Ansgar B. Siemer

Amyloid Fibrils of the HET-s(218–289) Prion Form a β Solenoid with a Triangular Hydrophobic Core

Prion and nonprion forms of proteins are believed to differ solely in their three-dimensional structure, which is therefore of paramount importance for the prion function. However, no atomic-resolution structure of the fibrillar state that is likely infectious has been reported to date. We present a structural model based on solid-state nuclear magnetic resonance restraints for amyloid fibrils from the prion-forming domain (residues 218 to 289) of the HET-s protein from the filamentous fungus Podospora anserina. On the basis of 134 intra- and intermolecular experimental distance restraints, we find that HET-s(218–289) forms a left-handed β solenoid, with each molecule forming two helical windings, a compact hydrophobic core, at least 23 hydrogen bonds, three salt bridges, and two asparagine ladders. The structure is likely to have broad implications for understanding the infectious amyloid state.

Correlation of structural elements and infectivity of the HET-s prion

Prions are believed to be infectious, self-propagating polymers of otherwise soluble, host-encoded proteins. This concept is now strongly supported by the recent findings that amyloid fibrils of recombinant prion proteins from yeast, Podospora anserina and mammals can induce prion phenotypes in the corresponding hosts. However, the structural basis of prion infectivity remains largely elusive because acquisition of atomic resolution structural properties of amyloid fibrils represents a largely unsolved technical challenge. HET-s, the prion protein of P. anserina, contains a carboxy-terminal prion domain comprising residues 218–289. Amyloid fibrils of HET-s(218–289) are necessary and sufficient for the induction and propagation of prion infectivity. Here, we have used fluorescence studies, quenched hydrogen exchange NMR and solid-state NMR to determine the sequence-specific positions of amyloid fibril secondary structure elements of HET-s(218–289). This approach revealed four β-strands constituted by two pseudo-repeat sequences, each forming a β-strand-turn-β-strand motif. By using a structure-based mutagenesis approach, we show that this conformation is the functional and infectious entity of the HET-s prion. These results correlate distinct structural elements with prion infectivity.

Fluoroalcohol-induced structural changes of proteins: some aspects of cosolvent-protein interactions

Abstract. The conformational transitions of bovine β-lactoglobulin A and phosphoglycerate kinase from yeast induced by hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) have been studied by dynamic light scattering and circular dichroism spectroscopy in order to elucidate the potential of fluoroalcohols to bring about structural changes of proteins. Moreover, pure fluoroalcohol-water mixed solvents were investigated to prove the relation between cluster formation and the effects on proteins. The results demonstrate that cluster formation is mostly an accompanying phenomenon because important structural changes of the proteins occur well below the critical concentration of fluoroalcohol at which the formation of clusters sets in. According to our light scattering experiments, the remarkable potential of HFIP is a consequence of extensive preferential binding. Surprisingly, preferential binding seems to play a vanishing role in the case of TFE. However, the comparable Stokes radii of both proteins in the highly helical state induced by either HFIP or TFE point to a similar degree of solvation in both mixed solvents. This shows that direct binding or an indirect mechanism must be equally taken into consideration to explain the effects of alcohols on proteins. The existence of a compact helical intermediate with non-native secondary structure on the transition of β-lactoglobulin A from the native to the highly helical state is clearly demonstrated.

Protein–ice interaction of an antifreeze protein observed with solid-state NMR

NMR on frozen solutions is an ideal method to study fundamental questions of macromolecular hydration, because the hydration shell of many biomolecules does not freeze together with bulk solvent. In the present study, we present previously undescribed NMR methods to study the interactions of proteins with their hydration shell and the ice lattice in frozen solution. We applied these methods to compare solvent interaction of an ice-binding type III antifreeze protein (AFP III) and ubiquitin a non-ice-binding protein in frozen solution. We measured 1H-1H cross-saturation and cross-relaxation to provide evidence for a molecular contact surface between ice and AFP III at moderate freezing temperatures of -35 °C. This phenomenon is potentially unique for AFPs because ubiquitin shows no such cross relaxation or cross saturation with ice. On the other hand, we detected liquid hydration water and strong water–AFP III and water–ubiquitin cross peaks in frozen solution using relaxation filtered 2H and HETCOR spectra with additional 1H-1H mixing. These results are consistent with the idea that ubiquitin is surrounded by a hydration shell, which separates it from the bulk ice. For AFP III, the water cross peaks indicate that only a portion of its hydration shell (i.e., at the ice-binding surface) is in contact with the ice lattice. The rest of AFP III’s hydration shell behaves similarly to the hydration shell of non-ice-interacting proteins such as ubiquitin and does not freeze together with the bulk water.

Protein linewidth and solvent dynamics in frozen solution NMR.

Solid-state NMR of proteins in frozen aqueous solution is a potentially powerful technique in structural biology, especially if it is combined with dynamic nuclear polarization signal enhancement strategies. One concern regarding NMR studies of frozen solution protein samples at low temperatures is that they may have poor linewidths, thus preventing high-resolution studies. To learn more about how the solvent shell composition and temperature affects the protein linewidth, we recorded 1H, 2H, and 13C spectra of ubiquitin in frozen water and frozen glycerol-water solutions at different temperatures. We found that the 13C protein linewidths generally increase with decreasing temperature. This line broadening was found to be inhomogeneous and independent of proton decoupling. In pure water, we observe an abrupt line broadening with the freezing of the bulk solvent, followed by continuous line broadening at lower temperatures. In frozen glycerol-water, we did not observe an abrupt line broadening and the NMR lines were generally narrower than for pure water at the same temperature. 1H and 2H measurements characterizing the dynamics of water that is in exchange with the protein showed that the 13C line broadening is relatively independent from the arrest of isotropic water motions.

