DNA comprises molecular information stored via genetic bases (G, C, T, A) and also epigenetic bas... more DNA comprises molecular information stored via genetic bases (G, C, T, A) and also epigenetic bases, principally 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Both genetic and epigenetic information are vital to our understanding of biology and disease states. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases typically fail to capture common C-to-T mutations or distinguish 5mC from 5hmC. Here, we present a single-base-resolution sequencing methodology that will simultaneously sequence complete genetics and complete epigenetics in a single workflow. The approach is non-destructive to DNA and provides a digital readout of bases, which we exemplify by simultaneous sequencing of G, C, T, A, 5mC and 5hmC; 6-Letter sequencing. We demonstrate sequencing of human genomic DNA and also cell-free DNA taken from a blood sample of a cancer patient. The approach is accurate...
DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vit... more DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases fail to capture common C-to-T mutations or distinguish 5-methylcytosine from 5-hydroxymethylcytosine. We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow. DNA is copied and bases are enzymatically converted. Coupled decoding of bases across the original and copy strand provides a phased digital readout. Methods are demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. Simultaneous, phased reading of genetic and epigenetic bases provides a more comp...
Mixed globular protein gels, or gel composites, based on heating solutions of proteins from milk ... more Mixed globular protein gels, or gel composites, based on heating solutions of proteins from milk have been investigated for many years, although the great majority of these studies have used rather crude whey isolates containing a fixed ratio of the principal components ...
... We thank John Pacey (King's College) and Anthony Weaver (Unilever Research) ... more ... We thank John Pacey (King's College) and Anthony Weaver (Unilever Research) for assistance with electron microscopy experiments; Dr. Xue-Feng Yuan, Dr. Yung Jong Lee, and Dr. Paul Royall (King's College ... 6) Clark, AH; Judge, FJ; Richards, JB; Stubbs, JM; Suggett, A. Int. ...
Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. D... more Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. Despite their importance in degenerative human diseases, the overall structure of amyloid fibrils remains unknown. High-resolution studies of model peptide assemblies have identified residues forming cross-β-strands and have revealed some details of local β-strand packing. However, little is known about the assembly contacts that define the fibril architecture. Here we present a set of threedimensional structures of amyloid fibrils formed from full-length β 2-microglobulin, a 99-residue protein involved in clinical amyloidosis. Our cryo-electron microscopy maps reveal a hierarchical fibril structure built from tetrameric units of globular density, with at least three different subunit interfaces in this homopolymeric assembly. These findings suggest a more complex superstructure for amyloid than hitherto suspected and prompt a re-evaluation of the defining features of the amyloid fold. Abbreviations FTIR, Fourier transform infrared; 3D, three-dimensional; EM, electron microscopy; β 2 m, β 2microglobulin; STEM, scanning transmission electron microscopy; MPL, mass per unit length
Dialysis related amyloidosis is a serious complication of longterm hemodialysis in which  2-micr... more Dialysis related amyloidosis is a serious complication of longterm hemodialysis in which  2-microglobulin ( 2 m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of  2 m amyloid fibrils, building on observations that macrophages are found in association with  2 m amyloid deposits in vivo and that these cells contain intra-lysosomal  2 m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric  2 m, whereupon it is sorted to lysosomes. At lysosomal pH  2 m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly  2 m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before  2 m fibrils can accumulate in what may represent an otherwise fibrillogenic environment.
Current Opinion in Colloid & Interface Science, 2000
... However, the gel state reflects the existence of a mega-molecule, which spans the size of its... more ... However, the gel state reflects the existence of a mega-molecule, which spans the size of its container, so 1 μm is still a factor >10 4 smaller. It would appear impossible to establish structure over the entire range of relevant distance scales. ...
