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. To address this, we leverage FragPipe and then process its output in FLiPPR. FLiPPR formalizes a specific data imputation heuristic that carefully uses missing data in LiP-MS experiments to report on the most significant structural changes. Moreover, FLiPPR introduces a data merging scheme and a protein-centric multiple hypothesis correction scheme, enabling processed LiP-MS data sets to be more robust and less redundant. These improvements strengthen statistical trends when previously published data are reanalyzed with the FragPipe/FLiPPR workflow. We hope that FLiPPR will lower the barrier for more users to adopt LiP-MS, standardize statistical procedures for LiP-MS data analysis, and systematize output to facilitate eventual larger-scale integration of LiP-MS data.

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. We synthesize each protein from its wildtype, fastest- and slowest-translating synonymous mRNAs in silico and calculate the ensemble-averaged interaction energy between the resulting dimers. We find synonymous mutations alter oligoribonuclease's dimer properties. Relative to wildtype, the dimer interaction energy becomes 4% and 10% stronger, respectively, when translated from its fastest- and slowest-translating mRNAs. Ribonuclease T dimerization, however, is insensitive to synonymous mutations. The structural and kinetic origin of these changes are misfolded states containing non-covalent lasso-entanglements, many of which structurally perturb the dimer interface, and whose probability of occurrence depends on translation speed. These entangled states are kinetic traps that persist for long time scales. Entanglements cause altered dimerization energies for oligoribonuclease, as there is a large association (odds ratio: 52) between the co-occurrence of non-native self-entanglements and weak-binding dimer conformations. Simulated at all-atom resolution, these entangled structures persist for long timescales, indicating the conclusions are independent of model resolution. Finally, we show that regions of the protein we predict to have changes in entanglement are also structurally perturbed during refolding, as detected by limited-proteolysis mass spectrometry. Thus, non-native changes in entanglement at dimer interfaces is a mechanism through which oligomer structure and stability can be altered.

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. To address this, we leverage the fast, sensitive, and accurate search and label-free quantification algorithms in FragPipe and then process its output in FLiPPR. FLiPPR formalizes a specific data imputation heuristic that carefully uses missing data in LiP-MS experiments to report on the most significant structural changes. Moreover, FLiPPR introduces a new data merging scheme (from ions to cut-sites) and a protein-centric multiple hypothesis correction scheme, collectively enabling processed LiP-MS datasets to be more robust and less redundant. These improvements substantially strengthen statistical trends when previously published data are reanalyzed with the FragPipe/FLiPPR workflow. As a final feature, FLiPPR facilitates the collection of structural metadata to identify correlations between experiments and structural features. We hope that FLiPPR will lower the barrier for more users to adopt LiP-MS, standardize statistical procedures for LiP-MS data analysis, and systematize output to facilitate eventual larger-scale integration of LiP-MS data.

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. Surprisingly, we discover that this translocation mechanism drives the silk proteins to assemble into fibers spontaneously on the cell surface, in a process we call secretion-catalyzed assembly (SCA). Secreted silk fibers form self-healing hydrogels with minimal processing. Alternatively, the fibers retained on the membrane provide a facile route to create engineered living materials from Bacillus cells. This work provides a blueprint to achieve autonomous assembly of protein biomaterials in useful morphologies directly from microbial factories.

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. Here, we demonstrate the synthesis and application of two heterobifunctional photo-cross-linkers that feature diazirines and N-hydroxy-succinimidyl carbamate groups, the latter of which unveil doubly fissile MS-cleavable linkages upon acyl transfer to protein targets. Moreover, these cross-linkers demonstrate high water-solubility and cell-permeability. Using these compounds, we demonstrate the feasibility of proteome-wide photo-cross-linking in cellulo. These studies elucidate a small portion of Escherichia coli's interaction network, albeit with residue-level resolution. With further optimization, these methods will enable the detection of protein quinary interaction networks in their native environment at residue-level resolution, and we expect that they will prove useful toward the effort to explore the molecular sociology of the cell.

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. The results show that foldability was likely a critical factor in the selection of the canonical alphabet. Unbranched aliphatic amino acids were purged from the proteinogenic alphabet despite their high prebiotic abundance because they generate polypeptides that are oversolubilized and have low packing efficiency. Surprisingly, we find that the inclusion of a short-chain basic amino acid also decreases polypeptides' secondary structure potential, for which we suggest a biophysical model. Our results support the view that, despite lacking basic residues, the early canonical alphabet was remarkably adaptive at supporting protein folding and explain why basic residues were only incorporated at a later stage of protein evolution.

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. The modelling involves coarse-grained simulations of protein synthesis and post-translational behaviour, all-atom simulations to test robustness and quantum mechanics/molecular mechanics calculations to characterize enzymatic function. We show that changes in codon translation rates induced by synonymous mutations cause shifts in co-translational and post-translational folding pathways that kinetically partition molecules into subpopulations that very slowly interconvert to the native, functional state. Structurally, these states resemble the native state, with localized misfolding near the active sites of the enzymes. These long-lived states exhibit reduced catalytic activity, as shown by their increased activation energies for the reactions they catalyse.

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. Here, we develop a limited proteolysis-mass spectrometry approach paired with an isotope-labeling strategy to globally monitor the structures of refolding Escherichia coli proteins in the cytosolic medium and with the chaperones, GroEL/ES (Hsp60) and DnaK/DnaJ/GrpE (Hsp70/40). GroEL can refold the majority (85%) of the E. coli proteins for which we have data and is particularly important for restoring acidic proteins and proteins with high molecular weight, trends that come to light because our assay measures the structural outcome of the refolding process itself, rather than binding or aggregation. For the most part, DnaK and GroEL refold a similar set of proteins, supporting the view that despite their vastly different structures, these two chaperones unfold misfolded states, as one mechanism in common. Finally, we identify a cohort of proteins that are intransigent to being refolded with either chaperone. We suggest that these proteins may fold most efficiently cotranslationally, and then remain kinetically trapped in their native conformations.

