The structural landscape of the immunoglobulin fold by large-scale de novo design.

De novo designing immunoglobulin-like frameworks that allow for functional loop diversification shows great potential for crafting antibody-like scaffolds with fully customizable structures and functions. In this work, we combined de novo parametric design with deep-learning methods for protein structure prediction and design to explore the structural landscape of 7-stranded immunoglobulin domains. After screening folding of nearly 4 million designs, we have assembled a structurally diverse library of ~50,000 immunoglobulin domains with high-confidence AlphaFold2 predictions and structures diverging from naturally occurring ones. The designed dataset enabled us to identify structural requirements for the correct folding of immunoglobulin domains, shed light on ß-sheet-ß-sheet rotational preferences and how these are linked to functional properties. Our approach eliminates the need for preset loop conformations and opens the route to large-scale de novo design of immunoglobulin-like frameworks.

Intermediate antiparallel beta structure in amyloid plaques revealed by infrared spectroscopic imaging.

Aggregation of amyloid beta (Aß) peptides into extracellular plaques is a hallmark of the molecular pathology of Alzheimer's disease (AD). Amyloid aggregates have been extensively studied in-vitro, and it is well known that mature amyloid fibrils contain an ordered parallel ß structure. The structural evolution from unaggregated peptide to fibrils can be mediated through intermediate structures that deviate significantly from mature fibrils, such as antiparallel ß-sheets. However, it is currently unknown if these intermediate structures exist in plaques, which limits the translation of findings from in-vitro structural characterizations of amyloid aggregates to AD. This arises from the inability to extend common structural biology techniques to ex-vivo tissue measurements. Here we report the use of infrared (IR) imaging, wherein we can spatially localize plaques and probe their protein structural distributions with the molecular sensitivity of IR spectroscopy. Analyzing individual plaques in AD tissues, we demonstrate that fibrillar amyloid plaques exhibit antiparallel ß-sheet signatures, thus providing a direct connection between in-vitro structures and amyloid aggregates in AD brain. We further validate results with IR imaging of in-vitro aggregates and show that antiparallel ß-sheet structure is a distinct structural facet of amyloid fibrils.

Chain Sliding versus ß-Sheet Formation upon Shearing Single α-Helical Coiled Coils.

Coiled coils (CCs) are key building blocks of biogenic materials and determine their mechanical response to large deformations. Of particular interest is the observation that CC-based materials display a force-induced transition from α-helices to mechanically stronger ß-sheets (αßT). Steered molecular dynamics simulations predict that this αßT requires a minimum, pulling speed-dependent CC length. Here, de novo designed CCs with a length between four to seven heptads are utilized to probe if the transition found in natural CCs can be mimicked with synthetic sequences. Using single-molecule force spectroscopy and molecular dynamics simulations, these CCs are mechanically loaded in shear geometry and their rupture forces and structural responses to the applied load are determined. Simulations at the highest pulling speed (0.01 nm ns-1 ) show the appearance of ß-sheet structures for the five- and six-heptad CCs and a concomitant increase in mechanical strength. The αßT is less probable at a lower pulling speed of 0.001 nm ns-1 and is not observed in force spectroscopy experiments. For CCs loaded in shear geometry, the formation of ß-sheets competes with interchain sliding. ß-sheet formation is only possible in higher-order CC assemblies or in tensile-loading geometries where chain sliding and dissociation are prohibited.

Hydrogen Bond Surrogate-Constrained Dynamic Antiparallel ß-Sheets.

Antiparallel ß-sheets are important secondary structures within proteins that equilibrate with random-coil states; however, little is known about the exact dynamics of this process. Here, the first dynamic ß-sheet models that mimic this equilibrium have been designed by using an H-bond surrogate that introduces constraint and torque into a tertiary amide bond. 2D NMR data sufficiently reveal the structure, kinetics, and thermodynamics of the folding process, thereby leading the way to similar analysis in isolated biologically relevant ß-sheets.

