NMR structure of the bovine prion protein

The NMR structures of the recombinant 217-residue polypeptide chain of the mature bovine prion protein, bPrP(23–230), and a C-terminal fragment, bPrP(121–230), include a globular domain extending from residue 125 to residue 227, a short flexible chain end of residues 228–230, and an N-terminal flexibly disordered “tail” comprising 108 residues for the intact protein and 4 residues for bPrP(121–230), respectively. The globular domain contains three α-helices comprising the residues 144–154, 173–194, and 200–226, and a short antiparallel β-sheet comprising the residues 128–131 and 161–164. The best-defined parts of the globular domain are the central portions of the helices 2 and 3, which are linked by the only disulfide bond in bPrP. Significantly increased disorder and mobility is observed for helix 1, the loop 166–172 leading from the β-strand 2 to helix 2, the end of helix 2 and the following loop, and the last turn of helix 3. Although there are characteristic local differences relative to the conformations of the murine and Syrian hamster prion proteins, the bPrP structure is essentially identical to that of the human prion protein. On the other hand, there are differences between bovine and human PrP in the surface distribution of electrostatic charges, which then appears to be the principal structural feature of the “healthy” PrP form that might affect the stringency of the species barrier for transmission of prion diseases between humans and cattle.

NMR structures of three single-residue variants of the human prion protein

The NMR structures of three single-amino acid variants of the C-terminal domain of the human prion protein, hPrP(121–230), are presented. In hPrP(M166V) and hPrP(R220K) the substitution is with the corresponding residue in murine PrP, and in hPrP(S170N) it is with the corresponding Syrian hamster residue. All three substitutions are in the surface region of the structure of the cellular form of PrP (PrPC) that is formed by the C-terminal part of helix 3, with residues 218–230, and a loop of residues 166–172. This molecular region shows high species variability and has been implicated in specific interactions with a so far not further characterized “protein X,” and it is related to the species barrier for transmission of prion diseases. As expected, the three variant hPrP(121–230) structures have the same global architecture as the previously determined wild-type bovine, human, murine, and Syrian hamster prion proteins, but with the present study two localized “conformational markers” could be related with single amino acid exchanges. These are the length and quality of definition of helix 3, and the NMR-observability of the residues in the loop 166–172. Poor definition of the C-terminal part of helix 3 is characteristic for murine PrP and has now been observed also for hPrP(R220K), and NMR observation of the complete loop 166–172 has so far been unique for Syrian hamster PrP and is now also documented for hPrP(S170N).

Elucidating the mechanism of familial amyloidosis– Finnish type: NMR studies of human gelsolin domain 2

Familial amyloidosis–Finnish type (FAF) results from a single mutation at residue 187 (D187N or D187Y) within domain 2 of the actin-regulating protein gelsolin. The mutation somehow allows a masked cleavage site to be exposed, leading to the first step in the formation of an amyloidogenic fragment. We have performed NMR experiments investigating structural and dynamic changes between wild-type (WT) and D187N gelsolin domain 2 (D2). On mutation, no significant structural or dynamic changes occur at or near the cleavage site. Areas in conformational exchange are observed between β-strand 4 and α-helix 1 and within the loop region following β-strand 5. Chemical shift differences are noted along the face of α-helix 1 that packs onto the β-sheet, suggesting an altered conformation. Conformational changes within these areas can have an effect on actin binding and may explain why D187N gelsolin is inactive. {1H-15N} nuclear Overhauser effect and chemical shift data suggest that the C-terminal tail of D187N gelsolin D2 is less structured than WT by up to six residues. In the crystal structure of equine gelsolin, the C-terminal tail of D2 lies across a large cleft between domains 1 and 2 where the masked cleavage site sits. We propose that the D187N mutation destabilizes the C-terminal tail of D2 resulting in a more exposed cleavage site leading to the first proteolysis step in the formation of the amyloidogenic fragment.

Three-dimensional domain swapping in p13suc1 occurs in the unfolded state and is controlled by conserved proline residues

p13suc1 has two native states, a monomer and a domain-swapped dimer. We show that their folding pathways are connected by the denatured state, which introduces a kinetic barrier between monomer and dimer under native conditions. The barrier is lowered under conditions that speed up unfolding, thereby allowing, to our knowledge for the first time, a quantitative dissection of the energetics of domain swapping. The monomer–dimer equilibrium is controlled by two conserved prolines in the hinge loop that connects the exchanging domains. These two residues exploit backbone strain to specifically direct dimer formation while preventing higher-order oligomerization. Thus, the loop acts as a loaded molecular spring that releases tension in the monomer by adopting its alternative conformation in the dimer. There is an excellent correlation between domain swapping and aggregation, suggesting they share a common mechanism. These insights have allowed us to redesign the domain-swapping propensity of suc1 from a fully monomeric to a fully dimeric protein.