Characterization of prion-like conformational changes of the neuronal isoform of Aplysia CPEB

The neuronal isoform of cytoplasmic polyadenylation element–binding protein (CPEB) is a regulator of local protein synthesis at synapses and is critical in maintaining learning-related synaptic plasticity in Aplysia. Previous studies indicate that the function of Aplysia CPEB can be modulated by conversion to a stable prion-like state, thus contributing to the stabilization of long-term memory on a molecular level. Here, we used biophysical methods to demonstrate that Aplysia CPEB, like other prions, undergoes a conformational switch from soluble α-helix–rich oligomer to β-sheet–rich fiber in vitro. Solid-state NMR analyses of the fibers indicated a relatively rigid N-terminal prion domain. The fiber form of Aplysia CPEB showed enhanced binding to target mRNAs as compared to the soluble form. Consequently, we propose a model for the Aplysia CPEB fibers that may have relevance for functional prions in general.Although significant knowledge of cellular and molecular mechanisms underlying the acquisition and early storage of implicit and explicit long-term memory has been gained, the mechanisms by which memories are maintained for long periods of time are still not fully understood. Because proteins normally have relatively short half-lives, of hours or days, the question remains: How can the change in molecular composition of a synapse be maintained for long periods of time, as is required for long-term memory? We previously found one answer to this conundrum in a work describing a prion-like regulator of local protein synthesis at the synapse in the marine snail Aplysia californica: the cytoplasmic polyadenylation element–binding protein Aplysia CPEB. This provided physiological evidence that the prion-like properties of Aplysia CPEB might explain the self-sustained, continuous molecular turnover at the synapse.

Solid-State NMR on a Type III Antifreeze Protein in the Presence of Ice

Antifreeze proteins (AFPs) are found in fish, insects, plants, and a variety of other organisms where they serve to prevent the growth of ice at subzero temperatures. Type III AFPs cloned from polar fishes have been studied extensively with X-ray crystallography, liquid-state NMR, and site directed mutagenesis and are, therefore, among the best characterized AFPs. A flat surface on the protein has previously been proposed to be the ice-binding site of type III AFP. The detailed nature of the ice binding remains controversial since it is not clear whether only polar or also hydrophobic residues are involved in ice binding and there is no structural information available of a type III AFP bound to ice. Here we present a high-resolution solid-state NMR study of a type III AFP (HPLC-12 isoform) in the presence of ice. The chemical-shift differences we detected between the frozen and the nonfrozen state agree well with the proposed ice-binding site. Furthermore, we found that the (1)H T(1) of HPLC-12 in frozen solution is very long compared to typical (1)H of proteins in the solid state as for example of ubiquitin in frozen solution.

Homonuclear Mixing Sequences for Perdeuterated Proteins

We tested the performance of several (13)C homonuclear mixing sequences on perdeuterated microcrystalline ubiquitin. All sequences were applied without (1)H decoupling and at relatively low MAS frequencies. We found that RFDR gave the highest overall transfer efficiency and that DREAM performs surprisingly well under these conditions being twice as efficient in the aliphatic region of the spectrum than the other mixing sequences tested.

Solid-State Nuclear Magnetic Resonance on the Static and Dynamic Domains of Huntingtin Exon-1 Fibrils.

Amyloid-like fibrils formed by huntingtin exon-1 (htt_ex1) are a hallmark of Huntingtons disease (HD). The structure of these fibrils is unknown, and determining their structure is an important step toward understanding the misfolding processes that cause HD. In HD, a polyglutamine (polyQ) domain in htt_ex1 is expanded to a degree that it gains the ability to form aggregates comprising the core of the resulting fibrils. Despite the simplicity of this polyQ sequence, the structure of htt_ex1 fibrils has been difficult to determine. This study provides a detailed structural investigation of fibrils formed by htt_ex1 using solid-state nuclear magnetic resonance (NMR) spectroscopy. We show that the polyQ domain of htt_ex1 forms the static amyloid core similar to polyQ model peptides. The Gln residues of this domain exist in two distinct conformations that are found in separate domains or monomers but are relatively close in space. The rest of htt_ex1 is relatively dynamic on an NMR time scale, especially the proline-rich C-terminus, which we found to be in a polyproline II helical and random coil conformation. We observed a similar dynamic C-terminus in a soluble form of htt_ex1, indicating that the conformation of this part of htt_ex1 is not changed upon its aggregation into an amyloid fibril. From these data, we propose a bottlebrush model for the fibrils formed by htt_ex1. In this model, the polyQ domains form the center and the proline-rich domains the bristles of the bottlebrush.

Identification and Structural Characterization of the N-terminal Amyloid Core of Orb2 isoform A

Orb2 is a functional amyloid that plays a key role in Drosophila long-term memory formation. Orb2 has two isoforms that differ in their N-termini. The N-terminus of the A isoform (Orb2A) that precedes its Q-rich prion-like domain has been shown to be important for Orb2 aggregation and long-term memory. However, besides the fact that it forms fibrillar aggregates, structural information of Orb2 is largely absent. To understand the importance of the N-terminus of Orb2A and its relation to the fibril core, we recorded solid-state NMR and EPR data on fibrils formed by the first 88 residues of Orb2A (Orb2A88). These data show that the N-terminus of Orb2A not only promotes the formation of fibrils, but also forms the fibril core of Orb2A88. This fibril core has an in-register parallel β-sheet structure and does not include the Q-rich, prion-like domain of Orb2. The Q-rich domain is part of the unstructured region, which becomes increasingly dynamic towards the C-terminus.