Jason Greenwald

Biology of Amyloid: Structure, Function, and Regulation

Amyloids are highly ordered cross-β sheet protein aggregates associated with many diseases including Alzheimers disease, but also with biological functions such as hormone storage. The cross-β sheet entity comprising an indefinitely repeating intermolecular β sheet motif is unique among protein folds. It grows by recruitment of the corresponding amyloid protein, while its repetitiveness can translate what would be a nonspecific activity as monomer into a potent one through cooperativity. Furthermore, the one-dimensional crystal-like repeat in the amyloid provides a structural framework for polymorphisms. This review summarizes the recent high-resolution structural studies of amyloid fibrils in light of their biological activities. We discuss how the unique properties of amyloids gives rise to many activities and further speculate about currently undocumented biological roles for the amyloid entity. In particular, we propose that amyloids could have existed in a prebiotic world, and may have been the first functional protein fold in living cells.

Protocols for the Sequential Solid‐State NMR Spectroscopic Assignment of a Uniformly Labeled 25 kDa Protein: HET‐s(1‐227)

The sequence‐specific resonance assignment of a protein forms the basis for studies of molecular structure and dynamics, as well as to functional assay studies by NMR spectroscopy. Here we present a protocol for the sequential 13C and 15N resonance assignment of uniformly [15N,13C]‐labeled proteins, based on a suite of complementary three‐dimensional solid‐state NMR spectroscopy experiments. It is directed towards the application to proteins with more than about 100 amino acid residues. The assignments rely on a walk along the backbone by using a combination of three experiments that correlate nitrogen and carbon spins, including the well‐dispersed Cβ resonances. Supplementary spectra that correlate further side‐chain resonances can be important for identifying the amino acid type, and greatly assist the assignment process. We demonstrate the application of this assignment protocol for a crystalline preparation of the N‐terminal globular domain of the HET‐s prion, a 227‐residue protein.

Structure–activity relationship of amyloid fibrils

Protein aggregation is a process in which proteins self‐associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as either amorphous or highly ordered, the most common form of the latter being amyloid fibrils. Amyloid fibrils composed of cross‐β‐sheet structure are the pathological hallmarks of several diseases including Alzheimers disease, but are also associated with functional states such as the fungal HET‐s prion. This review aims to summarize the recent high‐resolution structural studies of amyloid fibrils in light of their (potential) activities. We propose that the repetitive nature of the cross‐β‐sheet structure of amyloids is key for their multiple properties: the repeating motifs can translate a rather non‐specific interaction into a specific one through cooperativity.

The Molecular Organization of the Fungal Prion HET-s in Its Amyloid Form

The prion hypothesis states that it is solely the three-dimensional structure of the polypeptide chain that distinguishes the prion and nonprion forms of the protein. For HET-s, the atomic-resolution structure of the isolated prion domain HET-s(218-289), consisting of a highly ordered triangular cross-beta arrangement, is known. Here we present a solid-state NMR study of fibrils of the full-length HET-s prion in which we compare their spectra with spectra from isolated C-terminal prion domain fibrils and the crystalline N-terminal globular domain HET-s(1-227). The spectra reveal unequivocally that the highly ordered structure of the isolated prion domain HET-s(218-289) is conserved in the context of the full-length fibrils investigated here. However, the globular domain loses much of its tertiary structure while partly retaining its secondary structure, thus exhibiting behavior reminiscent of a molten globule. Flexible residues that may constitute the linker connecting the two domains are detected using INEPT (insensitive nuclei enhanced by polarization transfer) spectroscopy. Based on our data, we propose a structural model that is in line with a general model developed for amyloid fibrils built from a cross-beta core decorated with globular domains. The loss of structure in the HET-s globular domain sharply contrasts with the behavior observed for fibrils of Ure2p and suggests that there is considerable structural diversity in the fibrils of globular-domain-containing prions despite their similar appearances at the microscopic level.

The Mechanism of Toxicity in HET-S/HET-s Prion Incompatibility

A nontoxic functional prion activates toxicity in the HET-S/HET-s fungal heterokaryon incompatibility system by converting HET-S into a cytotoxic membrane protein.

On the possible amyloid origin of protein folds.

The diversity of protein folds is derived from the diversity of the underlying proteome. Such diversity must have originated from a so-called common ancestor: a hypothetical fold whose identity will, in all likelihood, never be known. Nonetheless, hypotheses exist to explain the evolution of protein folds. When formulating such hypotheses as done here, the entire repertoire of polypeptide structure, from well-defined tertiary structures and molten globule states to intrinsically disordered proteins and oligomeric aggregates, is worth considering. It is the aim of this short essay to discuss the hypothesis that one type of protein aggregate-the cross-β-sheet motif-was the first functional protein fold, that is, the common ancestor fold. Support for this hypothesis comes from the observations that (i) short peptides with simple amino acid sequences are able to form the cross-β-sheet structure, (ii) amyloids can be very stable under harsh conditions, (iii) amyloids can self-assemble in complex mixtures, (iv) amyloids have many potent activities that are attributable to the inherent repetitiveness of the structure, and (v) the proteomes of modern organisms appear to have evolved away from the more amyloidogenic sequences of older organisms, suggesting that amyloids were more ubiquitous earlier in the evolution of modern protein folds.

