuteration of IDPs can dramatically increase spectral resolution, a feature particularly important for the poorly dispersed IDP spectra, the impact of deuterium isotope shifts on random coil values has not yet been fully characterized. Very precise (2)H isotope shift measurements for (13)C(α), (13)C(β), (13)C', (15)N, and (1)H(N) have been obtained by using a mixed sample of protonated and uniformly perdeuterated α-synuclein, a protein with chemical shifts exceptionally close to random coil values. Decomposition of these isotope shifts into one-bond, two-bond and three-bond effects as well as intra- and sequential residue contributions shows that such an analysis, which ignores conformational dependence, is meaningful but does not fully describe the total isotope shift to within the precision of the measurements. Random coil (2)H isotope shifts provide an important starting point for analysis of such shifts in structural terms in folded proteins, where they are known to depend strongly on local geometry.

Impact of N-Terminal Acetylation of α-Synuclein on Its Random Coil and Lipid Binding Properties

N-Terminal acetylation of α-synuclein (aS), a protein implicated in the etiology of Parkinson’s disease, is common in mammals. The impact of this modification on the protein’s structure and dynamics in free solution and on its membrane binding properties has been evaluated by high-resolution nuclear magnetic resonance and circular dichroism (CD) spectroscopy. While no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in chemical shift perturbations of the first 12 residues of the protein that progressively decreased with the distance from the N-terminus. The directions of the chemical shift changes and small changes in backbone 3JHH couplings are consistent with an increase in the α-helicity of the first six residues of aS, although a high degree of dynamic conformational disorder remains and the helical structure is sampled <20% of the time. Chemical shift and 3JHH data for the intact protein are virtually indistinguishable from those recorded for the corresponding N-terminally acetylated and nonacetylated 15-residue synthetic peptides. An increase in α-helicity at the N-terminus of aS is supported by CD data on the acetylated peptide and by weak medium-range nuclear Overhauser effect contacts indicative of α-helical character. The remainder of the protein has chemical shift values that are very close to random coil values and indistinguishable between the two forms of the protein. No significant differences in the fibrillation kinetics were observed between acetylated and nonacetylated aS. However, the lipid binding properties of aS are strongly impacted by acetylation and exhibit distinct behavior for the first 12 residues, indicative of an initiation role for the N-terminal residues in an “initiation–elongation” process of binding to the membrane.

β-Hairpin Peptide That Targets Vascular Endothelial Growth Factor (VEGF) Receptors

VEGF receptors have been the target of intense research aimed to develop molecules able to inhibit or stimulate angiogenesis. Based on the x-ray structure of the complex placental growth factor-VEGF receptor 1(D2), we designed a VEGF receptor-binding peptide reproducing the placental growth factor β-hairpin region Gln(87)-Val(100) that is involved in receptor recognition. A conformational analysis showed that the designed peptide adopts the expected fold in pure water. Moreover, a combination of NMR interaction analysis and cell binding studies were used to demonstrate that the peptide targets VEGF receptors. The VEGF receptor 1(D2)-interacting residues were characterized at the molecular level, and they correspond to the residues recognizing the placental growth factor sequence Gln(87)-Val(100). Finally, the peptide biological activity was characterized in vitro and in vivo, and it showed a VEGF-like behavior. Indeed, the peptide activated VEGF-dependent intracellular pathways, induced endothelial cell proliferation and rescue from apoptosis, and promoted angiogenesis in vivo. This compound is one of the few peptides known with proangiogenic activity, which makes it a candidate for the development of a novel peptide-based drug for medical applications in therapeutic angiogenesis.

