Protein surface chemistry encodes an adaptive tolerance to desiccation.

Cellular desiccation - the loss of nearly all water from the cell - is a recurring stress in an increasing number of ecosystems that can drive protein unfolding and aggregation. For cells to survive, at least some of the proteome must resume function upon rehydration. Which proteins tolerate desiccation, and the molecular determinants that underlie this tolerance, are largely unknown.

FLiPPR: A Processor for Limited Proteolysis (LiP) Mass Spectrometry Data Sets Built on FragPipe.

Here, we present FLiPPR, or FragPipe LiP (limited proteolysis) Processor, a tool that facilitates the analysis of data from limited proteolysis mass spectrometry (LiP-MS) experiments following primary search and quantification in FragPipe. LiP-MS has emerged as a method that can provide proteome-wide information on protein structure and has been applied to a range of biological and biophysical questions. Although LiP-MS can be carried out with standard laboratory reagents and mass spectrometers, analyzing the data can be slow and poses unique challenges compared to typical quantitative proteomics workflows.

Synonymous Mutations Can Alter Protein Dimerization Through Localized Interface Misfolding Involving Self-entanglements.

Synonymous mutations in messenger RNAs (mRNAs) can reduce protein-protein binding substantially without changing the protein's amino acid sequence. Here, we use coarse-grain simulations of protein synthesis, post-translational dynamics, and dimerization to understand how synonymous mutations can influence the dimerization of two E. coli homodimers, oligoribonuclease and ribonuclease T.

FLiPPR: A Processor for Limited Proteolysis (LiP) Mass Spectrometry Datasets Built on FragPipe.

Here, we present FLiPPR, or FragPipe LiP (limited proteolysis) Processor, a tool that facilitates the analysis of data from limited proteolysis mass spectrometry (LiP-MS) experiments following primary search and quantification in FragPipe. LiP-MS has emerged as a method that can provide proteome-wide information on protein structure and has been applied to a range of biological and biophysical questions. Although LiP-MS can be carried out with standard laboratory reagents and mass spectrometers, analyzing the data can be slow and poses unique challenges compared to typical quantitative proteomics workflows.

Secretion-Catalyzed Assembly of Protein Biomaterials on a Bacterial Membrane Surface.

Protein-based biomaterials have played a key role in tissue engineering, and additional exciting applications as self-healing materials and sustainable polymers are emerging. Over the past few decades, recombinant expression and production of various fibrous proteins from microbes have been demonstrated; however, the resulting proteins typically must then be purified and processed by humans to form usable fibers and materials. Here, we show that the Gram-positive bacterium Bacillus subtilis can be programmed to secrete silk through its translocon via an orthogonal signal peptide/peptidase pair.

Progress toward Proteome-Wide Photo-Cross-Linking to Enable Residue-Level Visualization of Protein Structures and Networks In Vivo.

Cross-linking mass spectrometry (XL-MS) is emerging as a method at the crossroads of structural and cellular biology, uniquely capable of identifying protein-protein interactions with residue-level resolution and on the proteome-wide scale. With the development of cross-linkers that can form linkages inside cells and easily cleave during fragmentation on the mass spectrometer (MS-cleavable cross-links), it has become increasingly facile to identify contacts between any two proteins in complex samples, including in live cells or tissues. Photo-cross-linkers possess the advantages of high temporal resolution and high reactivity, thereby engaging all residue-types (rather than just lysine); nevertheless, photo-cross-linkers have not enjoyed widespread use and are yet to be employed for proteome-wide studies because their products are challenging to identify.

Early Selection of the Amino Acid Alphabet Was Adaptively Shaped by Biophysical Constraints of Foldability.

