Spatial propagation of protein polymerization.

We consider the spatial dependence of filamentous protein self-assembly. Through studying the cases where the spreading of aggregated material is dominated either by diffusion or by growth, we derive analytical results for the spatial evolution of filamentous protein aggregation, which we validate against Monte Carlo simulations. Moreover, we compare the predictions of our theory with experimental measurements of two systems for which we identify the propagation as either growth or diffusion controlled. Our results connect the macroscopic observables that characterize the spatial propagation of protein self-assembly with the underlying microscopic processes and provide physical limits on spatial propagation and prionlike behavior associated with protein aggregation.

Detection of transient protein folding populations by mass spectrometry.

Hydrogen-deuterium exchange measurements are becoming increasingly important in studies of the dynamics of protein molecules and, particularly, of their folding behavior. Electrospray ionization mass spectrometry (ESI-MS) has been used to obtain the distribution of masses within a population of protein molecules that had undergone hydrogen exchange in solution. This information is complementary to that from nuclear magnetic resonance spectroscopy (NMR) experiments, which measure the average occupancy of individual sites over the distribution of protein molecules. In experiments with hen lysozyme, a combination of ESI-MS and NMR was used to distinguish between alternative mechanisms of hydrogen exchange, providing insight into the nature and populations of transient folding intermediates. These results have helped to detail the pathways available to a protein during refolding.

Protein folding monitored at individual residues during a two-dimensional NMR experiment.

An approach is described to monitor directly at the level of individual residues the formation of structure during protein folding. A two-dimensional heteronuclear nuclear magnetic resonance (NMR) spectrum was recorded after the rapid initiation of the refolding of a protein labeled with nitrogen-15. The intensities and line shapes of the cross peaks in the spectrum reflected the kinetic time course of the folding events that occurred during the spectral accumulation. The method was used to demonstrate the cooperative nature of the acquisition of the native main chain fold of apo bovine alpha-lactalbumin. The general approach, however, should be applicable to the investigation of a wide range of chemical reactions.

Sleeping beauty mutase (sbm) is expressed and interacts with ygfd in Escherichia coli.

In Escherichia coli, a four-gene operon, sbm-ygfD-ygfG-ygfH, has been shown to encode a putative cobalamin-dependent pathway with the ability to produce propionate from succinate in vitro [Haller T, Buckel T, Retey J, Gerlt JA. Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli. Biochemistry 2000;39:4622-4629]. However, the operon was thought to be silent in vivo, illustrated by the eponym describing its first gene, "sleeping beauty mutase" (methylmalonyl-CoA mutase, MCM). Of the four genes described, only ygfD could not be assigned a function. In this study, we have evaluated the functional integrity of YgfD and Sbm and show that, indeed, both proteins are expressed in E. coli and that YgfD has GTPase activity. We show that YgfD and Sbm can be co-immunoprecipitated from E. coli extracts using antibody to either protein, demonstrating in vivo interaction, a result confirmed using a strain deleted for ygfD. We show further that, in vitro, purified His-tagged YgfD and Sbm behave as a monomer and dimer, respectively, and that they form a multi-subunit complex that is dependent on pre-incubation of YgfD with non-hydrolysable GTP, an outcome that was not affected by the state of Sbm, as holo- or apoenzyme. These studies reinforce a role for the in vivo interaction of YgfD and Sbm.

Chemical form of dietary L-carnitine affects plasma but not tissue carnitine concentrations in male Sprague-Dawley rats.

In Experiment 1, rats (n = 54) were randomly assigned to control or one of the four sources of l-Carnitine supplemented at either 100 or 200 micromol/kg/day and were allowed to acclimate for 14 days. Following a 12-h fast, plasma samples were obtained at 0, 5, 10, 15, 30, 60, 120, 240, 480 and 720 min after l-Carnitine feeding and assayed for free l-Carnitine concentration. Plasma-free l-Carnitine levels were affected by time after treatment intake (p < 0.0001) and l-Carnitine source (p < 0.0001). The time x source interaction was not statistically significant (p = 0.99). In Experiment 2, rats (n = 54) were randomly assigned to control or one of the four sources of l-Carnitine at either 100 or 200 micromol/kg/day and were acclimated as in experiment 1. Rats were sacrificed 120 min after feeding. Samples of liver and skeletal muscle were obtained and assayed for free l-Carnitine concentration. Neither skeletal muscle (p = 0.44) or liver (p = 0.59) tissue concentrations of l-Carnitine were affected by any l-Carnitine source as compared with the control. We conclude that some differences exist in plasma concentrations of free l-Carnitine following ingestion of different chemical forms of l-Carnitine. It is unclear if these differences in the circulating concentration of free l-Carnitine translate into any physiological differences for the animal. In this study, chemical form of l-Carnitine had no effect on skeletal muscle or liver tissue concentrations of l-Carnitine in young male Wistar rats.

