Initial conformational changes of human transthyretin under partially denaturing conditions.

Human transthyretin (TTR) is an amyloidogenic protein. The pathway of TTR amyloid formation has been proposed based on lines of evidence: TTR tetramer first dissociates into native monomers, which is shown to be a rate-limiting step in the formation of fibrils. Subsequently, the monomeric species partially unfold to form the aggregation intermediates. Once such intermediates are formed, the following self-assembly process is a downhill polymerization. Hence, tertiary structural changes within the monomers after the dissociation are essential for the amyloid formation. These tertiary structural changes can be facilitated by partial denaturation. To probe the conformational changes under the partially denaturing conditions, five independent trajectories were collected for the wild-type (WT) and its pathogenic variants at 300 and 350 K, resulting in simulations that totaled 59 ns. Under these conditions, L55P variant is more labile than the wild-type and V30M variant. We have observed that the D strand of WT-TTR is trapped in two local minima: the native conformation and the amyloidogenic fold that resembles the surface loop of residues 54-55 of L55P variant. In the tetrameric state, the F strand is bent with large separations at the F-F' interface. This strand becomes flatter in the monomeric state, which may facilitate the formation of new F-F' interface with possible prolonged hydrogen bonds and/or shift in beta-strand register in the fibril state. During the unfolding process, the anticorrelated motion between the strands H and G as well as the strands H and A pulls the H strand out of the inner sheet plane, leading to a more twisted inner sheet. Our simulation has provided important detailed structural information about the partially unfolded state of TTR that may be related to the amyloidogenic intermediates.

Related papers:

title: Therapeutic strategies for human amyloid diseases
abstract: Amyloid diseases are a large group of a much larger family of misfolding diseases. This group includes pathologies as diverse as Alzheimer's disease, immunoglobulin-light-chain disease, reactive amyloid disease and the familial amyloid polyneuropathies. These diseases are generally incurable at present, although some drugs are known to transiently slow the progression of Alzheimer's disease. As we increase our understanding of the causative mechanisms of these disorders, the likelihood of success for a given therapeutic strategy will become clearer. This review will look at small-molecule and macromolecular approaches for intervention in amyloid diseases other than Alzheimer's disease, although select examples from Alzheimer's disease will be discussed.

title: Transthyretin: a review from a structural perspective
abstract: Transthyretin (formerly called prealbumin) plays important physiological roles as a transporter of thyroxine and retinol-binding protein. X-ray structural studies have provided information on the active conformation of the protein and the site of binding of both ligands. Transthyretin is also one of the precursor proteins commonly found in amyloid deposits. Both wild-type and single-amino-acid-substituted variants have been identified in amyloid deposits, the variants being more amyloidogenic. Sequencing of the gene and the resulting production of a transgenic mouse model have resulted in progress toward solving the mechanism of amyloid formation and detecting the variant gene in individuals at risk.

title: Structure of prealbumin: secondary, tertiary and quaternary interactions determined by Fourier refinement at 1.8 A.
abstract: The principal elements of the secondary, tertiary and quaternary structure of the tetrameric human plasma prealbumin molecule have been determined by Fourier refinement of X-ray diffraction data at 1.8 A resolution. The subunit has an extensive β-structure composed of eight strands organised into two four-stranded sheets. There is also one short α-helix. The tertiary structure is largely determined by the association of the two β-sheets. Important contributions to the tertiary structure are made by three tyrosines and one aspartic acid involved in side-chain-main-chain interactions; a buried histidine associated with a group of internal water molecules; and a compact cluster of seven aromatic residues. Quaternary interactions occur at two sets of interfaces closely organised around two of the three molecular 2-fold axes. The exclusive monomer-monomer interface is chiefly concerned with antiparallel hydrogen bond interactions which extend the two four-stranded sheets in the monomers to eight-stranded sheets in the dimer. One of the sheet interactions includes water molecules and tyrosine hydroxyls in the hydrogen bond pattern. The dimers associate through both hydrophilic and hydrophobic interactions at interfaces that involve all four monomers.

title: The X-ray crystal structure refinements of normal human transthyretin and the amyloidogenic Val-30→Met variant to 1.7-Å resolution
abstract: The x-ray crystal structures of normal human transthyretin (prealbumin) and the amyloidogenic Val-30-Met variant have been refined at 1.7-A resolution to R-values of 0.168 and 0.179, respectively, for 19,882 and 20,362 reflections (Fobs > 2.0 sigma). Standard deviations for stereochemical parameters are 0.018 and 0.022 A for bond distances, 0.030 and 0.038 A for angle distances, and 0.035 and 0.070 A for planar 1-4 distances. The newly refined normal structure shows improvement over the original structure of Blake and Swan (Blake, C. C. F., and Swan, I. D. A. (1971)  in stereochemistry and in the conformation of the loop regions. Residues Arg-103, Thr-123, Asn-124, and Pro-125 have now been resolved, and residues 1-9 and 126-127 have been modeled with the aid of simulated annealing refinement. The functional form of transthyretin is a tetramer, having a cylindrical cavity which will bind thyroxine and an exterior binding site for the complex of retinol with retinol-binding protein. The monomer is a beta barrel flattened to become more like a sandwich with residue 30 in the interior. The methionyl for valyl substitution forces the beta sheets of the monomer as much as 1 A apart, resulting in a distortion of the thyroxine-binding cavity, in agreement with the independent observations that the Met-30 variant has low affinity for thyroxine.

title: The Crystal Structure of Amyloidogenic Leu55→ Pro Transthyretin Variant Reveals a Possible Pathway for Transthyretin Polymerization into Amyloid Fibrils*
abstract: The x-ray crystal structure of the amyloidogenic Leu55 → Pro transthyretin (TTR) variant, implicated as the causative agent in early-onset familial amyloidotic polyneuropathy (Jacobson, D. R., McFarlin, D. E., Kane, I., and Buxbaum,  (1992) Hum. Genet. 89, 353–356), has been solved by molecular replacement, refined at 2.7 Å to a R cryst value of 0.190 (F obs > 2.0ς), and compared with wild-type transthyretin to understand the molecular mechanism(s) involved in amyloidogenesis. Leu55 → Pro TTR crystallizes in space group C2, with eight monomers in the asymmetric unit, and the observed packing contacts are considerably different from those described for the wild-type protein. Refinement of the crystal structure shows that the proline for leucine substitution disrupts the hydrogen bonds between strands D and A, resulting in different interface contacts. Based on the assumption that the observed packing contacts may be significant for amyloidogenesis, a model for the TTR amyloid is proposed. It consists of a tubular structure with inner and outer diameters approximately of 30 and 100 Å and four monomers per cross-section.

