Fit for Purpose Approach To Evaluate Detection of Amino Acid Substitutions in Shotgun Proteomics.

Amino acid substitutions (AASs) alter proteins from their genome-expected sequences. Accumulation of substitutions in proteins underlies numerous diseases and antibiotic mechanisms. Accurate global detection of AASs and their frequencies is crucial for understanding these mechanisms. Shotgun proteomics provides an untargeted method for measuring AASs but introduces biases when extrapolating from the genome to identify AASs. To characterize these biases, we created a "ground-truth" approach using the similarities betweenEscherichia coli and Salmonella typhimurium to model the complexity of AAS detection. Shotgun proteomics on mixed lysates generated libraries representing ∼100,000 peptide-spectra and 4161 peptide sequences with a single AAS and defined stoichiometry. Identifying S. typhimurium peptide-spectra with only the E. coli genome resulted in 64.1% correctly identified library peptides. Specific AASs exhibit variable identification efficiencies. There was no inherent bias from the stoichiometry of the substitutions. Short peptides and AASs localized near peptide termini had poor identification efficiency. We identify a new class of "scissor substitutions" that gain or lose protease cleavage sites. Scissor substitutions also had poor identification efficiency. This ground-truth AAS library reveals various sources of bias, which will guide the application of shotgun proteomics to validate AAS hypotheses.

Competing stress-dependent oligomerization pathways regulate self-assembly of the periplasmic protease-chaperone DegP.

DegP is an oligomeric protein with dual protease and chaperone activity that regulates protein homeostasis and virulence factor trafficking in the periplasm of gram-negative bacteria. A number of oligomeric architectures adopted by DegP are thought to facilitate its function. For example, DegP can form a "resting" hexamer when not engaged to substrates, mitigating undesired proteolysis of cellular proteins. When bound to substrate proteins or lipid membranes, DegP has been shown to populate a variety of cage- or bowl-like oligomeric states that have increased proteolytic activity. Though a number of DegP's substrate-engaged structures have been robustly characterized, detailed mechanistic information underpinning its remarkable oligomeric plasticity and the corresponding interplay between these dynamics and biological function has remained elusive. Here, we have used a combination of hydrodynamics and NMR spectroscopy methodologies in combination with cryogenic electron microscopy to shed light on the apo-DegP self-assembly mechanism. We find that, in the absence of bound substrates, DegP populates an ensemble of oligomeric states, mediated by self-assembly of trimers, that are distinct from those observed in the presence of substrate. The oligomeric distribution is sensitive to solution ionic strength and temperature and is shifted toward larger oligomeric assemblies under physiological conditions. Substrate proteins may guide DegP toward canonical cage-like structures by binding to these preorganized oligomers, leading to changes in conformation. The properties of DegP self-assembly identified here suggest that apo-DegP can rapidly shift its oligomeric distribution in order to respond to a variety of biological insults.

Synonymous codon substitutions perturb cotranslational protein folding in vivo and impair cell fitness.

In the cell, proteins are synthesized from N to C terminus and begin to fold during translation. Cotranslational folding mechanisms are therefore linked to elongation rate, which varies as a function of synonymous codon usage. However, synonymous codon substitutions can affect many distinct cellular processes, which has complicated attempts to deconvolve the extent to which synonymous codon usage can promote or frustrate proper protein folding in vivo. Although previous studies have shown that some synonymous changes can lead to different final structures, other substitutions will likely be more subtle, perturbing predominantly the protein folding pathway without radically altering the final structure. Here we show that synonymous codon substitutions encoding a single essential enzyme lead to dramatically slower cell growth. These mutations do not prevent active enzyme formation; instead, they predominantly alter the protein folding mechanism, leading to enhanced degradation in vivo. These results support a model in which synonymous codon substitutions can impair cell fitness by significantly perturbing cotranslational protein folding mechanisms, despite the chaperoning provided by the cellular protein homeostasis network.

Properties of protein unfolded states suggest broad selection for expanded conformational ensembles.

