Choosing the optimal spectroscopic toolkit to understand protein function

Spectroscopy was one of the earliest methods used to study the properties and reactions of proteins, and remains one of the most powerful and widely used approaches to this day. A sometimes bewildering range of spectroscopies is now available, applicable to different sample states, timescales and indeed biological questions. This editorial describes some of the most relevant spectroscopic methods together with a selection of illustrative examples.</jats:p

Complex folding and misfolding effects of deer-specific amino acid substitutions in the β2-α2 loop of murine prion protein

The β2–α2 loop of PrPC is a key modulator of disease-associated prion protein misfolding. Amino acids that differentiate mouse (Ser169, Asn173) and deer (Asn169, Thr173) PrPC appear to confer dramatically different structural properties in this region and it has been suggested that amino acid sequences associated with structural rigidity of the loop also confer susceptibility to prion disease. Using mouse recombinant PrP, we show that mutating residue 173 from Asn to Thr alters protein stability and misfolding only subtly, whilst changing Ser to Asn at codon 169 causes instability in the protein, promotes oligomer formation and dramatically potentiates fibril formation. The doubly mutated protein exhibits more complex folding and misfolding behaviour than either single mutant, suggestive of differential effects of the β2–α2 loop sequence on both protein stability and on specific misfolding pathways. Molecular dynamics simulation of protein structure suggests a key role for the solvent accessibility of Tyr168 in promoting molecular interactions that may lead to prion protein misfolding. Thus, we conclude that ‘rigidity’ in the β2–α2 loop region of the normal conformer of PrP has less effect on misfolding than other sequence-related effects in this region

Dynamic Diagnosis of Familial Prion Diseases Supports the β2-α2 Loop as a Universal Interference Target

[Background] Mutations in the cellular prion protein associated to familial prion disorders severely increase the likelihood of its misfolding into pathogenic conformers. Despite their postulation as incompatible elements with the native fold, these mutations rarely modify the native state structure. However they variably have impact on the thermodynamic stability and metabolism of PrPC and on the properties of PrPSc aggregates. To investigate whether the pathogenic mutations affect the dynamic properties of the HuPrP(125-229) α-fold and find possible common patterns of effects that could help in prophylaxis we performed a dynamic diagnosis of ten point substitutions.[Methodology/Principal Findings] Using all-atom molecular dynamics simulations and novel analytical tools we have explored the effect of D178N, V180I, T183A, T188K, E196K, F198S, E200K, R208H, V210I and E211Q mutations on the dynamics of HuPrP(125-228) α-fold. We have found that while preserving the native state, all mutations produce dynamic changes which perturb the coordination of the α2-α3 hairpin to the rest of the molecule and cause the reorganization of the patches for intermolecular recognition, as the disappearance of those for conversion inhibitors and the emergence of an interaction site at the β2-α2 loop region.[Conclusions/Significance] Our results suggest that pathogenic mutations share a common pattern of dynamical alterations that converge to the conversion of the β2-α2 loop into an interacting region that can be used as target for interference treatments in genetic diseases.This work was supported in parts by grants BFU2009-07971 from the MICINN (MG), FundaciÃ3n Cien (MG); Fondazione Cariplo (GC) and AIRC (GC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.Peer reviewe

NMR Structure of the Human Prion Protein with the Pathological Q212P Mutation Reveals Unique Structural Features

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrPC) conformer, denoted as infectious scrapie isoform or PrPSc. In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrPSc in specific brain regions leading to neuronal cell degeneration and death. Here, we describe the NMR solution structure of the truncated recombinant human PrP from residue 90 to 231 carrying the Q212P mutation, which is believed to cause Gerstmann-Sträussler-Scheinker (GSS) syndrome, a familial prion disease. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90–124) and a globular domain (residues 125–231). The substitution of a glutamine by a proline at the position 212 introduces novel structural differences in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the β2–α2 loop region. This structure might provide new insights into the early events of conformational transition of PrPC into PrPSc. Indeed, the spontaneous formation of prions in familial cases might be due to the disruptions of the hydrophobic core consisting of β2–α2 loop and α3 helix

The Bacterial Fimbrial Tip Acts as a Mechanical Force Sensor

The subunits that constitute the bacterial adhesive complex located at the tip of the fimbria form a hook-chain that acts as a rapid force-sensitive anchor at high flow