title: Transthyretin slowly exchanges subunits under physiological conditions: A convenient chromatographic method to study subunit exchange in oligomeric proteins
abstract: Transthyretin (TTR) subunits were labeled with a charge‐modifying tag to evaluate the possibility of subunit exchange between tetramers under physiological conditions. Starting with a mixture of two TTR homotetramers, one having all subunits tagged at the N termini and the other composed of untagged subunits, heterotetramer formation as a function of time and temperature was evaluated using ion exchange chromatography. The data indicate that the subunit exchange can occur under native conditions at physiological pH in vitro, albeit slowly. Wild‐type TTR exchanges subunits on a timescale of days at 37°C and on a timescale of hours at 4°C. The familial amyloid polyneuropathy‐associated variant V30M exchanges subunits at the same rate as wild‐type TTR at 4°C but slower and less efficiently at 37°C. Small molecule tetramer stabilizers abolish TTR subunit exchange, supporting a dissociative mechanism.

title: Sequence-dependent denaturation energetics: A major determinant in amyloid disease diversity
abstract: Several misfolding diseases commence when a secreted folded protein encounters a partially denaturing microenvironment, enabling its self assembly into amyloid. Although amyloidosis is modulated by numerous environmental and genetic factors, single point mutations within the amyloidogenic protein can dramatically influence disease phenotype. Mutations that destabilize the native state predispose an individual to disease; however, thermodynamic stability alone does not reliably predict disease severity. Here we show that the rate of transthyretin (TTR) tetramer dissociation required for amyloid formation is strongly influenced by mutation (V30M, L55P, T119M, V122I), with rapid rates exacerbating and slow rates reducing amyloidogenicity. Although these rates are difficult to predict a priori, they notably influence disease penetrance and age of onset. L55P TTR exhibits severe pathology because the tetramer both dissociates quickly and is highly destabilized. Even though V30M and L55P TTR are similarly destabilized, the V30M disease phenotype is milder because V30M dissociates more slowly, even slower than wild type (WT). Although WT and V122I TTR have nearly equivalent tetramer stabilities, V122I cardiomyopathy, unlike WT cardiomyopathy, has nearly complete penetrance—presumably because of its 2-fold increase in dissociation rate. We show that the T119M homotetramer exhibits kinetic stabilization and therefore dissociates exceedingly slowly, likely explaining how it functions to protect V30M/T119M compound heterozygotes from disease. An understanding of how mutations influence both the kinetics and thermodynamics of misfolding allows us to rationalize the phenotypic diversity of amyloid diseases, especially when considered in concert with other genetic and environmental data.

title: Transthyretin aggregation under partially denaturing conditions is a downhill polymerization.
abstract: The deposition of fibrils and amorphous aggregates of transthyretin (TTR) in patient tissues is a hallmark of TTR amyloid disease, but the molecular details of amyloidogenesis are poorly understood. Tetramer dissociation is typically rate-limiting for TTR amyloid fibril formation, so we have used a monomeric variant of TTR (M-TTR) to study the mechanism of aggregation. Amyloid formation is often considered to be a nucleation-dependent process, where fibril growth requires the formation of an oligomeric nucleus that is the highest energy species on the pathway. According to this model, the rate of fibril formation should be accelerated by the addition of preformed aggregates or "seeds", which effectively bypasses the nucleation step. Herein, we demonstrate that M-TTR amyloidogenesis at low pH is a complex, multistep reaction whose kinetic behavior is incompatible with the expectations for a nucleation-dependent polymerization. M-TTR aggregation is not accelerated by seeding, and the dependence of the reaction timecourse is first-order on the M-TTR concentration, consistent either with a dimeric nucleus or with a nonnucleated process where each step is bimolecular and essentially irreversible. These studies suggest that amyloid formation by M-TTR under partially denaturing conditions is a downhill polymerization, in which the highest energy species is the native monomer. Our results emphasize the importance of therapeutic strategies that stabilize the TTR tetramer and may help to explain why more than eighty TTR variants are disease-associated. The differences between amyloid formation by M-TTR and other amyloidogenic peptides (such as amyloid beta-peptide and islet amyloid polypeptide) demonstrate that these polypeptides do not share a common aggregation mechanism, at least under the conditions examined thus far.

title: An engineered transthyretin monomer that is nonamyloidogenic, unless it is partially denatured.
abstract: Transthyretin (TTR) is a soluble human plasma protein that can be converted into amyloid by acid-mediated dissociation of the homotetramer into monomers. The pH required for disassembly also results in tertiary structural changes within the monomeric subunits. To understand whether these tertiary structural changes are required for amyloidogenicity, we created the Phe87Met/Leu110Met TTR variant (M-TTR) that is monomeric according to analytical ultracentrifugation and gel filtration analyses and nonamyloidogenic at neutral pH. Results from far- and near-UV circular dichroism spectroscopy, one-dimensional proton NMR spectroscopy, and X-ray crystallography, as well as the ability of M-TTR to form a complex with retinol binding protein, indicate that M-TTR forms a tertiary structure at pH 7 that is very similar if not identical to that found within the tetramer. Reducing the pH results in tertiary structural changes within the M-TTR monomer, rendering it amyloidogenic, demonstrating the requirement for partial denaturation. M-TTR exhibits stability toward acid and urea denaturation that is nearly identical to that characterizing wild-type (WT) TTR at low concentrations (0.01-0.1 mg/mL), where monomeric WT TTR is significantly populated at intermediate urea concentrations prior to the tertiary structural transition. However, the kinetics of denaturation and fibril formation are much faster for M-TTR than for tetrameric WT TTR, particularly at near-physiological concentrations, because of the barrier associated with the tetramer to folded monomer preequilibrium. These results demonstrate that the tetramer to folded monomer transition is insufficient for fibril formation; further tertiary structural changes within the monomer are required.