A DFT study of structure and stability of pleated and rippled cross-ß sheets with hydrophobic sidechains.

The rippled cross-ß sheet, a topography, in which mirror-image peptides are arranged with alternating chirality into a periodic two-dimensional network, is burgeoning as a new design principle for materials and biomedical applications. Experiments by the Schneider, Nilsson, and Raskatov labs have independently shown diverse racemic mixtures of aggregation-prone peptide of different sizes to favor the rippled over the pleated topography. Yet, systematic ab initio studies are lacking, and the field is yet to develop rules that would enable the design of new rippled cross-ß frameworks from first principles. Here, DFT calculations were performed on a set of model systems, designed to begin understanding the impact that bulky, hydrophobic sidechains have upon the formation of pleated and rippled cross-ß frameworks. It is hoped that this study will help stimulate the development of a predictive, general framework to enable rational design of rippled cross-ß sheets in the future.

Pathways of electron transfer and proton translocation in the action of superoxide dismutase dimer.

Superoxide dismutase, known to gain large rate enhancement on dimerization, forms a homodimer stabilized by hydrogen bonding between a number of internal water molecules and a few amino acid residues at the interface. Within each subunit the ß-sheets provide a sequence of delocalized π-electron units of peptide bonds alternating with hydrogen bonds referred as π-H pathway. These pathways in the two subunits in the dimer are interlinked through a chain of four water molecules bridged by hydrogen bonds at the interface. Connecting the two Cu-centers this π-H pathway can enable rapid electron transfer from one superoxide molecule to the other, crucial for the catalytic reaction and the high rate in the dimer. A proton relay of hydrogen-bonded water molecules in the dimer translocates protons to form the product, hydrogen peroxide.

Yeast Prions Compared to Functional Prions and Amyloids.

Saccharomyces cerevisiae is an occasional host to an array of prions, most based on self-propagating, self-templating amyloid filaments of a normally soluble protein. [URE3] is a prion of Ure2p, a regulator of nitrogen catabolism, while [PSI+] is a prion of Sup35p, a subunit of the translation termination factor Sup35p. In contrast to the functional prions, [Het-s] of Podospora anserina and [BETA] of yeast, the amyloid-based yeast prions are rare in wild strains, arise sporadically, have an array of prion variants for a single prion protein sequence, have a folded in-register parallel ß-sheet amyloid architecture, are detrimental to their hosts, arouse a stress response in the host, and are subject to curing by various host anti-prion systems. These characteristics allow a logical basis for distinction between functional amyloids/prions and prion diseases. These infectious yeast amyloidoses are outstanding models for the many common human amyloid-based diseases that are increasingly found to have some infectious characteristics.

A Structuring Repeat for Peptide Design: Long Beta Ribbons.

Beta sheets are inherently length-limited; adding residues to the ends of model ß-sheets does not necessarily grow the ß-sheet. Here, we present a method for extending ß-sheets to any length with a stabilizing repeat unit containing cross-strand Trp residues. Beta ribbons as long as 35 residues (approaching 100 Šin length) are reported and characterized.

Precise assembly of complex beta sheet topologies from de novo designed building blocks.

Design of complex alpha-beta protein topologies poses a challenge because of the large number of alternative packing arrangements. A similar challenge presumably limited the emergence of large and complex protein topologies in evolution. Here, we demonstrate that protein topologies with six and seven-stranded beta sheets can be designed by insertion of one de novo designed beta sheet containing protein into another such that the two beta sheets are merged to form a single extended sheet, followed by amino acid sequence optimization at the newly formed strand-strand, strand-helix, and helix-helix interfaces. Crystal structures of two such designs closely match the computational design models. Searches for similar structures in the SCOP protein domain database yield only weak matches with different beta sheet connectivities. A similar beta sheet fusion mechanism may have contributed to the emergence of complex beta sheets during natural protein evolution.

An integrated imaging probe design: the synthesis of (99m)Tc/Re-containing macrocyclic peptide scaffolds.