Whereas modern proteins rely on a quasi-universal repertoire of 20 canonical amino acids (AAs), numerous lines of evidence suggest that ancient proteins relied on a limited alphabet of 10 "early" AAs and that the 10 "late" AAs were products of biosynthetic pathways. However, many nonproteinogenic AAs were also prebiotically available, which begs two fundamental questions: Why do we have the current modern amino acid alphabet and would proteins be able to fold into globular structures as well if different amino acids comprised the genetic code? Here, we experimentally evaluate the solubility and secondary structure propensities of several prebiotically relevant amino acids in the context of synthetic combinatorial 25-mer peptide libraries. The most prebiotically abundant linear aliphatic and basic residues were incorporated along with or in place of other early amino acids to explore these alternative sequence spaces.

How synonymous mutations alter enzyme structure and function over long timescales.

The specific activity of enzymes can be altered over long timescales in cells by synonymous mutations that alter a messenger RNA molecule's sequence but not the encoded protein's primary structure. How this happens at the molecular level is unknown. Here, we use multiscale modelling of three Escherichia coli enzymes (type III chloramphenicol acetyltransferase, D-alanine-D-alanine ligase B and dihydrofolate reductase) to understand experimentally measured changes in specific activity due to synonymous mutations.

A proteome-wide map of chaperone-assisted protein refolding in a cytosol-like milieu.

The journey by which proteins navigate their energy landscapes to their native structures is complex, involving (and sometimes requiring) many cellular factors and processes operating in partnership with a given polypeptide chain's intrinsic energy landscape. The cytosolic environment and its complement of chaperones play critical roles in granting many proteins safe passage to their native states; however, it is challenging to interrogate the folding process for large numbers of proteins in a complex background with most biophysical techniques. Hence, most chaperone-assisted protein refolding studies are conducted in defined buffers on single purified clients.

DomainMapper: Accurate domain structure annotation including those with non-contiguous topologies.

Automated domain annotation is an important tool for structural informatics. These pipelines typically involve searching query sequences against hidden Markov model (HMM) profiles, yielding matches to profiles for various domains. However, domain annotation can be ambiguous or inaccurate when proteins contain domains with non-contiguous residue ranges, and especially when insertional domains are hosted within them.

Universal protein misfolding intermediates can bypass the proteostasis network and remain soluble and less functional.

Some misfolded protein conformations can bypass proteostasis machinery and remain soluble in vivo. This is an unexpected observation, as cellular quality control mechanisms should remove misfolded proteins. Three questions, then, are: how do long-lived, soluble, misfolded proteins bypass proteostasis? How widespread are such misfolded states? And how long do they persist? We address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E.

Intracellular Formation of a DNA Damage-Induced, Histone Post-Translational Modification Following Bleomycin Treatment.

Evaluating the significance of various forms of DNA damage is complicated by discoveries that some lesions inactivate repair enzymes or produce more deleterious forms of damage. Histone lysines within nucleosomes react with the commonly produced C4'-oxidized abasic site (C4-AP) to concomitantly yield an electrophilic modification (K) on lysine and DNA strand scission. We developed a chemoproteomic approach to identify K in HeLa cells.

Nucleosome recognition and DNA distortion by the Chd1 remodeler in a nucleotide-free state.

Chromatin remodelers are ATP-dependent enzymes that reorganize nucleosomes within all eukaryotic genomes. Here we report a complex of the Chd1 remodeler bound to a nucleosome in a nucleotide-free state, determined by cryo-EM to 2.3 Å resolution.

Peptides before and during the nucleotide world: an origins story emphasizing cooperation between proteins and nucleic acids.

Recent developments in Origins of Life research have focused on substantiating the narrative of an abiotic emergence of nucleic acids from organic molecules of low molecular weight, a paradigm that typically sidelines the roles of peptides. Nevertheless, the simple synthesis of amino acids, the facile nature of their activation and condensation, their ability to recognize metals and cofactors and their remarkable capacity to self-assemble make peptides (and their analogues) favourable candidates for one of the earliest functional polymers. In this mini-review, we explore the ramifications of this hypothesis.

Optimized Loopable Translation as a Platform for the Synthesis of Repetitive Proteins.