Oscillatory shear rheometry (mechanical spectroscopy) has been used to study the heat-set gelatio... more Oscillatory shear rheometry (mechanical spectroscopy) has been used to study the heat-set gelation of-lactoglobulin at pH 2. Modulus-concentration relationships were obtained by extrapolating cure data to infinite time. In terms of theory, these fail to provide a clear distinction between the fractal description of biopolymer gels and the classical random f-functional polycondensation branching theory (cascade) approach, though the latter is preferred. Critical exponents for the sol-gel transition, derived from these data, are also discussed. Where gel time-concentration results are concerned the fractal model makes no predictions, and the cascade approach in its simplest form must be rejected in favor of a more sophisticated version involving delivery of fibrils by nucleation and growth into the random aggregation process. Over the limited concentration range accessed experimentally, cure data for the different-lactoglobulin solutions, reduced to the universal form G′/G′ inf versus t/t gel , superimposed well for samples heated both at 80 and 75°C and for different batches of protein. Studies of the frequency responses of the fully cured gels confirm the validity of the gel description given to these materials, and a study of the temperature dependence of the frequency spectrum suggests a fall in the elastic component of the modulus as temperature decreases. This contrasts with what has been found for other heat-set globular protein gels such as those from serum albumin where the gel modulus increases at lower temperatures. The present results are in good agreement with more limited amounts of pH 2-lactoglobulin data published earlier, though some differences arise through a previous neglect of measurement "dead time".
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2005
Deriving a complete understanding of protein self-association into amyloid fibrils across multipl... more Deriving a complete understanding of protein self-association into amyloid fibrils across multiple distance and time scales is an enormous challenge. At small length scales, a detailed description of the partially folded protein ensemble that participates in self-assembly remains obscure. At larger length scales, amyloid fibrils are often heterogeneous, can form along multiple pathways, and are further complicated by phenomena such as phase-separation. Over the last 5 years, we have used an array of biophysical approaches in order to elucidate the structural and molecular mechanism of amyloid fibril formation, focusing on the all h-sheet protein, h 2-microglubulin (h 2 m). This protein forms amyloid deposits in the human disease Fdialysis-related amyloidosis_ (DRA). We have shown that under acidic conditions h 2 m rapidly associates in vitro to form amyloid-like fibrils that have different morphological properties, but which contain an underpinning cross-h structure. In this review, we discuss our current knowledge of the structure of these fibrils, as well as the structural, kinetic and thermodynamic relationship between fibrils with different morphologies. The results provide some of the first insights into the shape of the self-assembly free-energy landscape for this protein and highlight the parallel nature of the assembly process. We include a detailed description of the structure and dynamics of partially folded and acid unfolded species of h 2 m using NMR, and highlight regions thought to be important in early self-association events. Finally, we discuss briefly how knowledge of assembly mechanisms in vitro can be used to inform the design of therapeutic strategies for this, and other amyloid disorders, and we speculate on how the increasing power of biophysical approaches may lead to a fuller description of protein self-assembly into amyloid in the future.
Fibrils associated with amyloid disease are molecular assemblies of key biological importance, ye... more Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity.
Amyloid fibrils have historically been characterized by diagnostic dye-binding assays, their fibr... more Amyloid fibrils have historically been characterized by diagnostic dye-binding assays, their fibrillar morphology, and a ''cross-b'' x-ray diffraction pattern. Whereas the latter demonstrates that amyloid fibrils have a common b-sheet core structure, they display a substantial degree of morphological variation. One striking example is the remarkable ability of human apolipoprotein C-II amyloid fibrils to circularize and form closed rings. Here we explore in detail the structure of apoC-II amyloid fibrils using electron microscopy, atomic force microscopy, and x-ray diffraction studies. Our results suggest a model for apoC-II fibrils as ribbons ;2.1-nm thick and 13-nm wide with a helical repeat distance of 53 nm 6 12 nm. We propose that the ribbons are highly flexible with a persistence length of 36 nm. We use these observed biophysical properties to model the apoC-II amyloid fibrils either as wormlike chains or using a random-walk approach, and confirm that the probability of ring formation is critically dependent on the fibril flexibility. More generally, the ability of apoC-II fibrils to form rings also highlights the degree to which the common cross-b superstructure can, as a function of the protein constituent, give rise to great variation in the physical properties of amyloid fibrils.