Much attention is being paid to conformational biases in the ensembles of intrinsically disordered proteins. However, it is currently unknown whether or how conformational biases within the disordered ensembles of foldable proteins affect function in vivo. Recently, we demonstrated that water can be a good solvent for unfolded polypeptide chains, even those with a hydrophobic and charged sequence composition typical of folded proteins. These results run counter to the generally accepted model that protein folding begins with hydrophobicity-driven chain collapse. Here we investigate what other features, beyond amino acid composition, govern chain collapse. We found that local clustering of hydrophobic and/or charged residues leads to significant collapse of the unfolded ensemble of pertactin, a secreted autotransporter virulence protein from Bordetella pertussis, as measured by small angle X-ray scattering (SAXS). Sequence patterns that lead to collapse also correlate with increased intermolecular polypeptide chain association and aggregation. Crucially, sequence patterns that support an expanded conformational ensemble enhance pertactin secretion to the bacterial cell surface. Similar sequence pattern features are enriched across the large and diverse family of autotransporter virulence proteins, suggesting sequence patterns that favor an expanded conformational ensemble are under selection for efficient autotransporter protein secretion, a necessary prerequisite for virulence. More broadly, we found that sequence patterns that lead to more expanded conformational ensembles are enriched across water-soluble proteins in general, suggesting protein sequences are under selection to regulate collapse and minimize protein aggregation, in addition to their roles in stabilizing folded protein structures.

Multi-layer sequential network analysis improves protein 3D structural classification.

Protein structural classification (PSC) is a supervised problem of assigning proteins into pre-defined structural (e.g., CATH or SCOPe) classes based on the proteins' sequence or 3D structural features. We recently proposed PSC approaches that model protein 3D structures as protein structure networks (PSNs) and analyze PSN-based protein features, which performed better than or comparable to state-of-the-art sequence or other 3D structure-based PSC approaches. However, existing PSN-based PSC approaches model the whole 3D structure of a protein as a static (i.e., single-layer) PSN. Because folding of a protein is a dynamic process, where some parts (i.e., sub-structures) of a protein fold before others, modeling the 3D structure of a protein as a PSN that captures the sub-structures might further help improve the existing PSC performance. Here, we propose to model 3D structures of proteins as multi-layer sequential PSNs that approximate 3D sub-structures of proteins, with the hypothesis that this will improve upon the current state-of-the-art PSC approaches that are based on single-layer PSNs (and thus upon the existing state-of-the-art sequence and other 3D structural approaches). Indeed, we confirm this on 72 datasets spanning ~44 000 CATH and SCOPe protein domains.

Commonly used FRET fluorophores promote collapse of an otherwise disordered protein.

The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt in the absence of denaturant remain controversial. We developed an analysis procedure for small-angle X-ray scattering (SAXS) profiles and used it to demonstrate that even relatively hydrophobic IDPs remain nearly as expanded in water as they are in high denaturant concentrations. In contrast, as demonstrated here, most fluorescence resonance energy transfer (FRET) measurements have indicated that relatively hydrophobic IDPs contract significantly in the absence of denaturant. We use two independent approaches to further explore this controversy. First, using SAXS we show that fluorophores employed in FRET can contribute to the observed discrepancy. Specifically, we find that addition of Alexa-488 to a normally expanded IDP causes contraction by an additional 15%, a value in reasonable accord with the contraction reported in FRET-based studies. Second, using our simulations and analysis procedure to accurately extract both the radius of gyration (Rg) and end-to-end distance (Ree) from SAXS profiles, we tested the recent suggestion that FRET and SAXS results can be reconciled if the Rg and Ree are "uncoupled" (i.e., no longer simply proportional), in contrast to the case for random walk homopolymers. We find, however, that even for unfolded proteins, these two measures of unfolded state dimensions remain proportional. Together, these results suggest that improved analysis procedures and a correction for significant, fluorophore-driven interactions are sufficient to reconcile prior SAXS and FRET studies, thus providing a unified picture of the nature of unfolded polypeptide chains in the absence of denaturant.

Network analysis of synonymous codon usage.

MOTIVATION: Most amino acids are encoded by multiple synonymous codons, some of which are used more rarely than others. Analyses of positions of such rare codons in protein sequences revealed that rare codons can impact co-translational protein folding and that positions of some rare codons are evolutionarily conserved. Analyses of their positions in protein 3-dimensional structures, which are richer in biochemical information than sequences alone, might further explain the role of rare codons in protein folding. RESULTS: We model protein structures as networks and use network centrality to measure the structural position of an amino acid. We first validate that amino acids buried within the structural core are network-central, and those on the surface are not. Then, we study potential differences between network centralities and thus structural positions of amino acids encoded by conserved rare, non-conserved rare and commonly used codons. We find that in 84% of proteins, the three codon categories occupy significantly different structural positions. We examine protein groups showing different codon centrality trends, i.e. different relationships between structural positions of the three codon categories. We see several cases of all proteins from our data with some structural or functional property being in the same group. Also, we see a case of all proteins in some group having the same property. Our work shows that codon usage is linked to the final protein structure and thus possibly to co-translational protein folding. AVAILABILITY AND IMPLEMENTATION: https://nd.edu/∼cone/CodonUsage/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

How to Build a Complex, Functional Propeller Protein, From Parts.