title: Partial denaturation of transthyretin is sufficient for amyloid fibril formation in vitro.
abstract: Amyloid diseases are caused by the self-assembly of a given protein into an insoluble cross-beta-sheet quaternary structural form which is pathogenic. An understanding of the biochemical mechanism of amyloid fibril formation should prove useful in understanding amyloid disease. Toward this end, a procedure for the conversion of the amyloidogenic protein transthyretin into amyloid fibrils under conditions which mimic the acidic environment of a lysosome has been developed. Association of a structured transthyretin denaturation intermediate is sufficient for amyloid fibril formation in vitro. The rate of fibril formation is pH dependent with significant rates being observed at pHs accessible within the lysosome (3.6-4.8). Far-UV CD spectroscopic studies suggest that transthyretin retains its secondary structural features at pHs where fibrils are formed. Near-UV CD studies demonstrate that transthyretin has retained the majority of its tertiary structure during fibril formation as well. Near-UV CD analysis in combination with glutaraldehyde cross-linking studies suggests that a pH-mediated tetramer to monomer transition is operative in the pH range where fibril formation occurs. The rate of fibril formation decreases markedly at pHs below pH 3.6, consistent with denaturation to a monomeric TTR intermediate which has lost its native tertiary structure and capability to form fibrils. It is difficult to specify with certainty which quaternary structural form of transthyretin is the amyloidogenic intermediate at this time. These difficulties arise because the maximal rate of fibril formation occurs at pH 3.6 where tetramer, traces of dimer, and significant amounts of monomer are observed.(ABSTRACT TRUNCATED AT 250 WORDS)

title: Characterization of the transthyretin acid denaturation pathways by analytical ultracentrifugation: implications for wild-type, V30M, and L55P amyloid fibril formation.
abstract: Analytical ultracentrifugation methods were utilized to further characterize the acid denaturation pathways of wild-type, V30M, and L55P transthyretin (TTR) that generate intermediates leading to amyloid fibril formation and possibly the diseases senile systemic amyloidosis and familial amyloid polyneuropathy. Equilibrium and velocity methods were employed herein to characterize the TTR quaternary structural requirements for amyloid fibril formation. From neutral to slightly acidic conditions (pH 7.5-5.1), wild-type transthyretin (0.2-0.3 mg/mL, 100 mM KCl, 37 degrees C) exists as a tetramer and is incapable of fibril formation. Under more acidic conditions (pH 5 to 3.9), tetrameric wild-type TTR slowly dissociates to a monomer having an alternatively folded tertiary structure(s) that self-assembles at physiological concentration (0.2 mg/mL) into a ladder of quaternary structural intermediates of increasing molecular weight. These intermediates appear to be on the pathway of amyloid fibril formation, since they ultimately disappear when amyloid fibrils are observed. The V30M and L55P TTR variants exhibit similar acid denaturation pathways, with the exception that dissociation of the tetramer to the monomeric amyloidogenic intermediate occurs at a higher pH and to a much greater extent, allowing the quaternary structural intermediates to be readily observed by velocity methods. Partial denaturation and assembly of the monomeric amyloidogenic intermediate(s) occur at pH 5.4 for V30M and L55P TTR over a 72 h period, during which wild-type TTR maintains its normal tetrameric three-dimensional structure. Interestingly, the L55P and V30M familial amyloid polyneuropathy (FAP) associated variants form amyloid protofilaments at pH 7.5 (37 degrees C) after several weeks of incubation, suggesting that the activation barriers for TTR tetramer dissociation to the monomeric amyloidogenic intermediate are much lower for the FAP variants relative to wild-type TTR, which does not form amyloid or amyloid protofilaments under these conditions. This study establishes the key role of the monomeric amyloidogenic intermediate and its self-assembly into a ladder of quaternary structural intermediates for the formation of wild-type, V30M, and L55P transthyretin amyloid fibrils.

title: A comparative analysis of 23 structures of the amyloidogenic protein transthyretin.
abstract: Self-assembly of the human plasma protein transthyretin (TTR) into unbranched insoluble amyloid fibrils occurs as a result of point mutations that destabilize the molecule, leading to conformational changes. The tertiary structure of native soluble TTR and many of its disease-causing mutants have been determined. Several independent studies by X-ray crystallography have suggested structural differences between TTR variants which are claimed to be of significance for amyloid formation. As these changes are minor and not consistent between the studies, we have compared all TTR structures available at the protein data bank including three wild-types, three non-amyloidogenic mutants, seven amyloidogenic mutants and nine complexes. The reference for this study is a new 1.5 A resolution structure of human wild-type TTR refined to an R-factor/R-free of 18.6 %/21.6 %. The present findings are discussed in the light of the previous structural studies of TTR variants, and show the reported structural differences to be non-significant.

title: Intrinsic versus mutation dependent instability/flexibility: a comparative analysis of the structure and dynamics of wild-type transthyretin and its pathogenic variants.
abstract: Transthyretin (TTR) is one of the about 20 known human proteins associated with amyloidosis which is characterized by the accumulation of amyloid fibrils in tissues or extracellular matrix surrounding vital organs. Unlike Alzheimer's fibrils that comprise a fragment of a large precursor protein, TTR amyloid fibrils are composed of both full-length protein and fragments of the molecule. The native state of TTR is a homotetramer with eight beta-strands organized into a beta-sandwich in each monomer. To elucidate the structural reorganization mechanisms preceding amyloid formation, it is important to characterize the dynamic features of the wild-type native state as well as to reveal the influence of disease-associated mutations on the structure and dynamics. Molecular dynamics (MD) simulations complement X-ray crystallography and D-H exchange to capture the intrinsically unstable/flexible sites of the wild-type as well as the mutation dependent unstable sites of the pathogenic variants. Our results of MD simulations have shown that the Leu55-->Pro (L55P) mutation occurs in an intrinsically unstable site, leading to substantial local and global structural changes. This observation supports the early speculation that the C-strand-loop-D-strand rearrangement leads to the formation of amyloidogenic intermediates. In addition to the D strand, the alpha-helical region and the strands at the monomer-monomer interface are also intrinsically unstable. The central channel of L55P-TTR undergoes opening and closing fluctuations, which may provide an explanation for the fact that while the mutation is far from the channel, the mutant shows a substantial low binding affinity of thyroxine.