Understanding how proteins fold: the lysozyme story so far.

Hen lysozyme is one of the best characterized and most studied of all proteins. Recently, we have used a range of different methods to examine the events involved in the in vitro folding pathway of this protein. In this review we show that, by combining complementary techniques, it has been possible to piece together a detailed model for the folding of this enzyme. Important questions prompted by this work are highlighted and we then propose some ideas consistent with our data, as well as those of others, which we believe begin to provide insight into one of the most intriguing of structural problems in biology--how proteins can achieve their complex native forms from disordered denatured states.

Understanding protein folding via free-energy surfaces from theory and experiment.

The ability of protein molecules to fold into their highly structured functional states is one of the most remarkable evolutionary achievements of biology. In recent years, our understanding of the way in which this complex self-assembly process takes place has increased dramatically. Much of the reason for this advance has been the development of energy surfaces (landscapes), which allow the folding reaction to be described and visualized in a meaningful manner. Analysis of these surfaces, derived from the constructive interplay between theory and experiment, has led to the development of a unified mechanism for folding and a recognition of the underlying factors that control the rates and products of the folding process.

Real-time PCR detection of bacteria belonging to the Firmicutes Phylum.

Members of the bacterial Phylum Firmicutes occupy a wide range of habitats and can be either beneficial or detrimental in diverse settings, including food- and beverage-related industries. Firmicutes are responsible for the vast majority of beer-spoilage incidents and, as such, they have a substantial financial impact in the brewing industry. Rapid detection and identification of a bacterium as a Firmicutes is difficult due to widespread genetic transfer and genome reduction resulting in phenotypic diversity in these bacteria. Here we describe a real-time multiplex PCR to detect and differentiate Firmicutes associated with beer-spoilage from non-Firmicutes bacteria that may be present as benign environmental contaminants. A region of the 16S rRNA gene was identified and predicted to be highly conserved amongst, and essentially specific for, Firmicutes. A real-time PCR assay using a hydrolysis probe targeting this region of the 16S rRNA gene was experimentally shown to detect ten genera of Firmicutes known to be beer spoilers, but does not cross-react with eleven of twelve non-Firmicutes genera which can periodically appear in beer. Only one non-Firmicutes species, Zymomonas mobilis, weakly reacted with the Firmicutes probe. This rPCR assay has a standard curve that is linear over six orders of magnitude of DNA, with a quantitation limit of DNA from <10 bacteria. When used to detect bacteria present in beer, the assay was able to detect 50-100 colony forming units (CFU) of Firmicutes directly from 2.5 cm membranes used to filter 100 ml of contaminated beer. Through incorporation of a 4.7 cm filter and an overnight pre-enrichment incubation, the sensitivity was increased to 2.5-10 CFU per package of beer (341 ml). When multiplexed with a second hydrolysis probe targeting a universal region of the 16S rRNA gene, the assay reliably differentiates between Firmicutes and non-Firmicutes bacteria found in breweries.

Protein folding. Solid evidence for molten globules.

Novel experimental strategies are providing details of the structures of non-native states of proteins and shedding light on the concept of a "molten globule" and its relevance to protein folding.

Time-resolved biophysical methods in the study of protein folding.

Many of the biophysical techniques developed to characterize native proteins at equilibrium have now been adapted to the structural and thermodynamic characterization of transient intermediate populations during protein folding. Recent advances in these techniques, the use of novel methods of initiating refolding, and a convergence of theoretical and experimental approaches are leading to a detailed understanding of many aspects of the folding process.

Collapse and cooperativity in protein folding.

The folding of a polypeptide chain is associated both with compactness and cooperativity within local and global regions of the protein structure, and with the formation of the native-like molecular architecture. Recent experiments shed light on these issues and their relationships to the pathways of protein folding.

The fundamentals of protein folding: bringing together theory and experiment.

Experimental and theoretical studies together are providing insights into the mechanism by which proteins fold. Our present knowledge of the essential aspects of the folding reaction is outlined and some approaches, both theoretical and experimental, that are being developed to obtain a more detailed understanding of this complex process are described.

Structural Characteristics of α-Synuclein Oligomers.