By combining ancestral sequence reconstruction and in vitro evolution, Smock et al. identified single motifs that assemble into a functional five-bladed ß-propeller, and a likely route for conversion into the more complex, extant single chain fusion. Interestingly, although sequence diversification destabilized five-motif fusions, it also destabilized aggregation-prone intermediates, increasing the level of functional protein in vivo.

Widespread position-specific conservation of synonymous rare codons within coding sequences.

Synonymous rare codons are considered to be sub-optimal for gene expression because they are translated more slowly than common codons. Yet surprisingly, many protein coding sequences include large clusters of synonymous rare codons. Rare codons at the 5' terminus of coding sequences have been shown to increase translational efficiency. Although a general functional role for synonymous rare codons farther within coding sequences has not yet been established, several recent reports have identified rare-to-common synonymous codon substitutions that impair folding of the encoded protein. Here we test the hypothesis that although the usage frequencies of synonymous codons change from organism to organism, codon rarity will be conserved at specific positions in a set of homologous coding sequences, for example to tune translation rate without altering a protein sequence. Such conservation of rarity-rather than specific codon identity-could coordinate co-translational folding of the encoded protein. We demonstrate that many rare codon cluster positions are indeed conserved within homologous coding sequences across diverse eukaryotic, bacterial, and archaeal species, suggesting they result from positive selection and have a functional role. Most conserved rare codon clusters occur within rather than between conserved protein domains, challenging the view that their primary function is to facilitate co-translational folding after synthesis of an autonomous structural unit. Instead, many conserved rare codon clusters separate smaller protein structural motifs within structural domains. These smaller motifs typically fold faster than an entire domain, on a time scale more consistent with translation rate modulation by synonymous codon usage. While proteins with conserved rare codon clusters are structurally and functionally diverse, they are enriched in functions associated with organism growth and development, suggesting an important role for synonymous codon usage in organism physiology. The identification of conserved rare codon clusters advances our understanding of distinct, functional roles for otherwise synonymous codons and enables experimental testing of the impact of synonymous codon usage on the production of functional proteins.

Folding the proteome.

Protein folding is an essential prerequisite for protein function and hence cell function. Kinetic and thermodynamic studies of small proteins that refold reversibly were essential for developing our current understanding of the fundamentals of protein folding mechanisms. However, we still lack sufficient understanding to accurately predict protein structures from sequences, or the effects of disease-causing mutations. To date, model proteins selected for folding studies represent only a small fraction of the complexity of the proteome and are unlikely to exhibit the breadth of folding mechanisms used in vivo. We are in urgent need of new methods - both theoretical and experimental - that can quantify the folding behavior of a truly broad set of proteins under in vivo conditions. Such a shift in focus will provide a more comprehensive framework from which to understand the connections between protein folding, the molecular basis of disease, and cell function and evolution.

Multiple driving forces required for efficient secretion of autotransporter virulence proteins.

Autotransporter (AT) proteins are a broad class of virulence proteins from Gram-negative bacterial pathogens that require their own C-terminal transmembrane domain to translocate their N-terminal passenger across the bacterial outer membrane (OM). But given the unavailability of ATP or a proton gradient across the OM, it is unknown what energy source(s) drives this process. Here we used a combination of computational and experimental approaches to quantitatively compare proposed AT OM translocation mechanisms. We show directly for the first time that when translocation was blocked an AT passenger remained unfolded in the periplasm. We demonstrate that AT secretion is a kinetically controlled, non-equilibrium process coupled to folding of the passenger and propose a model connecting passenger conformation to secretion kinetics. These results reconcile seemingly contradictory reports regarding the importance of passenger folding as a driving force for OM translocation but also reveal that another energy source is required to initiate translocation.

Of linkers and autochaperones: an unambiguous nomenclature to identify common and uncommon themes for autotransporter secretion.