title: Structure of a new polymorphic monoclinic form of human transthyretin at 3 A resolution reveals a mixed complex between unliganded and T4-bound tetramers of TTR.
abstract: The crystal structure of a new polymorphic form of human transthyretin (hTTR) with a lattice containing a unique assembly of apo hTTR and TTR-T(4) complex has been determined to 3 A resolution. The monoclinic form of human TTR reported here crystallizes in space group P2(1), with unit-cell parameters a = 76.7 (6), b = 96.7 (8), c = 81.7 (4) A, beta = 106.8 (4) degrees. The asymmetric unit contains two tetramers of transthyretin related by the non-crystallographic symmetry (NCS) operation of a 90.28 degrees rotation between two hTTR molecules around an axis close to crystallographic z. The r.m.s. difference between the two tetramers calculated from their C(alpha) positions is 0.48 A. The structure was refined using 15.0-3.0 A resolution data to R = 22.9% and R(free) = 28.9% for reflections F > 0.0sigma(F), and R = 19.7% and R(free) = 25.8% for reflections F > 3.0sigma(F). The intermolecular interactions involve the tips of alpha-helices and loops around Arg21, Glu61 and Ser100 of all monomers. The electron-density maps revealed residual thyroxine (T(4)) bound in only one of the two unique tetrameric TTR molecules, with an occupancy of 53%, while the second tetramer is unliganded. One thyroxine ligand is bound in a way similar to the orientations described for the orthorhombic form of the hTTR-T(4) complex. The T(4) bound in the second site is positioned similar to 3',5'-dinitro-N-acetyl-L-thyronine in its hTTR complex. Differences in the size of the central channel defined by the D, A, G and H beta-strands of two monomeric subunits are observed between the apo TTR and T(4)-bound tetramer. The averaged distances between Ala108 C(alpha) and its equivalent measured across each binding site are 12.34 A for the T(4)-bound and 10.96 A for the unliganded TTR tetramer, respectively. The observed differences might reflect the mechanics of the ligand binding in the channel and possibly explain the observed negative cooperativity effect for ligand binding.

title: The beta-strand D of transthyretin trapped in two discrete conformations.
abstract: Conformational changes in native and variant forms of the human plasma protein transthyretin (TTR) induce several types of amyloid diseases. Biochemical and structural studies have mapped the initiation site of amyloid formation onto residues at the outer C and D beta-strands and their connecting loop. In this study, we characterise an engineered variant of transthyretin, Ala108Tyr/Leu110Glu, which is kinetically and thermodynamically more stable than wild-type transthyretin, and as a consequence less amyloidogenic. Crystal structures of the mutant were determined in two space groups, P2(1)2(1)2 and C2, from crystals grown in the same crystallisation set-up. The structures are identical with the exception for residues Leu55-Leu58, situated at beta-strand D and the following DE loop. In particular, residues Leu55-His56 display large shifts in the C2 structure. There the direct hydrogen bonding between beta-strands D and A has been disrupted and is absent, whereas the beta-strand D is present in the P2(1)2(1)2 structure. This difference shows that from a mixture of metastable TTR molecules, only the molecules with an intact beta-strand D are selected for crystal growth in space group P2(1)2(1)2. The packing of TTR molecules in the C2 crystal form and in the previously determined amyloid TTR (ATTR) Leu55Pro crystal structure is close-to-identical. This packing arrangement is therefore not unique in amyloidogenic mutants of TTR.

title: Why is Leu55→Pro55 transthyretin variant the most amyloidogenic: Insights from molecular dynamics simulations of transthyretin monomers
abstract: Transthyretin (TTR) is one of the known human amyloidogenic proteins. Its native state is a homotetramer with each monomer having a β‐sandwich structure. Strong experimental evidence suggests that TTR dissociates into monomeric intermediates and that the monomers subsequently self‐assemble to form amyloid deposits and insoluble fibrils. However, details on the early steps along the pathway of TTR amyloid formation are unclear, although various experimental approaches with resolutions at the molecular or residue level have provided some clues. It is highly likely that the stability and flexibility of monomeric TTR play crucial roles in the early steps of amyloid formation; thereby, it is essential to characterize initial conformational changes of TTR monomers. In this article we probe the possibility that the differences in the monomeric forms of wild‐type (WT) TTR and its variants are responsible for differential amyloidogenesis. We begin with the simulations of WT, Val30→Met (V30M), and Leu55→Pro (L55P) TTR monomers. Nanosecond time scale molecular dynamics simulations at 300 K were performed using AMBER. The results indicate that the L55P‐TTR monomer undergoes substantial structural changes relative to fluctuations observed in the WT and V30M TTR monomers. The observation supports earlier speculation that the L55P mutation may lead to disruption of the β‐sheet structure through the disorder of the “edge strands” that might facilitate amyloidogenesis.

title: Semianalytical treatment of solvation for molecular mechanics and dynamics
abstract: sorption results9 revealed that the iron surface was mostly covered by promoter oxides of AI, Ca, and K. Postreaction XPS results also revealed a C( Is) XPS peak of weak to moderate intensity centered at 284.1-283.7 eV. This binding energy approaches those (ca. 283.5 eV) reported for iron cat bide^.^^*'^ More convincing evidence for carbide formation was obtained from TPHT results collected after reaction studies like those displayed in Figure 1 in which methane was the only product. After reaction at temperatures below 340 OC, only small amounts of reactive carbon could be distinguished with maximum methane desorption rates near 300 OC. However, for higher reaction temperatures, large amounts of methane were produced with a maximum rate just above 400 OC. Since XPS results revealed only small amounts of carbonaceous residue on top of the catalyst surface, this reactive carbon must be associated with carbiding of the catalyst. Consequently, it appears that the active carbon incorporation catalyst is carbided iron. This conclusion is well supported by bulk carbon to iron stoichiometries of 0.1-0.25 estimated from the TPHT peak areas which were adequate to represent 40-60'36 conversion to bulk carbides such as Fe,C or FeSC2. Moreover, preliminary results from studies using bona fide iron carbides have shown similar catalytic b e h a ~ i o r .