Oligomeric forms of amyloid aggregates have been detected in the brains and tissues of patients suffering from neurodegenerative disorders such as Parkinson's disease, and it is widely thought that such species are key pathogenic agents in the development and spreading of the disease; however, the study of these species has been proven to be extremely challenging, primarily as a result of their intrinsically transient nature and high levels of heterogeneity. Identifying the structural nature and the details of the mechanisms of formation and interconversion of individual oligomeric species, particularly those with high toxicity, is of fundamental importance not only for understanding the mechanisms of protein misfolding and amyloid aggregation but also for the identification of diagnostic and therapeutic targets. In this review, we will focus on the current knowledge of the multitude of oligomeric forms of α-synuclein that have been reported to date, with particular emphasis on their structural features and possible relationship to other amyloid species, in order to build a clearer understanding of the types of oligomeric species that accumulate during the aggregation of α-synuclein and to develop a comprehensive picture of the misfolding behavior of the protein.

Solution structure of a peptide fragment of human alpha-lactalbumin in trifluoroethanol: a model for local structure in the molten globule.

BACKGROUND: At low pH, human alpha-lactalbumin forms a partly folded molten globule state that contains a non-native clustering of the side chains of Tyr103, Trp104 and His107. In order to understand the conformation of this region of the protein in the molten globule state, we investigated the structure of a peptide corresponding to residues 101-110 of human alpha-lactalbumin in trifluoroethanol. RESULTS: We determined the structure of the 101-110 peptide from an NMR data set of 145 nuclear Overhauser effects and nine 3JHN alpha coupling constants, using an ensemble calculation approach to take into account the possibilities of conformational averaging of the data. The backbone of residues 3-10 in the peptide adopts a series of turns, that involving residues 5-8 being the best defined, while the side chains of residues 1, 3, 4, 5, 6 and 7 form a hydrophobic cluster. CONCLUSIONS: The peptide conformation differs from that previously determined for residues 101-110 in crystal structures of native alpha-lactalbumin determined at both high and low pH, particularly in the relative orientations of the side chains. The series of turns seen in the peptide could, however, be related to the alpha-helical structure seen for residues 104-111 in crystals at high pH, and may be important in the molten globule state for bringing the peptide chain into a compact conformation where favourable interactions between the side chains can occur.

The crystal structure of the catalytic domain of human urokinase-type plasminogen activator.

BACKGROUND: Urokinase-type plasminogen activator (u-PA) promotes fibrinolysis by catalyzing the conversion of plasminogen to the active protease plasmin via the cleavage of a peptide bond. When localized to the external cell surface it contributes to tissue remodelling and cellular migration; inhibition of its activity impedes the spread of cancer. u-PA has three domains: an N-terminal receptor-binding growth factor domain, a central kringle domain and a C-terminal catalytic protease domain. The biological roles of the fibrinolytic enzymes render them therapeutic targets, however, until now no structure of the protease domain has been available. Solution of the structure of the u-PA serine protease was undertaken to provide such data. RESULTS: The crystal structure of the catalytic domain of recombinant, non-glycosylated human u-PA, complexed with the inhibitor Glu-Gly-Arg chloromethyl ketone (EGRcmk), has been determined at a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 22.4% on all data (20.4% on data > 3 sigma). The enzyme has the expected topology of a trypsin-like serine protease. CONCLUSIONS: The enzyme has an S1 specificity pocket similar to that of trypsin, a restricted, less accessible, hydrophobic S2 pocket and a solvent-accessible S3 pocket which is capable of accommodating a wide range of residues. The EGRcmk inhibitor binds covalently at the active site to form a tetrahedral hemiketal structure. Although the overall structure is similar to that of homologous serine proteases, at six positions insertions of extra residues in loop regions create unique surface areas. One of these loop regions is highly mobile despite being anchored by the disulphide bridge which is characteristic of a small subset of serine proteases namely tissuetype plasminogen activator, Factor XII and Complement Factor I.

Molecular characterisation of a thermoactive beta-1,3-glucanase from Oerskovia xanthineolytica.

Molecular characterisation of a lytic thermoactive beta-1,3-glucanase from Oerskovia xanthineolytica LL-G109 has been performed. A molecular mass of 27 195.6 +/- 1.3 Da and an isoelectric point of 4.85 were determined by electrospray mass spectrometry and from its titration curve, respectively. Its thermoactivity profile shows it to be a heat-stable enzyme with a temperature optimum of 65 degrees C. The secondary structure content of the protein was estimated by circular dichroism to be approx. 25% alpha-helix, 7% random coil, and 68% beta-sheet and beta-turn structure. Nuclear magnetic resonance spectra confirm the high content of beta-structure. Furthermore, the presence of a compact hydrophobic core is indicated by the presence of slowly exchanging amide hydrogens and the enzyme's relatively high resistance to proteolysis. The N-terminal sequences of the intact protein and of a tryptic peptide each exhibit significant similarity to family 16 of glycosyl hydrolases whose overall fold is known to contain almost exclusively beta-sheets and surface loops. Moreover, the sequenced tryptic peptide appears to encompass residues of the Oerskovia xanthineolytica glucanase active site, since it contains a portion of the family 16 active-site motif E-[L/I/V]-D-[L/I/V]-E.