Despite its importance in biological phenomena, a comprehensive understanding of the mechanism of... more Despite its importance in biological phenomena, a comprehensive understanding of the mechanism of amyloid formation remains elusive. Here, we use atomic force microscopy to map the formation of b 2-microglobulin amyloid fibrils with distinct morphologies and persistence lengths, when protein concentration, pH and ionic strength are varied. Using the resulting state-diagrams, we demonstrate the existence of two distinct competitive pathways of assembly, which define an energy landscape that rationalises the sensitivity of fibril morphology on the solution conditions. Importantly, we show that semi-flexible (worm-like) fibrils, which form rapidly during assembly, are kinetically trapped species, formed via a non-nucleated pathway that is explicitly distinct from that leading to the formation of the relatively rigid long-straight fibrils classically associated with amyloid. These semi-flexible fibrils also share an antibody epitope common to other protein oligomers that are known to be toxic species linked to human disease. The results demonstrate the heterogeneity of amyloid assembly, and have important implications for our understanding of the importance of oligomeric states in amyloid disease, the origins of prion strains, and the development of therapeutic strategies.
Early detection of colorectal cancer (CRC) will improve survival rates. We created a classifier t... more Early detection of colorectal cancer (CRC) will improve survival rates. We created a classifier to detect CRC, based on 5-hydroxymethylcytosine levels in cell free DNA isolated from blood samples of 2198 individuals. Our classifier discriminated CRC samples from controls with an area under the receiver operating characteristic curve (AUC) of 90% (sensitivity was 55% at 95% specificity). Performance was similar for early stage 1 (AUC 89%) and late stage 4 CRC (AUC 94%). Performance was independent of the proportion of tumor-DNA in the cell free DNA. We expanded the classifier to include information about cell free DNA fragment size and abundance across the genome. Overall performance was similar (AUC 91%), with gains in sensitivity (63% at 95% specificity). The 5-hydroxymethylcytosine signal allows detection of CRC, even in cell free DNA samples with undetectable tumor DNA. Including 5-hydroxymethylcytosine in multi-analyte screening, will improve sensitivity for early-stage cancer.
As a prelude to experimental and theoretical work on the mechanical properties of fibrillar beta-... more As a prelude to experimental and theoretical work on the mechanical properties of fibrillar beta-lactoglobulin gels, this paper reports the structural characterization of beta-lactoglobulin fibrils by electron and atomic force microscopy (AFM), infrared and Raman spectroscopy, and powder X-ray diffraction. Aggregates formed by incubation of beta-lactoglobulin in various alcohol-water mixtures at pH 2, and in water-trifluoroethanol (TFE) at pH 7, were found to be wormlike (approximately 7 nm in width and <500 nm in length), with a "string-of-beads" appearance. Longer (approximately 7 nm in width, and >1 microm in length), smoother, and seemingly stiffer fibrils formed on heating aqueous beta-lactoglobulin solutions at pH 2 and low ionic strength, although there was little evidence for the higher-order structures common in most amyloid-forming systems. Time-lapse AFM also revealed differences in the formation of these two fibril types: thermally induced aggregation occurring more cooperatively, in keeping with a nucleation and growth process. Only short stiff-rods (<20 nm in length) formed on heating beta-lactoglobulin at pH 7, and only complex three-dimensional "amorphous"aggregates in alcohols other than TFE at this pH. Studies of all of the pH 2 fibrils from beta-lactoglobulin, by Raman and infrared spectroscopy confirmed beta-sheet as mediating the aggregation process. Interestingly, however, some evidence for de novo helix formation for the solvent-induced systems was obtained, although it remains to be seen whether this is actually incorporated into the fibril-structure. In contrast to other amyloid systems, X-ray powder diffraction provided no evidence for extensive repeating "crystalline" structures for any of the pH 2 beta-lactoglobulin fibrils. In relation to amyloid, the lactoglobulin fibrils bear more resemblance to protofilaments than to higher-order fibril structures, these latter appearing more convincingly for thermally induced insulin fibrils (pH 2) also included in the AFM study.