title: The acid-mediated denaturation pathway of transthyretin yields a conformational intermediate that can self-assemble into amyloid.
abstract: Transthyretin (TTR) amyloid fibril formation is observed during partial acid denaturation and while refolding acid-denatured TTR, implying that amyloid fibril formation results from the self-assembly of a conformational intermediate. The acid denaturation pathway of TTR has been studied in detail herein employing a variety of biophysical methods to characterize the intermediate(s) capable of amyloid fibril formation. At physiological concentrations, tetrameric TTR remains associated from pH 7 to pH 5 and is incapable of amyloid fibril formation. Tetrameric TTR dissociates to a monomer in a process that is dependent on both pH and protein concentration below pH 5. The extent of amyloid fibril formation correlates with the concentration of the TTR monomer having an altered, but defined, tertiary structure over the pH range of 5.0-3.9. The inherent Trp fluorescence-monitored denaturation curve of TTR exhibits a plateau over the pH range where amyloid fibril formation is observed (albeit at a higher concentration), implying that a steady-state concentration of the amyloidogenic intermediate with an altered tertiary structure is being detected. Interestingly, 1-anilino-8-naphthalenesulfonate fluorescence is at a minimum at the pH associated with maximal amyloid fibril formation (pH 4.4), implying that the amyloidogenic intermediate does not have a high extent of hydrophobic surface area exposed, consistent with a defined tertiary structure. Transthyretin has two Trp residues in its primary structure, Trp-41 and Trp-79, which are conveniently located far apart in the tertiary structure of TTR. Replacement of each Trp with Phe affords two single Trp containing variants which were used to probe local pH-dependent tertiary structural changes proximal to these chromophores. The pH-dependent fluorescence behavior of the Trp-79-Phe mutant strongly suggests that Trp-41 is located near the site of the tertiary structural rearrangement that occurs in the formation of the monomeric amyloidogenic intermediate, likely involving the C-strand-loop-D-strand region. Upon further acidification of TTR (below pH 4.4), the structurally defined monomeric amyloidogenic intermediate begins to adopt alternative conformations that are not amyloidogenic, ultimately forming an A-state conformation below pH 3 which is also not amyloidogenic. In summary, analytical equilibrium ultracentrifugation, SDS-PAGE, far- and near-UV CD, fluorescence, and light scattering studies suggest that the amyloidogenic intermediate is a monomeric predominantly beta-sheet structure having a well-defined tertiary structure.

title: A glimpse of a possible amyloidogenic intermediate of transthyretin
abstract: Studies have indicated that partially unfolded states occur under conditions that favor amyloid formation by transthyretin (TTR), as well as other amyloidogenic proteins. In this study, we used hydrogen exchange measurements to show that there is selective destabilization of one half of the β-sandwich structure of TTR under such conditions. This provides more direct information about conformational fluctuations than previously available, and will facilitate design of future experiments to probe the intermediates critical to amyloid formation.

title: AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules
abstract: We describe the development, current features, and some directions for future development of the AMBER package of computer programs. This package has evolved from a program that was constructed to do Assisted Model Building and Energy Refinement to a group of programs embodying a number of the powerful tools of modern computational chemistry-molecular dynamics and free energy calculations.

title: All-atom structure prediction and folding simulations of a stable protein.
abstract: We present results from all-atom, fully unrestrained ab initio folding simulations for a stable protein with nontrivial secondary structure elements and a hydrophobic core. The construct, "trpcage", is a 20-residue sequence optimized by the Andersen group at University of Washington and is currently the smallest protein that displays two-state folding properties. Compared over the well-defined regions of the experimental structure, our prediction has a remarkably low 0.97 A Calpha root-mean-square-deviation (rmsd) and 1.4 A for all heavy atoms. The simulated structure family displays additional features that are suggested by experimental data, yet are not evident in the family of NMR-derived structures.

title: Exploring protein native states and large‐scale conformational changes with a modified generalized born model
abstract: Implicit solvation models provide, for many applications, a reasonably accurate and computationally effective way to describe the electrostatics of aqueous solvation. Here, a popular analytical Generalized Born (GB) solvation model is modified to improve its accuracy in calculating the solvent polarization part of free energy changes in large‐scale conformational transitions, such as protein folding. In contrast to an earlier GB model (implemented in the AMBER‐6 program), the improved version does not overstabilize the native structures relative to the finite‐difference Poisson–Boltzmann continuum treatment. In addition to improving the energy balance between folded and unfolded conformers, the algorithm (available in the AMBER‐7 and NAB molecular modeling packages) is shown to perform well in more than 50 ns of native‐state molecular dynamics (MD) simulations of thioredoxin, protein‐A, and ubiquitin, as well as in a simulation of Barnase/Barstar complex formation. For thioredoxin, various combinations of input parameters have been explored, such as the underlying gas‐phase force fields and the atomic radii. The best performance is achieved with a previously proposed modification to the torsional potential in the Amber ff99 force field, which yields stable native trajectories for all of the tested proteins, with backbone root‐mean‐square deviations from the native structures being ∼1.5 Å after 6 ns of simulation time. The structure of Barnase/Barstar complex is regenerated, starting from an unbound state, to within 1.9 Å relative to the crystal structure of the complex. Proteins 2004;55:000–000.

title: Theory and applications of the generalized born solvation model in macromolecular simulations
abstract: Generalized Born (GB) models provide an attractive way to include some thermodynamic aspects of aqueous solvation into simulations that do not explicitly model the solvent molecules. Here we discuss our recent experience with this model, presenting in detail the way it is implemented and parallelized in the AMBER molecular modeling code. We compare results using the GB model (or GB plus a surface‐area based “hydrophobic” term) to explicit solvent simulations for a 10 base‐pair DNA oligomer, and for the 108‐residue protein thioredoxin. A slight modification of our earlier suggested parameters makes the GB results more like those found in explicit solvent, primarily by slightly increasing the strength of NHO and NHN internal hydrogen bonds. Timing and energy stability results are reported, with an eye toward using these model for simulations of larger macromolecular systems and longer time scales.