A single G-to-C change causes human centromere TGGAA repeats to fold back into hairpins.

Recently, we established that satellite III (TGGAA)n tandem repeats, which occur at the centromeres of human chromosomes, pair with themselves to form an unusual "self-complementary" antiparallel duplex containing (GGA)2 motifs in which two unpaired guanines from opposite strands intercalate between sheared G.A base pairs. In separate studies, we have also established that the GCA triplet does not form bimolecular (GCA)2 motifs but instead promotes the formation of hairpins containing a GCA-turn motif in which the loop contains a single cytidine closed by a sheared G.A pair. Since TGCAA is the most frequent variant of TGGAA found in satellite III repeats, we reasoned that the potential of this variant to form GCA-turn miniloop fold-back structures might be an important factor in modulating the local structure in natural (TGGAA)n repeats. We report here the NMR-derived solution structure of the heptadecadeoxynucleotide (G)TGGAATGCAATGGAA(C) in which a central TGCAA pentamer is flanked by two TGGAA pentamers. This 17-mer forms a rather unusual and very stable hairpin structure containing eight base pairs in the stem, only four of which are Watson-Crick pairs, and a loop consisting of a single cytidine residue. The stem contains a (GGA)2 motif with intercalative 14G/4G stacking between two sheared G.A base pairs; the loop end of the stem consists of a sheared 8G.10A closing pair with the cytosine base of the 9C loop stacked on 8G. The remarkable stability of this unusual hairpin structure (Tm = 63 degrees C) suggests that it probably plays an important role in modulating the folding of satellite III (TGGAA)n repeats at the centromere.

A modified gambler's ruin model of polyethylene chains in the amorphous region.

Polyethylene chains in the amorphous region between two crystalline lamellae M unit apart are modeled as random walks with one-step memory on a cubic lattice between two absorbing boundaries. These walks avoid the two preceding steps, though they are not true self-avoiding walks. Systems of difference equations are introduced to calculate the statistics of the restricted random walks. They yield that the fraction of loops is (2M - 2)/(2M + 1), the fraction of ties 3/(2M + 1), the average length of loops 2M - 0.5, the average length of ties 2/3M2 + 2/3M - 4/3, the average length of walks equals 3M - 3, the variance of the loop length 16/15M3 + O(M2), the variance of the tie length 28/45M4 + O(M3), and the variance of the walk length 2M3 + O(M2).

Molecular basis for dysfunction of some mutant forms of methylmalonyl-CoA mutase: deductions from the structure of methionine synthase.

Inherited defects in the gene for methylmalonyl-CoA mutase (EC 5.4.99.2) result in the mut forms of methylmalonic aciduria. mut- mutations lead to the absence of detectable mutase activity and are not corrected by excess cobalamin, whereas mut- mutations exhibit residual activity when exposed to excess cobalamin. Many of the mutations that cause methylmalonic aciduria in humans affect residues in the C-terminal region of the methylmalonyl-CoA mutase. This portion of the methylmalonyl-CoA mutase sequence can be aligned with regions in other B12 (cobalamin)-dependent enzymes, including the C-terminal portion of the cobalamin-binding region of methionine synthase. The alignments allow the mutations of human methylmalonyl-CoA mutase to be mapped onto the structure of the cobalamin-binding fragment of methionine synthase from Escherichia coli (EC 2.1.1.13), which has recently been determined by x-ray crystallography. In this structure, the dimethylbenzimidazole ligand to the cobalt in free cobalamin has been displaced by a histidine ligand, and the dimethylbenzimidazole nucleotide "tail" is thrust into a deep hydrophobic pocket in the protein. Previously identified mut0 and mut- mutations (Gly-623 --> Arg, Gly-626 --> Cys, and Gly-648 --> Asp) of the mutase are predicted to interfere with the structure and/or stability of the loop that carries His-627, the presumed lower axial ligand to the cobalt of adenosylcobalamin. Two mutants that lead to severe impairment (mut0) are Gly-630 --> Glu and Gly-703 --> Arg, which map to the binding site for the dimethylbenzimidazole nucleotide substituent of adenosylcobalamin. The substitution of larger residues for glycine is predicted to block the binding of adenosylcobalamin.

The wing of the enhancer-binding domain of Mu phage transposase is flexible and is essential for efficient transposition.