The expression of long proteins with repetitive amino acid sequences often presents a challenge in recombinant systems. To overcome this obstacle, we report a genetic construct that circularizes mRNA by rearranging the topology of a group I self-splicing intron from T4 bacteriophage, thereby enabling "loopable" translation. Using a fluorescence-based assay to probe the translational efficiency of circularized mRNAs, we identify several conditions that optimize protein expression from this system.

Nonrefoldability is Pervasive Across the Proteome.

Decades of research on protein folding have primarily focused on a subset of small proteins that can reversibly refold from a denatured state. However, these studies have generally not been representative of the complexity of natural proteomes, which consist of many proteins with complex architectures and domain organizations. Here, we introduce an experimental approach to probe protein refolding kinetics for whole proteomes using mass spectrometry-based proteomics.

An error prone PCR method for small amplicons.

Error-prone PCR (epPCR) is a commonly employed approach in molecular biology, especially in directed evolution, to generate libraries of DNA molecules with broad mutational spectrums. Though commonly applied to mutagenize protein coding sequences of several hundreds or thousands of basepairs, we found that commonly used protocols were not suitable for small (<100 bp) amplicons. Here we report a modified error-prone PCR protocol utilizing a Touchdown approach and employing only commercially available components, that should be broadly useful for the researcher interested in concentrating mutations into a small region of plasmid DNA.

SurA is a cryptically grooved chaperone that expands unfolded outer membrane proteins.

The periplasmic chaperone network ensures the biogenesis of bacterial outer membrane proteins (OMPs) and has recently been identified as a promising target for antibiotics. SurA is the most important member of this network, both due to its genetic interaction with the β-barrel assembly machinery complex as well as its ability to prevent unfolded OMP (uOMP) aggregation. Using only binding energy, the mechanism by which SurA carries out these two functions is not well-understood.

A Preorganized Electric Field Leads to Minimal Geometrical Reorientation in the Catalytic Reaction of Ketosteroid Isomerase.

Electrostatic interactions play a pivotal role in enzymatic catalysis and are increasingly modeled explicitly in computational enzyme design; nevertheless, they are challenging to measure experimentally. Using vibrational Stark effect (VSE) spectroscopy, we have measured electric fields inside the active site of the enzyme ketosteroid isomerase (KSI). These studies have shown that these fields can be unusually large, but it has been unclear to what extent they specifically stabilize the transition state (TS) relative to a ground state (GS).

Rapid discovery and evolution of orthogonal aminoacyl-tRNA synthetase-tRNA pairs.

A central challenge in expanding the genetic code of cells to incorporate noncanonical amino acids into proteins is the scalable discovery of aminoacyl-tRNA synthetase (aaRS)-tRNA pairs that are orthogonal in their aminoacylation specificity. Here we computationally identify candidate orthogonal tRNAs from millions of sequences and develop a rapid, scalable approach-named tRNA Extension (tREX)-to determine the in vivo aminoacylation status of tRNAs. Using tREX, we test 243 candidate tRNAs in Escherichia coli and identify 71 orthogonal tRNAs, covering 16 isoacceptor classes, and 23 functional orthogonal tRNA-cognate aaRS pairs.

Local and Global Electric Field Asymmetry in Photosynthetic Reaction Centers.

The origin of unidirectional electron transfer in photosynthetic reaction centers (RCs) has been widely discussed. Despite the high level of structural similarity between the two branches of pigments that participate in the initial electron transfer steps of photosynthesis, electron transfer only occurs along one branch. One possible explanation for this functional asymmetry is the differences in the electrostatic environment between the active and the inactive branches arising from the charges and dipoles of the organized protein structure.

Controlling orthogonal ribosome subunit interactions enables evolution of new function.

Orthogonal ribosomes are unnatural ribosomes that are directed towards orthogonal messenger RNAs in Escherichia coli, through an altered version of the 16S ribosomal RNA of the small subunit. Directed evolution of orthogonal ribosomes has provided access to new ribosomal function, and the evolved orthogonal ribosomes have enabled the encoding of multiple non-canonical amino acids into proteins. The original orthogonal ribosomes shared the pool of 23S ribosomal RNAs, contained in the large subunit, with endogenous ribosomes.