1H nuclear magnetic resonance studies of the interaction of urea with hen lysozyme. Origins of the conformational change induced in hen lysozyme by N-acetylglucosamine oligosaccharides.

The interaction between hen lysozyme and urea has been investigated using 1H nuclear magnetic resonance spectroscopy. Chemical shift changes for resonances of a number of residues in the vicinity of the active site of the protein have been observed in the presence of urea prior to denaturation. These shifts are similar to those induced in the hen lysozyme spectrum by the specific binding of N-acetylglucosamine (GlcNAc) in site C of the active site cleft, indicating that urea and GlcNAc induce a similar conformational change in the enzyme. This implies that the conformational changes experienced by the enzyme on the binding of GlcNAc oligosaccharides are the consequence of interactions, possibly hydrogen bonding, involving the N-acetyl group of the sugar residue bound in site C, rather than the result of contacts between the protein and the pyranose rings of the oligosaccharides. This suggests that hen lysozyme employs an induced fit type mechanism to discriminate for N-acetylated saccharides as substrates.

Chemical dissection and reassembly of amyloid fibrils formed by a peptide fragment of transthyretin.

We have examined the chemical dissection and subsequent reassembly of fibrils formed by a ten-residue peptide to probe the forces that drive the formation of amyloid. The peptide, TTR(10-19), encompasses the A strand of the inner beta-sheet structure that lines the thyroid hormone binding site of the human plasma protein transthyretin. When dissolved in water under low pH conditions the peptide readily forms amyloid fibrils. Electron microscopy of these fibrils indicates the presence of long (>1000 nm) rigid structures of uniform diameter (approximately 14 nm). Addition of urea (3 M) to preformed fibrils disrupts these rigid structures. The partially disrupted fibrils form flexible ribbon-like arrays, which are composed of a number of clearly visible protofilaments (3-4 nm diameter). These protofilaments are highly stable, and resist denaturation in 6 M urea at 75 degrees C over a period of hours. High concentrations (>50%, v/v) of 2,2,2-trifluoroethanol also dissociate TTR(10-19) fibrils to the constituent protofilaments, but these slowly dissociate to monomeric, soluble peptides with extensive alpha-helical structure. Dilution of the denaturant or co-solvent at the stage when dissociation to protofilaments has occurred results in the efficient reassembly of fibrils. These results indicate that assembly of fibrils from protofilaments involves relatively weak and predominantly hydrophobic interactions, whereas assembly of peptides into protofilaments involves both electrostatic and hydrophobic forces, resulting in a highly stable and compact structures.

A new two-disulphide intermediate in the refolding of reduced bovine pancreatic trypsin inhibitor.

The apparently complete refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) is shown to produce a mixture of two species. One of these is native BPTI, but the other lacks the disulphide bond between cysteines 30 and 51. The latter species has a folded conformation very like that of native BPTI, and is oxidized by air to native BPTI on warming in aqueous solution. The two unreactive cysteine thiol groups appear to be buried in the interior of the molecule, which restricts access by reagents that can alkylate them or oxidize them to form the disulphide bond. The implications of this intermediate and its conformation for the understanding of protein folding are discussed.

Fast and slow tracks in lysozyme folding: insight into the role of domains in the folding process.

The folding of lysozyme involves parallel events in which hydrogen exchange kinetics indicate the development of persistent structure at very different rates. We have monitored directly the kinetics of formation of the native molecule by the binding of a fluorescently labelled inhibitor, MeU-diNAG (4-methylumbelliferyl-N,N'-diacetyl-beta-D-chitobioside). The data show that native character monitored in this way also develops with different timescales. Although the rate determining step on the slow pathway (approximately 75% of molecules at pH 5.5, 20 degrees C) can be attributed to the need to reorganise structure formed early in the folding process, the data indicate that the rate determining step on the fast track (involving approximately 25% of molecules) involves the docking of the two constituent domains of the protein. In the fast folding track the data are consistent with a model in which each domain forms persistent structure prior to their docking in a locally cooperative manner on a timescale comparable to the folding of small single domain proteins.