A tetramer of the Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Juxtaposition of the recombination sites is accomplished by the assembly of a stable synaptic complex of MuA protein and Mu DNA. This initial critical step is facilitated by the transient binding of the N-terminal domain of MuA to an enhancer DNA element within the Mu genome (called the internal activation sequence, IAS). Recently we solved the three-dimensional solution structure of the enhancer-binding domain of Mu phage transposase (residues 1-76, MuA76) and proposed a model for its interaction with the IAS element. Site-directed mutagenesis coupled with an in vitro transposition assay has been used to assess the validity of the model. We have identified five residues on the surface of MuA that are crucial for stable synaptic complex formation but dispensable for subsequent events in transposition. These mutations are located in the loop (wing) structure and recognition helix of the MuA76 domain of the transposase and do not seriously perturb the structure of the domain. Furthermore, in order to understand the dynamic behavior of the MuA76 domain prior to stable synaptic complex formation, we have measured heteronuclear 15N relaxation rates for the unbound MuA76 domain. In the DNA free state the backbone atoms of the helix-turn-helix motif are generally immobilized whereas the residues in the wing are highly flexible on the pico- to nanosecond time scale. Together these studies define the surface of MuA required for enhancement of transposition in vitro and suggest that a flexible loop in the MuA protein required for DNA recognition may become structurally ordered only upon DNA binding.

Construction and characterization of an azurin analog for the purple copper site in cytochrome c oxidase.

A protein analog of a purple copper center has been constructed from a recombinant blue copper protein (Pseudomonas aeruginosa azurin) by replacing the loop containing the three ligands to the blue copper center with the corresponding loop of the CuA center in cytochrome c oxidase (COX) from Paracoccus denitrificans. The electronic absorption in the UV and visible region (UV-vis) and electron paramagnetic resonance (EPR) spectra of this analog are remarkably similar to those of the native CuA center in COX from Paracoccus denitrificans. The above spectra can be obtained upon addition of a mixture of Cu2+ and Cu+. Addition of Cu2+ only results in a UV-vis spectrum consisting of absorptions from both a purple copper center and a blue copper center. This spectrum can be converted to the spectrum of a pure purple copper by a prolonged incubation in the air, or by addition of excess ascorbate. The azurin mutant reported here is an example of an engineered purple copper center with the A480/A530 ratio greater than 1 and with no detectable hyperfines, similar to those of the CuA sites in COX of bovine heart and of Paracoccus denitrificans.

Complex flexibility of the transforming growth factor beta superfamily.

The transforming growth factors beta (TGF-beta s) are important modulators of growth and differentiation. They are intermolecular disulfide-bonded homodimeric molecules. The monomer fold has a conserved cystine knot and lacks a hydrophobic core. The biological specificity of a given member of the family is believed to be determined by the conformational flexibility of the variable loop regions of the monomer. The monomer subunit assembly in the dimer is stabilized mainly by hydrophobic contacts and a few hydrogen bonds. Since these interactions are nondirectional, we examined subunit assemblies of TGF-beta by using conformational analysis. The different subunit assemblies in TGF-beta 2 dimer were characterized in terms of the intersubunit disulfide torsion. Our analyses show that the subunit assemblies fall into two states: the crystallographically observed gauche+conformation and the previously not reported gauche--conformation, both having almost identical interaction energies. Furthermore, there is significant flexibility in the subunit assembly within the gauche+ and the gauche- states of the disulfide bond. The monomer subunit assembly is independent of the variations about the loop regions. The variations in the loop regions, coupled with flexibility in the monomer assembly, lead to a complex flexibility in the dimer of the TGF-beta superfamily. For the TGF-beta superfamily, the cystine knot acts as a scaffold and complex flexibility provides for biological selectivity. Complex flexibility might provide an explanation for the diverse range of biological activities that these important molecules display.

Swapping structural determinants of ribonucleases: an energetic analysis of the hinge peptide 16-22.

Bovine seminal ribonuclease (BS-RNase) is a homodimeric enzyme strictly homologous to the pancreatic ribonuclease (RNase A). Native BS-RNase is an equilibrium mixture of two distinct dimers differing in the interchange of the N-terminal segments and in their biological properties. The loop 16-22 plays a fundamental role on the relative stability of the two isomers. Both the primary and tertiary structures of the RNase A differ substantially from those of the seminal ribonuclease in the loop region 16-22. To analyze the possible stable conformations of this loop in both enzymes, structure predictions have been attempted, according to a procedure described by Palmer and Scheraga [Palmer, K. A. & Scheraga, H. A. (1992) J. Comput. Chem. 13, 329-350]. Results compare well with experimental x-ray structures and clarify the structural determinants that are responsible for the swapping of the N-terminal domains and for the peculiar properties of BS-RNase. Minimal modifications of RNase A sequence needed to form a stable swapped dimer are also predicted.