Narrowing SWNT diameter distribution using size-separated ferritin-based Fe catalysts.

Sensors and devices made from single-walled carbon nanotubes (SWNTs) are most often electrically probed through metal leads contacting the semiconducting SWNTs (s-SWNTs). Contact barriers in general and Schottky barriers (SBs) in particular are usually obtained at a metal-semiconductor interface. The unique one-dimensional structure (1D) of SWNTs allows tailoring of the SB heights through the contact metal type and the size of the s-SWNT bandgap. A large workfunction reduces the SB height (e.g. using Pd as the metal contact material). The bandgap of an SWNT is inversely proportional to its diameter. Ohmic contacts--the preferable choice--are achieved for s-SWNTs with diameters greater than 2 nm on Pd metal leads. SWNT device reproducibility, on the other hand, requires a narrow distribution of the SWNT diameters. Here, we present a method to fabricate SWNTs with a large and adjustable mean diameter (1.9-2.4 nm) and very narrow diameter distribution (+/- 0.27 nm at mean diameter 1.9 nm). The results are achieved through a size separation of the ferritin catalyst particles by sedimentation velocity centrifugation prior to their use in the chemical vapor deposition (CVD) formation of SWNTs.

Amyloid Aggregates Arise from Amino Acid Condensations under Prebiotic Conditions

Current theories on the origin of life reveal significant gaps in our understanding of the mechanisms that allowed simple chemical precursors to coalesce into the complex polymers that are needed to sustain life. The volcanic gas carbonyl sulfide (COS) is known to catalyze the condensation of amino acids under aqueous conditions, but the reported di-, tri-, and tetra-peptides are too short to support a regular tertiary structure. Here, we demonstrate that alanine and valine, two of the proteinogenic amino acids believed to have been among the most abundant on a prebiotic earth, can polymerize into peptides and subsequently assemble into ordered amyloid fibers comprising a cross-β-sheet quaternary structure following COS-activated continuous polymerization of as little as 1u2005mm amino acid. Furthermore, this spontaneous assembly is not limited to pure amino acids, since mixtures of glycine, alanine, aspartate, and valine yield similar structures.

Towards Prebiotic Catalytic Amyloids Using High Throughput Screening

Enzymes are capable of directing complex stereospecific transformations and of accelerating reaction rates many orders of magnitude. As even the simplest known enzymes comprise thousands of atoms, the question arises as to how such exquisite catalysts evolved. A logical predecessor would be shorter peptides, but they lack the defined structure and size that are apparently necessary for enzyme functions. However, some very short peptides are able to assemble into amyloids, thereby forming a well-defined tertiary structure called the cross-β-sheet, which bestows unique properties upon the peptides. We have hypothesized that amyloids could have been the catalytically active precursor to modern enzymes. To test this hypothesis, we designed an amyloid peptide library that could be screened for catalytic activity. Our approach, amenable to high-throughput methodologies, allowed us to find several peptides and peptide mixtures that form amyloids with esterase activity. These results indicate that amyloids, with their stability in a wide range of conditions and their potential as catalysts with low sequence specificity, would indeed be fitting precursors to modern enzymes. Furthermore, our approach can be efficiently expanded upon in library size, screening conditions, and target activity to yield novel amyloid catalysts with potential applications in aqueous-organic mixtures, at high temperature and in other extreme conditions that could be advantageous for industrial applications.

Crystal Structures of T. b. rhodesiense Adenosine Kinase Complexed with Inhibitor and Activator: Implications for Catalysis and Hyperactivation

Background The essential purine salvage pathway of Trypanosoma brucei bears interesting catalytic enzymes for chemotherapeutic intervention of Human African Trypanosomiasis. Unlike mammalian cells, trypanosomes lack de novo purine synthesis and completely rely on salvage from their hosts. One of the key enzymes is adenosine kinase which catalyzes the phosphorylation of ingested adenosine to form adenosine monophosphate (AMP) utilizing adenosine triphosphate (ATP) as the preferred phosphoryl donor. Methods and Findings Here, we present the first structures of Trypanosoma brucei rhodesiense adenosine kinase (TbrAK): the structure of TbrAK in complex with the bisubstrate inhibitor P1,P5-di(adenosine-5′)-pentaphosphate (AP5A) at 1.55 Å, and TbrAK complexed with the recently discovered activator 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) at 2.8 Å resolution. Conclusions The structural details and their comparison give new insights into substrate and activator binding to TbrAK at the molecular level. Further structure-activity relationship analyses of a series of derivatives of compound 1 support the observed binding mode of the activator and provide a possible mechanism of action with respect to their activating effect towards TbrAK.