title: Continuum solvent molecular dynamics study of flexibility in interleukin-8.
abstract: Generalized Born continuum solvent methods have been shown to provide a reasonable description of the equilibrium thermodynamics of aqueous solvation in a variety of applications to peptides, proteins, and nucleic acids. Here we study the performance of these methods in molecular dynamics simulations of interleukin-8, comparing nanosecond-length explicit solvent simulations with those using the generalized Born model. In general, the simulations show similar results, although movement away from the initial NMR-determined structure and average fluctuations about the mean are slightly higher for the continuum solvent results. In both simulations, the two helices that are packed on top of the core sheet move closer together, resulting in a structure that more closely resembles the X-ray structure. Principal-component (quasiharmonic) analysis is used to analyze the motions of these helices in both of the simulations and in the NMR ensemble of structures. Prospects for making more general use of continuum solvent models in protein dynamics simulations are discussed.

title: Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes
abstract: A numerical algorithm integrating the 3N Cartesian equations of motion of a system of N points subject to holonomic constraints is formulated. The relations of constraint remain perfectly fulfilled at each step of the trajectory despite the approximate character of numerical integration. The method is applied to a molecular dynamics simulation of a liquid of 64 n-butane molecules and compared to a simulation using generalized coordinates. The method should be useful for molecular dynamics calculations on large molecules with internal degrees of freedom.

title: Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features
abstract: For a successful analysis of the relation between amino acid sequence and protein structure, an unambiguous and physically meaningful definition of secondary structure is essential. We have developed a set of simple and physically motivated criteria for secondary structure, programmed as a pattern‐recognition process of hydrogen‐bonded and geometrical features extracted from x‐ray coordinates. Cooperative secondary structure is recognized as repeats of the elementary hydrogen‐bonding patterns “turn” and “bridge.” Repeating turns are “helices,” repeating bridges are “ladders,” connected ladders are “sheets.” Geometric structure is defined in terms of the concepts torsion and curvature of differential geometry. Local chain “chirality” is the torsional handedness of four consecutive Cα positions and is positive for right‐handed helices and negative for ideal twisted β‐sheets. Curved pieces are defined as “bends.” Solvent “exposure” is given as the number of water molecules in possible contact with a residue. The end result is a compilation of the primary structure, including SS bonds, secondary structure, and solvent exposure of 62 different globular proteins. The presentation is in linear form: strip graphs for an overall view and strip tables for the details of each of 10.925 residues. The dictionary is also available in computer‐readable form for protein structure prediction work.

title: Twist and shear in beta-sheets and beta-ribbons.
abstract: The structures of the beta-sheets and the beta-ribbons have been analysed using high-resolution protein structure data. Systematic asymmetries measured in both parallel and antiparallel beta-structures include the sheet twist and the strand shear. In order to determine the origin of these asymmetries, numerous interactions and correlations were examined. The strongest correlations are observed for residues in antiparallel beta-sheets and beta-ribbons that form non-H-bonded pairs. For these residues, the sheet twist is correlated to the backbone phi angle but not to the psi angle. Our analysis supports the existence of an inter-strand C(alpha)H(alpha)...O weak H-bond, which, together with the CO...HN H-bond, constitutes a bifurcated H-bond that links neighbouring beta-strands. Residues of beta-sheets and beta-ribbons in high-resolution protein structures form a distinct region of the Ramachandran plot, which is determined by the formation of the bifurcated H-bond, the formation of an intra-strand O...H(alpha) non-bonded polar interaction, and an intra-strand O...C(beta) steric clash. Using beta-strands parameterised by phi-psi values from the allowed beta-sheet region of the Ramachandran plot, the shear and the right-hand twist can be reproduced in a simple model of the antiparallel and parallel beta-ribbon that models the bifurcated H-bonds specifically. The conformations of interior residues of beta-sheets are shown to be subsets of the conformations of residues of beta-ribbons.

title: Tissue damage in the amyloidoses: Transthyretin monomers and nonnative oligomers are the major cytotoxic species in tissue culture.
abstract: The transthyretin (TTR) amyloidoses are human diseases in which the misfolded TTR protein aggregates in tissues with subsequent visceral, peripheral, and autonomic nerve dysfunction. Recent reports have stressed the importance of oligomeric intermediates as major cytotoxic species in various forms of amyloidogenesis. We have examined the cytotoxic effects of several quaternary structural states of wild-type and variant TTR proteins on cells of neural lineage. TTR amyloid fibrils and soluble aggregates >100 kDa were not toxic. Incubation of TTR under the conditions of the cell assay and analysis by size-exclusion chromatography and SDS/PAGE reveal that monomeric TTR or relatively small, rapidly formed aggregates of a maximum size of six subunits were the major cytotoxic species. Small molecules that stabilize the native tetrameric state were shown to prevent toxicity. The studies are consistent with a model in which the misfolded TTR monomer rapidly aggregates to form transient low molecular mass assemblies (<100 kDa) that are highly cytotoxic in tissue culture.

title: Biophysical analysis of normal transthyretin: Implications for fibril formation in senile systemic amyloidosis
abstract: Transthyretin (TTR) is a plasma protein that transports thyroid hormone andretinot binding protein-vitamin A complex. Eighty-four variants of TTR have been identified and seventy-four are associated with familial amyloidotic polyneuropathy. Normal TTR is the major protein found in the fibrillar deposits in the heart at time of autopsy of individuals with senile systemic amyloidosis. The mechanism by which normally soluble TTR deposits as organ-damaging, insoluble, pathological fibrils late in life is unknown. Understanding the mechanism of fibrillogenesis of normal TTR is critical to the design of clinical treatments aimed at retardation, prevention, or reversal of fibril deposition. We have employed a biophysical approach to explore the hypothesis that an instability in a particular secondary or tertiary structure plays a role in the ability of normal TTR to form fibrils at physiological pH. Using far UV circular dichroic (CD) spectroscopy as a function of temperature we have identified simultaneous, cooperative, reversible structural changes in the β-sheet and α-helical regions. The flexible short, surface-located loops undergo an irreversible conformational change at a lower temperature. Spectra before and after heating are different, particularly in the wavelength region associated with these loops, strongly suggesting that the major portion of TTR returns to its initial conformation while the loops do not. Near UV CD reveals partially reversible and irreversible changes in tertiary structure. Using calorimetry to directly measure the enthalpy associated with these changes, two peaks are observed, with further analysis suggesting conformational intermediates. Precipitates from heated samples reveal pre-fibrillar morphology by negative stain electron microscopy. These biophysical studies suggest that heat-induced conformational rearrangements enable normal TTR to assemble into prefibrils at physiological pH.