Submitted for the MAR11 Meeting of The American Physical Society Deciphering evolutionary instruc... more Submitted for the MAR11 Meeting of The American Physical Society Deciphering evolutionary instructions for specifying protein fold and function WALRAJ GOSAL, RAMA RANGANATHAN, UT Southwestern Medical Center-Classical studies show that proteins have evolved to fold into functional native states that are, at best, only marginally stable through weak noncovalent interactions encoded by their primary sequences. How such fold and functional information is stored in a single amino acid sequence remains elusive. Using the statistical analysis of covariation between pairs of amino acids at all positions in a protein, here we identify groups of a few key physically-interconnected residues, which we term sectors. What information about the fold and function is captured by sectors? Using simulated-annealing Monte Carlo, we introduce variation in the sequence of a single member of the PDZ family in a manner that either preserves or disrupts sector correlations. Experimentally we show that function is specifically retained in designed proteins that obey sector correlations, and strikingly, even in the absence of a native state. Thus, we suggest that native-state stability is not a fundamental requirement for function, and is encoded in the sequence in an idiosyncratic manner in the PDZ family.
Oscillatory shear rheometry has been used to study the gelation of beta-lactoglobulin at ambient ... more Oscillatory shear rheometry has been used to study the gelation of beta-lactoglobulin at ambient in 50% v/v trifluoroethanol (TFE)/pH 7 aqueous buffer and in 50% v/v ethanol (EtOH)/water at pH 2. In contrast to what was found on heating aqueous solutions at pH 2 (Part 2 of this series), a more expected "chemical gelation"-like profile was found with modulus components G' and G' ' crossing over as the gels formed and then with G' ' passing through a maximum. In addition, for the EtOH system, there was a significant modulus increase at long time, suggestive of a more complex two-step aggregation scheme. Modulus-concentration relationships were obtained for both systems by extrapolating cure data to infinite time. For the TFE gels, this data was accurately described by classical branching theory, although it could also be approximated by a constant power--law relationship. Only the latter described the modulus--concentration data for the gels in ethanol, but there were problems here of greater frequency dependence of the modulus values and much less certain extrapolation. Gel times for the TFE systems showed higher power laws in the concentration than could be explained by the branching theory in its simplest form being similar, in this respect, to the heat-set systems at pH 2. Such power laws were harder to establish for the EtOH gels as for these there was evidence of gel time divergence close to a critical concentration. Reduced G'/G'inf versus t/tgel data were difficult to interpret for the gels in ethanol, but for the TFE system they were consistent with previous results for the heat-set gels and approximated master curve superposition. The frequency and temperature dependences of the final gel moduli were also studied. In general, the networks induced by alcohols appeared more flexible than those obtained by heating.
DNA comprises molecular information stored via genetic bases (G, C, T, A) and also epigenetic bas... more DNA comprises molecular information stored via genetic bases (G, C, T, A) and also epigenetic bases, principally 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Both genetic and epigenetic information are vital to our understanding of biology and disease states. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases typically fail to capture common C-to-T mutations or distinguish 5mC from 5hmC. Here, we present a single-base-resolution sequencing methodology that will simultaneously sequence complete genetics and complete epigenetics in a single workflow. The approach is non-destructive to DNA and provides a digital readout of bases, which we exemplify by simultaneous sequencing of G, C, T, A, 5mC and 5hmC; 6-Letter sequencing. We demonstrate sequencing of human genomic DNA and also cell-free DNA taken from a blood sample of a cancer patient. The approach is accurate...
DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vit... more DNA comprises molecular information stored in genetic and epigenetic bases, both of which are vital to our understanding of biology. Most DNA sequencing approaches address either genetics or epigenetics and thus capture incomplete information. Methods widely used to detect epigenetic DNA bases fail to capture common C-to-T mutations or distinguish 5-methylcytosine from 5-hydroxymethylcytosine. We present a single base-resolution sequencing methodology that sequences complete genetics and the two most common cytosine modifications in a single workflow. DNA is copied and bases are enzymatically converted. Coupled decoding of bases across the original and copy strand provides a phased digital readout. Methods are demonstrated on human genomic DNA and cell-free DNA from a blood sample of a patient with cancer. The approach is accurate, requires low DNA input and has a simple workflow and analysis pipeline. Simultaneous, phased reading of genetic and epigenetic bases provides a more comp...
Mixed globular protein gels, or gel composites, based on heating solutions of proteins from milk ... more Mixed globular protein gels, or gel composites, based on heating solutions of proteins from milk have been investigated for many years, although the great majority of these studies have used rather crude whey isolates containing a fixed ratio of the principal components ...
... We thank John Pacey (King's College) and Anthony Weaver (Unilever Research) ... more ... We thank John Pacey (King's College) and Anthony Weaver (Unilever Research) for assistance with electron microscopy experiments; Dr. Xue-Feng Yuan, Dr. Yung Jong Lee, and Dr. Paul Royall (King's College ... 6) Clark, AH; Judge, FJ; Richards, JB; Stubbs, JM; Suggett, A. Int. ...
Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. D... more Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. Despite their importance in degenerative human diseases, the overall structure of amyloid fibrils remains unknown. High-resolution studies of model peptide assemblies have identified residues forming cross-β-strands and have revealed some details of local β-strand packing. However, little is known about the assembly contacts that define the fibril architecture. Here we present a set of threedimensional structures of amyloid fibrils formed from full-length β 2-microglobulin, a 99-residue protein involved in clinical amyloidosis. Our cryo-electron microscopy maps reveal a hierarchical fibril structure built from tetrameric units of globular density, with at least three different subunit interfaces in this homopolymeric assembly. These findings suggest a more complex superstructure for amyloid than hitherto suspected and prompt a re-evaluation of the defining features of the amyloid fold. Abbreviations FTIR, Fourier transform infrared; 3D, three-dimensional; EM, electron microscopy; β 2 m, β 2microglobulin; STEM, scanning transmission electron microscopy; MPL, mass per unit length
Dialysis related amyloidosis is a serious complication of longterm hemodialysis in which  2-micr... more Dialysis related amyloidosis is a serious complication of longterm hemodialysis in which  2-microglobulin ( 2 m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of  2 m amyloid fibrils, building on observations that macrophages are found in association with  2 m amyloid deposits in vivo and that these cells contain intra-lysosomal  2 m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric  2 m, whereupon it is sorted to lysosomes. At lysosomal pH  2 m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly  2 m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before  2 m fibrils can accumulate in what may represent an otherwise fibrillogenic environment.
Current Opinion in Colloid & Interface Science, 2000
... However, the gel state reflects the existence of a mega-molecule, which spans the size of its... more ... However, the gel state reflects the existence of a mega-molecule, which spans the size of its container, so 1 μm is still a factor >10 4 smaller. It would appear impossible to establish structure over the entire range of relevant distance scales. ...