Autotransporter (AT) proteins provide a diverse array of important virulence functions to Gram-negative bacterial pathogens, and have also been adapted for protein surface display applications. The 'autotransporter' moniker refers to early models that depicted these proteins facilitating their own translocation across the bacterial outer membrane. Although translocation is less autonomous than originally proposed, AT protein segments upstream of the C-terminal transmembrane ß-barrel have nevertheless consistently been found to contribute to efficient translocation and/or folding of the N-terminal virulence region (the 'passenger'). However, defining the precise secretion functions of these AT regions has been complicated by the use of multiple overlapping and ambiguous terms to define AT sequence, structural, and functional features, including 'autochaperone', 'linker' and 'junction'. Moreover, the precise definitions and boundaries of these features vary among ATs and even among research groups, leading to an overall murky picture of the contributions of specific features to translocation. Here we propose a unified, unambiguous nomenclature for AT structural, functional and conserved sequence features, based on explicit criteria. Applied to 16 well-studied AT proteins, this nomenclature reveals new commonalities for translocation but also highlights that the autochaperone function is less closely associated with a conserved sequence element than previously believed.

Expanding Anfinsen's principle: contributions of synonymous codon selection to rational protein design.

Anfinsen's principle asserts that all information required to specify the structure of a protein is encoded in its amino acid sequence. However, during protein synthesis by the ribosome, the N-terminus of the nascent chain can begin to fold before the C-terminus is available. We tested whether this cotranslational folding can alter the folded structure of an encoded protein in vivo, versus the structure formed when refolded in vitro. We designed a fluorescent protein consisting of three half-domains, where the N- and C-terminal half-domains compete with each other to interact with the central half-domain. The outcome of this competition determines the fluorescence properties of the resulting folded structure. Upon refolding after chemical denaturation, this protein produced equimolar amounts of the N- and C-terminal folded structures, respectively. In contrast, translation in Escherichia coli resulted in a 2-fold enhancement in the formation of the N-terminal folded structure. Rare synonymous codon substitutions at the 5' end of the C-terminal half-domain further increased selection for the N-terminal folded structure. These results demonstrate that the rate at which a nascent protein emerges from the ribosome can specify the folded structure of a protein.

An alternative outer membrane secretion mechanism for an autotransporter protein lacking a C-terminal stable core.

Autotransporter (AT) proteins are a broad class of virulence factors from Gram-negative pathogens. AT outer membrane (OM) secretion appears simple in many regards, yet the mechanism that enables transport of the central AT 'passenger' across the OM remains unclear. OM secretion efficiency for two AT passengers is enhanced by approximately 20 kDa stable core at the C-terminus of the passenger, but studies on a broader range of AT proteins are needed in order to determine whether a stability difference between the passenger N- and C-terminus represents a truly common mechanistic feature. Yersinia pestis YapV is homologous to Shigella flexneri IcsA, and like IcsA, YapV recruits mammalian neural Wiskott-Aldrich syndrome protein (N-WASP). In vitro, the purified YapV passenger is functional and rich in ß-sheet structure, but lacks a approximately 20 kDa C-terminal stable core. However, the N-terminal 49 residues of the YapV passenger globally destabilize the entire YapV passenger, enhancing its OM secretion efficiency. These results indicate that the contributions of AT passenger sequences to OM secretion efficiency extend beyond a C-terminal stable core, and highlight a role of the passenger N-terminus in reducing passenger stability in order to facilitate OM secretion of some AT proteins.

Structure-based prediction reveals capping motifs that inhibit ß-helix aggregation.

The parallel ß-helix is a geometrically regular fold commonly found in the proteomes of bacteria, viruses, fungi, archaea, and some vertebrates. ß-helix structure has been observed in monomeric units of some aggregated amyloid fibers. In contrast, soluble ß-helices, both right- and left-handed, are usually "capped" on each end by one or more secondary structures. Here, an in-depth classification of the diverse range of ß-helix cap structures reveals subtle commonalities in structural components and in interactions with the ß-helix core. Based on these uncovered commonalities, a toolkit of automated predictors was developed for the two distinct types of cap structures. In vitro deletion of the toolkit-predicted C-terminal cap from the pertactin ß-helix resulted in increased aggregation and the formation of soluble oligomeric species. These results suggest that ß-helix cap motifs can prevent specific, ß-sheet-mediated oligomeric interactions, similar to those observed in amyloid formation.

How hydrophobicity, side chains, and salt affect the dimensions of disordered proteins.