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. Here, we present DomainMapper, an algorithm that accurately assigns a unique domain structure annotation to a query sequence, including those with complex topologies. We validate our domain assignments using the AlphaFold database and confirm that non-contiguity is pervasive (10.74% of all domains in yeast and 4.52% in human). Using this resource, we find that certain folds have strong propensities to be non-contiguous or insertional across the Tree of Life. DomainMapper is freely available and can be ran as a single command-line function.

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. coli proteins. We predict that half of proteins exhibit misfolded subpopulations that bypass molecular chaperones, avoid aggregation, and will not be rapidly degraded, with some misfolded states persisting for months or longer. The surface properties of these misfolded states are native-like, suggesting they will remain soluble, while self-entanglements make them long-lived kinetic traps. In terms of function, we predict that one-third of proteins can misfold into soluble less-functional states. For the heavily entangled protein glycerol-3-phosphate dehydrogenase, limited-proteolysis mass spectrometry experiments interrogating misfolded conformations of the protein are consistent with the structural changes predicted by our simulations. These results therefore provide an explanation for how proteins can misfold into soluble conformations with reduced functionality that can bypass proteostasis, and indicate, unexpectedly, this may be a wide-spread phenomenon.

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 (KMP) on lysine and DNA strand scission. We developed a chemoproteomic approach to identify KMP in HeLa cells. More than 60 000 KMP-modified histones are produced per cell. Using LC-MS/MS, we detected KMP at 17 of the 57 lysine residues distributed throughout the four core histone proteins. Therefore, KMP constitutes a DNA damage-induced, nonenzymatic histone post-translational modification. KMP formation suggests that downstream processes resulting from DNA damage could have ramifications on 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. The remodeler stimulates the nucleosome to absorb an additional nucleotide on each strand at two different locations: on the tracking strand within the ATPase binding site and on the guide strand one helical turn from the ATPase motor. Remarkably, the additional nucleotide on the tracking strand is associated with a local transformation toward an A-form geometry, explaining how sequential ratcheting of each DNA strand occurs. The structure also reveals a histone-binding motif, ChEx, which can block opposing remodelers on the nucleosome and may allow Chd1 to participate in histone reorganization during transcription.

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. Diverse lines of research in molecular biology, bioinformatics, geochemistry, biophysics and astrobiology provide clues about the progression and early evolution of proteins, and lend credence to the idea that early peptides served many central prebiotic roles before they were encodable by a polynucleotide template, in a putative 'peptide-polynucleotide stage'. For example, early peptides and mini-proteins could have served as catalysts, compartments and structural hubs. In sum, we shed light on the role of early peptides and small proteins before and during the nucleotide world, in which nascent life fully grasped the potential of primordial proteins, and which has left an imprint on the idiosyncratic properties of extant proteins.

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 in vivo 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. Our data suggested that translation of circularized mRNAs could be limited primarily by the rate of ribosomal initiation; therefore, using a modified error-prone PCR method, we generated a library that concentrated mutations into the initiation region of circularized mRNA and discovered mutants that generated markedly higher expression levels. Combining our rational improvements with those discovered through directed evolution, we report a loopable translator that achieves protein expression levels within 1.5-fold of the levels of standard vectorial translation. In summary, our work demonstrates loopable translation as a promising platform for the creation of large peptide chains, with potential utility in the development of novel protein materials.

Nonrefoldability is Pervasive Across the E. coli 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. Our study covers the majority of the soluble E. coli proteome expressed during log-phase growth, and among this group, we find that one-third of the E. coli proteome is not intrinsically refoldable on physiological time scales, a cohort that is enriched with certain fold-types, domain organizations, and other biophysical features. We also identify several properties and fold-types that are correlated with slow refolding on the minute time scale. Hence, these results illuminate when exogenous factors and processes, such as chaperones or cotranslational folding, might be required for efficient protein folding.

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. It will also be useful for achieving very high mutational loads on a standard-sized amplicon.

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 ß-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. Here, we use a combination of photo-crosslinking, mass spectrometry, solution scattering, and molecular modeling techniques to elucidate the key structural features that define how SurA solubilizes uOMPs. Our experimental data support a model in which SurA binds uOMPs in a groove formed between the core and P1 domains. This binding event results in a drastic expansion of the rest of the uOMP, which has many biological implications. Using these experimental data as restraints, we adopted an integrative modeling approach to create a sparse ensemble of models of a SurA•uOMP complex. We validated key structural features of the SurA•uOMP ensemble using independent scattering and chemical crosslinking data. Our data suggest that SurA utilizes three distinct binding modes to interact with uOMPs and that more than one SurA can bind a uOMP at a time. This work demonstrates that SurA operates in a distinct fashion compared to other chaperones in the OMP biogenesis network.

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). In the following, we use crystallography and computational modeling to show that KSI's intrinsic electric field is nearly perfectly oriented to stabilize the geometry of its reaction's TS. Moreover, we find that this electric field adjusts the orientation of its substrate in the ground state so that the substrate needs to only undergo minimal structural changes upon activation to its TS. This work provides evidence that the active site electric field in KSI is preorganized to facilitate catalysis and provides a template for how electrostatic preorganization can be measured in enzymatic systems.