Oscillatory shear rheometry (mechanical spectroscopy) has been used to study the heat-set gelatio... more Oscillatory shear rheometry (mechanical spectroscopy) has been used to study the heat-set gelation of-lactoglobulin at pH 2. Modulus-concentration relationships were obtained by extrapolating cure data to infinite time. In terms of theory, these fail to provide a clear distinction between the fractal description of biopolymer gels and the classical random f-functional polycondensation branching theory (cascade) approach, though the latter is preferred. Critical exponents for the sol-gel transition, derived from these data, are also discussed. Where gel time-concentration results are concerned the fractal model makes no predictions, and the cascade approach in its simplest form must be rejected in favor of a more sophisticated version involving delivery of fibrils by nucleation and growth into the random aggregation process. Over the limited concentration range accessed experimentally, cure data for the different-lactoglobulin solutions, reduced to the universal form G′/G′ inf versus t/t gel , superimposed well for samples heated both at 80 and 75°C and for different batches of protein. Studies of the frequency responses of the fully cured gels confirm the validity of the gel description given to these materials, and a study of the temperature dependence of the frequency spectrum suggests a fall in the elastic component of the modulus as temperature decreases. This contrasts with what has been found for other heat-set globular protein gels such as those from serum albumin where the gel modulus increases at lower temperatures. The present results are in good agreement with more limited amounts of pH 2-lactoglobulin data published earlier, though some differences arise through a previous neglect of measurement "dead time".
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2005
Deriving a complete understanding of protein self-association into amyloid fibrils across multipl... more Deriving a complete understanding of protein self-association into amyloid fibrils across multiple distance and time scales is an enormous challenge. At small length scales, a detailed description of the partially folded protein ensemble that participates in self-assembly remains obscure. At larger length scales, amyloid fibrils are often heterogeneous, can form along multiple pathways, and are further complicated by phenomena such as phase-separation. Over the last 5 years, we have used an array of biophysical approaches in order to elucidate the structural and molecular mechanism of amyloid fibril formation, focusing on the all h-sheet protein, h 2-microglubulin (h 2 m). This protein forms amyloid deposits in the human disease Fdialysis-related amyloidosis_ (DRA). We have shown that under acidic conditions h 2 m rapidly associates in vitro to form amyloid-like fibrils that have different morphological properties, but which contain an underpinning cross-h structure. In this review, we discuss our current knowledge of the structure of these fibrils, as well as the structural, kinetic and thermodynamic relationship between fibrils with different morphologies. The results provide some of the first insights into the shape of the self-assembly free-energy landscape for this protein and highlight the parallel nature of the assembly process. We include a detailed description of the structure and dynamics of partially folded and acid unfolded species of h 2 m using NMR, and highlight regions thought to be important in early self-association events. Finally, we discuss briefly how knowledge of assembly mechanisms in vitro can be used to inform the design of therapeutic strategies for this, and other amyloid disorders, and we speculate on how the increasing power of biophysical approaches may lead to a fuller description of protein self-assembly into amyloid in the future.
Fibrils associated with amyloid disease are molecular assemblies of key biological importance, ye... more Fibrils associated with amyloid disease are molecular assemblies of key biological importance, yet how cells respond to the presence of amyloid remains unclear. Cellular responses may not only depend on the chemical composition or molecular properties of the amyloid fibrils, but their physical attributes such as length, width, or surface area may also play important roles. Here, we report a systematic investigation of the effect of fragmentation on the structural and biological properties of amyloid fibrils. In addition to the expected relationship between fragmentation and the ability to seed, we show a striking finding that fibril length correlates with the ability to disrupt membranes and to reduce cell viability. Thus, despite otherwise unchanged molecular architecture, shorter fibrillar samples show enhanced cytotoxic potential than their longer counterparts. The results highlight the importance of fibril length in amyloid disease, with fragmentation not only providing a mechanism by which fibril load can be rapidly increased but also creating fibrillar species of different dimensions that can endow new or enhanced biological properties such as amyloid cytotoxicity.