Biosynthetic potential of the global ocean microbiome

Natural microbial communities are phylogenetically and metabolically diverse. In addition to underexplored organismal groups, this diversity encompasses a rich discovery potential for ecologically and biotechnologically relevant enzymes and biochemical compounds. However, studying this diversity to identify genomic pathways for the synthesis of such compounds and assigning them to their respective hosts remains challenging. The biosynthetic potential of microorganisms in the open ocean remains largely uncharted owing to limitations in the analysis of genome-resolved data at the global scale. Here we investigated the diversity and novelty of biosynthetic gene clusters in the ocean by integrating around 10,000 microbial genomes from cultivated and single cells with more than 25,000 newly reconstructed draft genomes from more than 1,000 seawater samples. These efforts revealed approximately 40,000 putative mostly new biosynthetic gene clusters, several of which were found in previously unsuspected phylogenetic groups. Among these groups, we identified a lineage rich in biosynthetic gene clusters ('Candidatus Eudoremicrobiaceae') that belongs to an uncultivated bacterial phylum and includes some of the most biosynthetically diverse microorganisms in this environment. From these, we characterized the phospeptin and pythonamide pathways, revealing cases of unusual bioactive compound structure and enzymology, respectively. Together, this research demonstrates how microbiomics-driven strategies can enable the investigation of previously undescribed enzymes and natural products in underexplored microbial groups and environments

Biosynthetic potential of the global ocean microbiome

8 pages, 4 figures, supplementary information https://doi.org/10.1038/s41586-022-04862-3.-- This Article is contribution number 130 of Tara OceansNatural microbial communities are phylogenetically and metabolically diverse. In addition to underexplored organismal groups1, this diversity encompasses a rich discovery potential for ecologically and biotechnologically relevant enzymes and biochemical compounds2,3. However, studying this diversity to identify genomic pathways for the synthesis of such compounds4 and assigning them to their respective hosts remains challenging. The biosynthetic potential of microorganisms in the open ocean remains largely uncharted owing to limitations in the analysis of genome-resolved data at the global scale. Here we investigated the diversity and novelty of biosynthetic gene clusters in the ocean by integrating around 10,000 microbial genomes from cultivated and single cells with more than 25,000 newly reconstructed draft genomes from more than 1,000 seawater samples. These efforts revealed approximately 40,000 putative mostly new biosynthetic gene clusters, several of which were found in previously unsuspected phylogenetic groups. Among these groups, we identified a lineage rich in biosynthetic gene clusters (‘Candidatus Eudoremicrobiaceae’) that belongs to an uncultivated bacterial phylum and includes some of the most biosynthetically diverse microorganisms in this environment. From these, we characterized the phospeptin and pythonamide pathways, revealing cases of unusual bioactive compound structure and enzymology, respectively. Together, this research demonstrates how microbiomics-driven strategies can enable the investigation of previously undescribed enzymes and natural products in underexplored microbial groups and environmentsThis work was supported by funding from the ETH and the Helmut Horten Foundation; the Swiss National Science Foundation (SNSF) through project grants 205321_184955 to S.S., 205320_185077 to J.P. and the NCCR Microbiomes (51NF40_180575) to S.S.; by the Gordon and Betty Moore Foundation (https://doi.org/10.37807/GBMF9204) and the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 101000392 (MARBLES) to J.P.; by an ETH research grant ETH-21 18-2 to J.P.; and by the Peter and Traudl Engelhorn Foundation and by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 897571 to C.C.F. S.L.R. was supported by an ETH Zurich postdoctoral fellowship 20-1 FEL-07. M.L., L.M.C. and G.Z. were supported by EMBL Core Funding and the German Research Foundation (DFG, Deutsche Forschungsgemeinschaft, project no. 395357507, SFB 1371 to G.Z.). M.B.S. was supported by the NSF grant OCE#1829831. C.B. was supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement Diatomic, no. 835067). S.G.A. was supported by the Spanish Ministry of Economy and Competitiveness (PID2020-116489RB-I00). M.K. and H.M. were funded by the SNSF grant 407540_167331 as part of the Swiss National Research Programme 75 ‘Big Data’. M.K., H.M. and A.K. are also partially funded by ETH core funding (to G. Rätsch)With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)Peer reviewe