Characteristics of Human Lysozyme and Its Disease‐Related Mutants in their Unfolded States

Characteristics of Human Lysozyme and Its Disease‐Related Mutants in their Unfolded States

Backbone assignment and secondary structure of the PsbQ protein from Photosystem II

PsbQ is one of the extrinsic proteins situated on the lumenal surface of photosystem II (PSII) in the higher plants and green algae. Its three-dimensional structure was determined by X-ray crystallography with exception of the residues 14-33. To obtain further details about its structure and potentially its dynamics, we approached the problem by NMR. In this paper we report (1)H, (15)N, and (13)C NMR assignments for the PsbQ protein. The very challenging oligo-proline stretches could be assigned using (13)C-detected NMR experiments that enabled the assignments of twelve out of the thirteen proline residues of PsbQ. The identification of PsbQ secondary structure elements on the basis of our NMR data was accomplished with the programs TALOS+, web server CS23D and CS-Rosetta. To obtain additional secondary structure information, three-bond H(N)-H(α) J-coupling constants and deviation of experimental (13)C(α) and (13)C(β) chemical shifts from random coil values were determined. The resulting "consensus" secondary structure of PsbQ compares very well with the resolved regions of the published X-ray crystallographic structure and gives a first estimate of the structure of the "missing link" (i.e. residues 14-33), which will serve as the basis for the further investigation of the structure, dynamics and interactions.

Backbone1H,15N and13C Resonance Assignment and Secondary Structure Prediction of HP0062 (O24902_HELPY) from Helicobacter pylori

HP0062 is an 86 residue hypothetical protein from Helicobacter pylori strain 26695. HP0062 was identified ESAT-6/WXG100 superfamily protein based on structure and sequence alignment and also contains leucine zipper domain sequence. Here, we report the sequence-specific backbone resonance assignment of HP0062. About 97.7% of all <TEX>$^1H_N,\;^{15}N,\;^{13}C_{\alpha},\;^{13}C_{\beta}\;and\;^{13}C=O$</TEX> resonances were assigned unambiguously. We could predict the secondary structure of HP0062 by analyzing the deviation of the <TEX>$^{13}C_{alpha}\;and\;^{13}C_{\beta}$</TEX> chemical shifts from their respective random coil values. Secondary structure prediction shows that HP0062 consist of two <TEX>${\alpha}$</TEX>-helices. This study is a prerequisite for determining the solution structure of HP0062 and can be used for the study on interaction between HP0062 and DNA and other Helicobacter pylori proteins.

Engineering a β‐Helical d,l‐Peptide for Folding in Polar Media

Beta helices--helices formed by alternating D,L-peptides and stabilized by beta-sheet hydrogen bonding--are found naturally in only a handful of highly hydrophobic peptides. This paper explores the scope of beta-helical structure by presenting the first design and biophysical characterization of a hydrophilic D,L-peptide, 1, that forms a beta helix in methanol. The design of 1 is based on the beta-hairpin/beta helix--a new supersecondary that had been characterized previously only for hydrophobic peptides in nonpolar solvents. Incorporating polar residues in 1 provided solubility in methanol, in which the peptide adopts the expected beta-hairpin/beta-helical structure, as evidenced by CD, analytical ultracentrifugation (AUC), NMR spectroscopy, and NMR-based structure calculations. Upon titration with water (at constant peptide concentration), the structure in methanol (1 m) transitions cooperatively to an extended conformation (1 w) resembling a cyclic beta-hairpin; observation of an isodichroic point in the solvent-dependent CD spectra indicates that this transition is a two-state process. In contrast, neither 1 m nor 1 w show cooperative thermal melting; instead, their structures appear intact at temperatures as high as 65 degrees C; this observation suggests that steric constraint is dominant in stabilizing these structures. Finally, the (1)H NMR C alphaH spectroscopic resonances of 1 m are downfield-shifted with respect to random-coil values, a hitherto unreported property for beta helices that appears to be a general feature of these structures. These results show for the first time that an appropriately designed beta-helical peptide can fold stably in a polar solvent; furthermore, the structural and spectroscopic data reported should prove useful in the future design and characterization of water-soluble beta helices.

The Functional Curli Amyloid Is Not Based on In-register Parallel β-Sheet Structure

The extracellular curli proteins of Enterobacteriaceae form fibrous structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid. Moreover we demonstrate that curli fibrils possess the cross-beta structure that distinguishes protein amyloid. However, solid state NMR experiments indicate that curli structure is not based on an in-register parallel beta-sheet architecture, which is common to many human disease-associated amyloids and the yeast prion amyloids. Solid state NMR and electron microscopy data are consistent with a beta-helix-like structure but are not sufficient to establish such a structure definitively.