Amyloid fibrils have historically been characterized by diagnostic dye-binding assays, their fibr... more Amyloid fibrils have historically been characterized by diagnostic dye-binding assays, their fibrillar morphology, and a ''cross-b'' x-ray diffraction pattern. Whereas the latter demonstrates that amyloid fibrils have a common b-sheet core structure, they display a substantial degree of morphological variation. One striking example is the remarkable ability of human apolipoprotein C-II amyloid fibrils to circularize and form closed rings. Here we explore in detail the structure of apoC-II amyloid fibrils using electron microscopy, atomic force microscopy, and x-ray diffraction studies. Our results suggest a model for apoC-II fibrils as ribbons ;2.1-nm thick and 13-nm wide with a helical repeat distance of 53 nm 6 12 nm. We propose that the ribbons are highly flexible with a persistence length of 36 nm. We use these observed biophysical properties to model the apoC-II amyloid fibrils either as wormlike chains or using a random-walk approach, and confirm that the probability of ring formation is critically dependent on the fibril flexibility. More generally, the ability of apoC-II fibrils to form rings also highlights the degree to which the common cross-b superstructure can, as a function of the protein constituent, give rise to great variation in the physical properties of amyloid fibrils.
Despite its importance in biological phenomena, a comprehensive understanding of the mechanism of... more Despite its importance in biological phenomena, a comprehensive understanding of the mechanism of amyloid formation remains elusive. Here, we use atomic force microscopy to map the formation of b 2-microglobulin amyloid fibrils with distinct morphologies and persistence lengths, when protein concentration, pH and ionic strength are varied. Using the resulting state-diagrams, we demonstrate the existence of two distinct competitive pathways of assembly, which define an energy landscape that rationalises the sensitivity of fibril morphology on the solution conditions. Importantly, we show that semi-flexible (worm-like) fibrils, which form rapidly during assembly, are kinetically trapped species, formed via a non-nucleated pathway that is explicitly distinct from that leading to the formation of the relatively rigid long-straight fibrils classically associated with amyloid. These semi-flexible fibrils also share an antibody epitope common to other protein oligomers that are known to be toxic species linked to human disease. The results demonstrate the heterogeneity of amyloid assembly, and have important implications for our understanding of the importance of oligomeric states in amyloid disease, the origins of prion strains, and the development of therapeutic strategies.
Early detection of colorectal cancer (CRC) will improve survival rates. We created a classifier t... more Early detection of colorectal cancer (CRC) will improve survival rates. We created a classifier to detect CRC, based on 5-hydroxymethylcytosine levels in cell free DNA isolated from blood samples of 2198 individuals. Our classifier discriminated CRC samples from controls with an area under the receiver operating characteristic curve (AUC) of 90% (sensitivity was 55% at 95% specificity). Performance was similar for early stage 1 (AUC 89%) and late stage 4 CRC (AUC 94%). Performance was independent of the proportion of tumor-DNA in the cell free DNA. We expanded the classifier to include information about cell free DNA fragment size and abundance across the genome. Overall performance was similar (AUC 91%), with gains in sensitivity (63% at 95% specificity). The 5-hydroxymethylcytosine signal allows detection of CRC, even in cell free DNA samples with undetectable tumor DNA. Including 5-hydroxymethylcytosine in multi-analyte screening, will improve sensitivity for early-stage cancer.