Imperial legacy? The EU's Developmental Model and the Crisis of the European Periphery. CES Papers - Open Forum #19, 2013-2014

The high hopes for rapid convergence of Eastern and Southern EU member states are increasingly being disappointed. With the onset of the Eurocrisis convergence has given way to divergence in the southern members, and many Eastern members have made little headway in closing the development gap. The EU´s performance compares unfavourably with East Asian success cases as well as with Western Europe´s own rapid catch-up to the USA after 1945. Historical experience indicates that successful catch up requires that less-developed economies to some extent are allowed to free-ride on an open international economic order. However, the EU´s model is based on the principle of a level-playing field, which militates against such a form of economic integration. The EU´s developmental model thus contrasts with the various strategies that have enabled successful catch up of industrial latecomers. Instead the EU´s current approach is more and more reminiscent of the relations between the pre-1945 European empires and their dependent territories. One reason for this unfortunate historical continuity is that the EU appears to have become entangled in its own myths. In the EU´s own interpretation, European integration is a peace project designed to overcome the almost continuous warfare that characterised the Westphalian system. As the sovereign state is identified as the root cause of all evil, any project to curtail its room of manoeuvre must ultimately benefit the common good. Yet, the existence of a Westphalian system of nation states is a myth. Empires and not states were the dominant actors in the international system for at least the last three centuries. If anything, the dawn of the age of the sovereign state in Western Europe occurred after 1945 with the disintegration of the colonial empires and thus historically coincided with the birth of European integration.

Evidence-based medicine is affordable: The cost-effectiveness of current compared with optimal treatment in rheumatoid and osteoarthritis

Objective. To determine the cost-effectiveness of averting the burden of disease. We used secondary population data and metaanalyses of various government-funded services and interventions to investigate the costs and benefits of various levels of treatment for rheumatoid arthritis (RA) and osteoarthritis (OA) in adults using a burden of disease framework. Method. Population burden was calculated for both diseases in the absence of any treatment as years lived with disability (YLD), ignoring the years of life lost. We then estimated the proportion of burden averted with current interventions, the proportion that could be averted with optimally implemented cut-rent evidence-based guidelines, and the direct treatment cost-effectiveness ratio in dollars per YLD averted for both treatment levels. Results. The majority of people with arthritis sought medical treatment. Current treatment for RA averted 26% of the burden, with a cost-effectiveness ratio of $19,000 per YLD averted. Optimal, evidence-based treatment would avert 48% of the burden. with a cost-effectiveness ratio of $12,000 per YLD averted. Current treatment of OA in Australia averted 27% of the burden, with a cost-effectiveness ratio of $25,000 per YLD averted. Optimal, evidence-based treatment would avert 39% of the burden, with an unchanged cost-effectiveness ratio of $25,000 per YLD averted. Conclusion. While the precise dollar costs in each country will differ, the relativities at this level of coverage should remain the same. There is no evidence that closing the gap between evidence and practice would result in a drop in efficiency.

Molecular dynamics simulations of the hydrophobin SC3 at a hydrophobic/hydrophilic interface

Hydrophobins are small (similar to 100 aa) proteins that have an important role in the growth and development of mycelial fungi. They are surface active and, after secretion by the fungi, self-assemble into amphipathic membranes at hydrophobic/hydrophilic interfaces, reversing the hydrophobicity of the surface. In this study, molecular dynamics simulation techniques have been used to model the process by which a specific class I hydrophobin, SC3, binds to a range of hydrophobic/ hydrophilic interfaces. The structure of SC3 used in this investigation was modeled based on the crystal structure of the class II hydrophobin HFBII using the assumption that the disulfide pairings of the eight conserved cysteine residues are maintained. The proposed model for SC3 in aqueous solution is compact and globular containing primarily P-strand and coil structures. The behavior of this model of SC3 was investigated at an air/water, an oil/water, and a hydrophobic solid/water interface. It was found that SC3 preferentially binds to the interfaces via the loop region between the third and fourth cysteine residues and that binding is associated with an increase in a-helix formation in qualitative agreement with experiment. Based on a combination of the available experiment data and the current simulation studies, we propose a possible model for SC3 self-assembly on a hydrophobic solid/water interface.

Functional split and crosslinking of the membrane domain of the β subunit of proton-translocating transhydrogenase from Escherichia coli

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an α subunit with the NAD(H)-binding domain I and a β subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the α and β subunits. The interface in domain II between the α and the β subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the α subunit and loops connecting the nine transmembrane helices in the β subunit. However, to investigate the organization of the nine transmembrane helices in the β subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type α subunit and the two new peptides β1 and β2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD + by NADPH, the cyclic reduction of 3-acetylpyridine-NAD + by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities. These high activities suggest that the α subunit was normally folded, followed by a concerted folding of β1 + β2. Cross-linking of a βS105C-βS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same β subunit has been demonstrated.