ß-Sheets account for over 30 % of all secondary structural conformations found in proteins. The intramolecular hydrogen bonding that exists between the two peptide strands is imperative in maintaining this secondary structure. With the proper design, cyclic peptides may act as scaffolds emulating active ß-sheet regions, enabling investigation of their importance in molecular recognition and protein aggregation. Starting from Fmoc-Lys(Fmoc)-OH, macrocyclic peptides were synthesized on a solid support, with peptide-chain elongation extending from both the alpha and epsilon amines of the lysine. The branching peptides were cyclized with a pyridyl tridentate chelation core followed by coordination using [(99m) Tc/Re(CO)3 (H2 O)3 ](+) . Variable temperature (1) H NMR spectroscopy studies were performed, demonstrating that intramolecular hydrogen bonding exists between the two sides of the uncoordinated macrocyclic peptide scaffolds. Additionally, computational modelling and circular dichroism spectroscopic analysis revealed that the peptide backbone exists in a similar conformation both before and after metal coordination. The ability to seamlessly incorporate a tridentate chelation core into the backbone of a macrocyclic peptide, without disrupting the secondary structure, can greatly assist in the design of metal-centric peptidomimetic imaging agents. This novel integrated imaging probe approach may facilitate the investigation into protein-protein interactions using macrocyclic ß-sheet scaffolds.

Diphenylacetylene-linked peptide strands induce bidirectional ß-sheet formation.

In the search for synthetic mimics of protein secondary structures relevant to the mediation of protein-protein interactions, we have synthesized a series of tetrasubstituted diphenylacetylenes that display ß-sheet structures in two directions. Extensive X-ray crystallographic and NMR solution phase studies are consistent with these proteomimetics adopting sheet structures, displaying both hydrophobic and hydrophilic amino acid side chains.

Students' understanding of primary and secondary protein structure: drawing secondary protein structure reveals student understanding better than simple recognition of structures.

The interdisciplinary nature of biochemistry courses requires students to use both chemistry and biology knowledge to understand biochemical concepts. Research that has focused on external representations in biochemistry has uncovered student difficulties in comprehending and interpreting external representations in addition to a fragmented understanding of fundamental biochemistry concepts. This project focuses on students' understanding of primary and secondary protein structure and drawings (representations) of hydrogen-bonding in alpha helices and beta sheets. Analysis demonstrated that students can recognize and identify primary protein structure concepts when given a polypeptide. However, when asked to draw alpha helices and beta sheets and explain the role of hydrogen bonding their drawings students exhibited a fragmented understanding that lacked coherence. Faculty are encouraged to have students draw molecular level representations to make their mental models more explicit, complete, and coherent. This is in contrast to recognition and identification tasks, which do not adequately probe mental models and molecular level understanding.

ß-Bulges: extensive structural analyses of ß-sheets irregularities.

ß-Sheets are quite frequent in protein structures and are stabilized by regular main-chain hydrogen bond patterns. Irregularities in ß-sheets, named ß-bulges, are distorted regions between two consecutive hydrogen bonds. They disrupt the classical alternation of side chain direction and can alter the directionality of ß-strands. They are implicated in protein-protein interactions and are introduced to avoid ß-strand aggregation. Five different types of ß-bulges are defined. Previous studies on ß-bulges were performed on a limited number of protein structures or one specific family. These studies evoked a potential conservation during evolution. In this work, we analyze the ß-bulge distribution and conservation in terms of local backbone conformations and amino acid composition. Our dataset consists of 66 times more ß-bulges than the last systematic study (Chan et al. Protein Science 1993, 2:1574-1590). Novel amino acid preferences are underlined and local structure conformations are highlighted by the use of a structural alphabet. We observed that ß-bulges are preferably localized at the N- and C-termini of ß-strands, but contrary to the earlier studies, no significant conservation of ß-bulges was observed among structural homologues. Displacement of ß-bulges along the sequence was also investigated by Molecular Dynamics simulations.