As a prelude to experimental and theoretical work on the mechanical properties of fibrillar beta-... more As a prelude to experimental and theoretical work on the mechanical properties of fibrillar beta-lactoglobulin gels, this paper reports the structural characterization of beta-lactoglobulin fibrils by electron and atomic force microscopy (AFM), infrared and Raman spectroscopy, and powder X-ray diffraction. Aggregates formed by incubation of beta-lactoglobulin in various alcohol-water mixtures at pH 2, and in water-trifluoroethanol (TFE) at pH 7, were found to be wormlike (approximately 7 nm in width and <500 nm in length), with a "string-of-beads" appearance. Longer (approximately 7 nm in width, and >1 microm in length), smoother, and seemingly stiffer fibrils formed on heating aqueous beta-lactoglobulin solutions at pH 2 and low ionic strength, although there was little evidence for the higher-order structures common in most amyloid-forming systems. Time-lapse AFM also revealed differences in the formation of these two fibril types: thermally induced aggregation occurring more cooperatively, in keeping with a nucleation and growth process. Only short stiff-rods (<20 nm in length) formed on heating beta-lactoglobulin at pH 7, and only complex three-dimensional "amorphous"aggregates in alcohols other than TFE at this pH. Studies of all of the pH 2 fibrils from beta-lactoglobulin, by Raman and infrared spectroscopy confirmed beta-sheet as mediating the aggregation process. Interestingly, however, some evidence for de novo helix formation for the solvent-induced systems was obtained, although it remains to be seen whether this is actually incorporated into the fibril-structure. In contrast to other amyloid systems, X-ray powder diffraction provided no evidence for extensive repeating "crystalline" structures for any of the pH 2 beta-lactoglobulin fibrils. In relation to amyloid, the lactoglobulin fibrils bear more resemblance to protofilaments than to higher-order fibril structures, these latter appearing more convincingly for thermally induced insulin fibrils (pH 2) also included in the AFM study.
Submitted for the MAR11 Meeting of The American Physical Society Deciphering evolutionary instruc... more Submitted for the MAR11 Meeting of The American Physical Society Deciphering evolutionary instructions for specifying protein fold and function WALRAJ GOSAL, RAMA RANGANATHAN, UT Southwestern Medical Center-Classical studies show that proteins have evolved to fold into functional native states that are, at best, only marginally stable through weak noncovalent interactions encoded by their primary sequences. How such fold and functional information is stored in a single amino acid sequence remains elusive. Using the statistical analysis of covariation between pairs of amino acids at all positions in a protein, here we identify groups of a few key physically-interconnected residues, which we term sectors. What information about the fold and function is captured by sectors? Using simulated-annealing Monte Carlo, we introduce variation in the sequence of a single member of the PDZ family in a manner that either preserves or disrupts sector correlations. Experimentally we show that function is specifically retained in designed proteins that obey sector correlations, and strikingly, even in the absence of a native state. Thus, we suggest that native-state stability is not a fundamental requirement for function, and is encoded in the sequence in an idiosyncratic manner in the PDZ family.
Oscillatory shear rheometry has been used to study the gelation of beta-lactoglobulin at ambient ... more Oscillatory shear rheometry has been used to study the gelation of beta-lactoglobulin at ambient in 50% v/v trifluoroethanol (TFE)/pH 7 aqueous buffer and in 50% v/v ethanol (EtOH)/water at pH 2. In contrast to what was found on heating aqueous solutions at pH 2 (Part 2 of this series), a more expected "chemical gelation"-like profile was found with modulus components G' and G' ' crossing over as the gels formed and then with G' ' passing through a maximum. In addition, for the EtOH system, there was a significant modulus increase at long time, suggestive of a more complex two-step aggregation scheme. Modulus-concentration relationships were obtained for both systems by extrapolating cure data to infinite time. For the TFE gels, this data was accurately described by classical branching theory, although it could also be approximated by a constant power--law relationship. Only the latter described the modulus--concentration data for the gels in ethanol, but there were problems here of greater frequency dependence of the modulus values and much less certain extrapolation. Gel times for the TFE systems showed higher power laws in the concentration than could be explained by the branching theory in its simplest form being similar, in this respect, to the heat-set systems at pH 2. Such power laws were harder to establish for the EtOH gels as for these there was evidence of gel time divergence close to a critical concentration. Reduced G'/G'inf versus t/tgel data were difficult to interpret for the gels in ethanol, but for the TFE system they were consistent with previous results for the heat-set gels and approximated master curve superposition. The frequency and temperature dependences of the final gel moduli were also studied. In general, the networks induced by alcohols appeared more flexible than those obtained by heating.
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Papers by Walraj Gosal