title: Probing Solvent Accessibility of Transthyretin Amyloid by Solution NMR Spectroscopy*
abstract: The human plasma protein transthyretin (TTR) may form fibrillar protein deposits that are associated with both inherited and idiopathic amyloidosis. The present study utilizes solution nuclear magnetic resonance spectroscopy, in combination with hydrogen/deuterium exchange, to determine residue-specific solvent protection factors within the fibrillar structure of the clinically relevant variant, TTRY114C. This novel approach suggests a fibril core comprised of the six β-strands, A-B-E-F-G-H, which retains a native-like conformation. Strands C and D are dislocated from their native edge region and become solvent-exposed, leaving a new interface involving strands A and B open for intermolecular interactions. Our results further support a native-like intermolecular association between strands F-F′ and H-H′ with a prolongation of these β-strands and, interestingly, with a possible shift in β-strand register of the subunit assembly. This finding may explain previous observations of a monomeric intermediate preceding fibril formation. A structural model based on our results is presented.

title: The "edge strand" hypothesis: Prediction and test of a mutational "hot-spot" on the transthyretin molecule associated with FAP amyloidogenesis
abstract: The amyloid fibrils of familial amyloidotic polyneuropathy (FAP) are composed of genetic variants of transthyretin (TTR). Over 40 amino-acid variants have been identified associated with amyloidosis. We have performed an analysis of the effect of the different substitutions, such as location and changes in side-chain volume, hydrophilicity and charge on the TTR molecule. We have also analyzed the distribution of variants along the peptide chain. While we could demonstrate no pattern of change associated with the bulk properties of the amyloidogenic variant residues, the distribution analysis suggested a significant correlation with the position of variant residues along the polypeptide chain. We identified a significant peak in the distribution of variants which, in the folded protein chain, was associated with the edge strands (residues 45-58) of the two s-sheets that form the structural framework of the TTR molecule. In contrast, the regions of the molecule associated with the subunit interactions are almost devoid of amyloidogenic variants. To test this finding, we constructed two variant TTRs using recombinant techniques, one with a triple substitution and one with a triple deletion in the edge strand region. On expression, the variants spontaneously formed material that produced a characteristic redshift upon Congo red staining, was composed of fibrils 80-110A in diameter and gave s-structure X-ray patterns suggesting the formation of amyloid fibrils. We conclude that the effect of amyloidogenic variants may be to alter the structure or stability of the edge strands of the s-sheets of TTR, thus promoting the growth of inter-molecular s-sheet structures characteristic of amyloid.

title: Anatomy of an amyloidogenic intermediate: conversion of beta-sheet to alpha-sheet structure in transthyretin at acidic pH.
abstract: The homotetramer of transthyretin (TTR) dissociates into a monomeric amyloidogenic intermediate that self-assembles into amyloid fibrils at low pH. We have performed molecular dynamics simulations of monomeric TTR at neutral and low pH at physiological (310 K) and very elevated temperature (498 K). In the low-pH simulations at both temperatures, one of the two beta-sheets (strands CBEF) becomes disrupted, and alpha-sheet structure forms in the other sheet (strands DAGH). alpha-sheet is formed by alternating alphaL and alphaR residues, and it was first proposed by Pauling and Corey. Overall, the simulations are in agreement with the available experimental observations, including solid-state NMR results for a TTR-peptide amyloid. In addition, they provide a unique explanation for the results of hydrogen exchange experiments of the amyloidogenic intermediate-results that are difficult to explain with beta-structure. We propose that alpha-sheet may represent a key pathological conformation during amyloidogenesis.

title: Pauling and Corey's alpha-pleated sheet structure may define the prefibrillar amyloidogenic intermediate in amyloid disease.
abstract: Transthyretin, beta(2)-microglobulin, lysozyme, and the prion protein are four of the best-characterized proteins implicated in amyloid disease. Upon partial acid denaturation, these proteins undergo conformational change into an amyloidogenic intermediate that can self-assemble into amyloid fibrils. Many experiments have shown that pH-mediated changes in structure are required for the formation of the amyloidogeneic intermediate, but it has proved impossible to characterize these conformational changes at high resolution using experimental means. To probe these conformational changes at atomic resolution, we have performed molecular dynamics simulations of these proteins at neutral and low pH. In low-pH simulations of all four proteins, we observe the formation of alpha-pleated sheet secondary structure, which was first proposed by L. Pauling and R. B. Corey [(1951) ]. In all beta-sheet proteins, transthyretin and beta(2)-microglobulin, alpha-pleated sheet structure formed over the strands that are highly protected in hydrogen-exchange experiments probing amyloidogenic conditions. In lysozyme and the prion protein, alpha-sheets formed in the specific regions of the protein implicated in the amyloidogenic conversion. We propose that the formation of alpha-pleated sheet structure may be a common conformational transition in amyloidosis.

title: How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules?
abstract: In this study, we present conformational energies for a molecular mechanical model (Parm99) developed for organic and biological molecules using the restrained electrostatic potential (RESP) approach to derive the partial charges. This approach uses the simple “generic” force field model (Parm94), and attempts to add a minimal number of extra Fourier components to the torsional energies, but doing so only when there is a physical justification. The results are quite encouraging, not only for the 34‐molecule set that has been studied by both the highest level ab initiomodel (GVB/LMP2) and experiment, but also for the 55‐molecule set for which high‐quality experimental data are available. Considering the 55 molecules studied by all the force field models for which there are experimental data, the average absolute errors (AAEs) are 0.28 (this model), 0.52 (MM3), 0.57 (CHARMm [MSI]), and 0.43 kcal/mol (MMFF). For the 34‐molecule set, the AAEs of this model versus experiment and ab initio are 0.28 and 0.27 kcal/mol, respectively. This is a lower error than found with MM3 and CHARMm, and is comparable to that found with MMFF (0.31 and 0.22 kcal/mol). We also present two examples of how well the torsional parameters are transferred from the training set to the test set. The absolute errors of molecules in the test set are only slightly larger than in the training set (differences of <0.1 kcal/mol). Therefore, it can be concluded that a simple “generic” force field with a limited number of specific torsional parameters can describe intra‐ and intermolecular interactions, although all comparison molecules were selected from our 82‐compound training set. We also show how this effective two‐body model can be extended for use with a nonadditive force field (NAFF), both with and without lone pairs. Without changing the torsional parameters, the use of more accurate charges and polarization leads to an increase in average absolute error compared with experiment, but adjustment of the parameters restores the level of agreement found with the additive model. After reoptimizing the Ψ, Φ torsional parameters in peptides using alanine dipeptide (6 conformational pairs) and alanine tetrapeptide (11 conformational pairs), the new model gives better energies than the Cornell et al. (  force field. The average absolute error of this model for high‐level ab initio calculation is 0.82 kcal/mol for alanine dipeptide and tetrapeptide as compared with 1.80 kcal/mol for the Cornell et al. model. For nucleosides, the new model also gives improved energies compared with the Cornell et al. model. To optimize force field parameters, we developed a program called parmscan, which can iteratively scan the torsional parameters in a systematic manner and finally obtain the best torsional potentials. Besides the organic molecules in our test set, parmscan was also successful in optimizing the Ψ, Φ torsional parameters in peptides to significantly improve agreement between molecular mechanical and high‐level ab initio energies.

title: Deuterium-proton exchange on the native wild-type transthyretin tetramer identifies the stable core of the individual subunits and indicates mobility at the subunit interface.
abstract: Transthyretin is a human protein capable of amyloid formation that is believed to cause several types of amyloid disease, depending on the sequence deposited. Previous studies have demonstrated that wild-type transthyretin (TTR), although quite stable, forms amyloid upon dissociation from its native tetrameric form into monomers with an altered conformation. Many naturally occurring single-site variants of TTR display decreased stability in vitro, manifested by the early onset familial amyloid diseases in vivo. Only subtle structural changes were observed in X-ray crystallographic structures of these disease associated variants. In this study, the stability of the wild-type TTR tetramer was investigated at the residue-resolution level by monitoring (2)H-H exchange via NMR spectroscopy. The measured protection factors for slowly-exchanging amide hydrogen atoms reveal a stable core consisting of strands A, B, E, F, and interestingly, the loop between strands A and B. In addition, the faster exchange of amide groups from residues at the subunit interfaces suggests unexpected mobility in these regions. This information is crucial for future comparisons between disease-associated and wild-type tetramers. Such studies can directly address the regions of TTR that become destabilized as a consequence of single amino acid substitutions, providing clues to aspects of TTR amyloidogenesis.

title: Identification of a subunit interface in transthyretin amyloid fibrils: evidence for self-assembly from oligomeric building blocks.
abstract: Amyloid and prion diseases appear to stem from the conversion of normally folded proteins into insoluble, fiber-like assemblies. Despite numerous structural studies, a detailed molecular characterization of amyloid fibrils remains elusive. In particular, models of amyloid fibrils proposed thus far have not adequately defined the constituent protein subunit interactions. To further our understanding of amyloid structure, we employed thiol-specific cross-linking and site-directed spin labeling to identify specific protein-protein associations in transthyretin (TTR) amyloid fibrils. We find that certain cysteine mutants of TTR, when dimerized by chemical cross-linkers, still form fibers under typical in vitro fibrillogenic conditions. In addition, site-directed spin labeling of many residues at the natural dimer interface reveals that their spatial proximity is preserved in the fibrillar state even in the absence of cross-linking constraints. Here, we present the first view of a subunit interface in TTR fibers and show that it is very similar to one of the natural dimeric interchain associations evident in the structure of soluble TTR. The results clarify varied models of amyloidogenesis by demonstrating that transthyretin amyloid fibrils may assemble from oligomeric protein building blocks rather than structurally rearranged monomers.

title: Collective motions in proteins: A covariance analysis of atomic fluctuations in molecular dynamics and normal mode simulations
abstract: A method is described for identifying collective motions in proteins from molecular dynamics trajectories or normal mode simulations. The method makes use of the covariances of atomic positional fluctuations. It is illustrated by an analysis of the bovine pancreatic trypsin inhibitor. Comparison of the covariance and cross‐correlation matrices shows that the relative motions have many similar features in the different simulations. Many regions of the protein, especially regions of secondary structure, move in a correlated manner. Anharmonic effects, which are included in the molecular dynamics simulations but not in the normal analysis, are of some importance in determining the larger scale collective motions, but not the more local fluctuations. Comparisons of molecular dynamics simulations in the present and absence of solvent indicate that the environment is of significance for the long‐range motions.

title: Calculation of ensembles of structures representing the unfolded state of an SH3 domain.
abstract: The N-terminal SH3 domain of drk (drkN SH3 domain) exists in equilibrium between a folded (F(exch)) and an unfolded (U(exch)) form under non-denaturing conditions. In order to further our previous descriptions of the U(exch) state, we have developed a protocol for calculating ensembles of structures, based on experimental spectroscopic data, which broadly represent the unfolded state. A large number of unfolding trajectories were generated, starting from the folded state structure of the protein, in order to provide a reasonable sampling of the conformational space accessible to this sequence. Unfolded state ensembles have been "calculated" using a newly developed program ENSEMBLE, which optimizes the population weights assigned to each structure based on experimental properties of the U(exch) state. Pseudo-energy terms for nuclear Overhauser effects, J-coupling constants, (13)C chemical shifts, translational diffusion coefficients and tryptophan ring burial based on NMR and fluorescence data have been implemented. The population weight assignment procedure was performed for different starting ensembles. Small numbers of structures (<60) dominate the final ensembles compared to the total number in the starting ensembles, suggesting that the drkN SH3 domain U(exch) state can be described by a limited number of lower-energy conformations. The calculated U(exch) state ensembles are much more compact than a "random coil" chain, with significant native-like residual structure observed. In particular, a sizable population of conformers having the n-src loop and distal beta-hairpin structures exist in the calculated U(exch) state ensembles, and Trp36 is involved in a large number of interactions, both native and non-native.