Despite the generally accepted role of the hydrophobic effect as the driving force for folding, many intrinsically disordered proteins (IDPs), including those with hydrophobic content typical of foldable proteins, behave nearly as self-avoiding random walks (SARWs) under physiological conditions. Here, we tested how temperature and ionic conditions influence the dimensions of the N-terminal domain of pertactin (PNt), an IDP with an amino acid composition typical of folded proteins. While PNt contracts somewhat with temperature, it nevertheless remains expanded over 10-58°C, with a Flory exponent, ν, >0.50. Both low and high ionic strength also produce contraction in PNt, but this contraction is mitigated by reducing charge segregation. With 46% glycine and low hydrophobicity, the reduced form of snow flea anti-freeze protein (red-sfAFP) is unaffected by temperature and ionic strength and persists as a near-SARW, ν ~ 0.54, arguing that the thermal contraction of PNt is due to stronger interactions between hydrophobic side chains. Additionally, red-sfAFP is a proxy for the polypeptide backbone, which has been thought to collapse in water. Increasing the glycine segregation in red-sfAFP had minimal effect on ν. Water remained a good solvent even with 21 consecutive glycine residues (ν > 0.5), and red-sfAFP variants lacked stable backbone hydrogen bonds according to hydrogen exchange. Similarly, changing glycine segregation has little impact on ν in other glycine-rich proteins. These findings underscore the generality that many disordered states can be expanded and unstructured, and that the hydrophobic effect alone is insufficient to drive significant chain collapse for typical protein sequences.

The Effects of Codon Usage on Protein Structure and Folding.

The rate of protein synthesis is slower than many folding reactions and varies depending on the synonymous codons encoding the protein sequence. Synonymous codon substitutions thus have the potential to regulate cotranslational protein folding mechanisms, and a growing number of proteins have been identified with folding mechanisms sensitive to codon usage. Typically, these proteins have complex folding pathways and kinetically stable native structures. Kinetically stable proteins may fold only once over their lifetime, and thus, codon-mediated regulation of the pioneer round of protein folding can have a lasting impact. Supporting an important role for codon usage in folding, conserved patterns of codon usage appear in homologous gene families, hinting at selection. Despite these exciting developments, there remains few experimental methods capable of quantifying translation elongation rates and cotranslational folding mechanisms in the cell, which challenges the development of a predictive understanding of how biology uses codons to regulate protein folding. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

CHARMING: Harmonizing synonymous codon usage to replicate a desired codon usage pattern.

There is a growing appreciation that synonymous codon usage, although historically regarded as phenotypically silent, can instead alter a wide range of mechanisms related to functional protein production, a term we use here to describe the net effect of transcription (mRNA synthesis), mRNA half-life, translation (protein synthesis) and the probability of a protein folding correctly to its active, functional structure. In particular, recent discoveries have highlighted the important role that sub-optimal codons can play in modifying co-translational protein folding. These results have drawn increased attention to the patterns of synonymous codon usage within coding sequences, particularly in light of the discovery that these patterns can be conserved across evolution for homologous proteins. Because synonymous codon usage differs between organisms, for heterologous gene expression it can be desirable to make synonymous codon substitutions to match the codon usage pattern from the original organism in the heterologous expression host. Here we present CHARMING (for Codon HARMonizING), a robust and versatile algorithm to design mRNA sequences for heterologous gene expression and other related codon harmonization tasks. CHARMING can be run as a downloadable Python script or via a web portal at http://www.codons.org.

Cotranslational folding increases GFP folding yield.

Protein sequences evolved to fold in cells, including cotranslational folding of nascent polypeptide chains during their synthesis by the ribosome. The vectorial (N- to C-terminal) nature of cotranslational folding constrains the conformations of the nascent polypeptide chain in a manner not experienced by full-length chains diluted out of denaturant. We are still discovering to what extent these constraints affect later, posttranslational folding events. Here we directly address whether conformational constraints imposed by cotranslational folding affect the partitioning between productive folding to the native structure versus aggregation. We isolated polyribosomes from Escherichia coli cells expressing GFP, analyzed the nascent chain length distribution to determine the number of nascent chains that were long enough to fold to the native fluorescent structure, and calculated the folding yield for these nascent chains upon ribosome release versus the folding yield of an equivalent concentration of full-length, chemically denatured GFP polypeptide chains. We find that the yield of native fluorescent GFP is dramatically higher upon ribosome release of nascent chains versus dilution of full-length chains from denaturant. For kinetically trapped native structures such as GFP, folding correctly the first time, immediately after release from the ribosome, can lead to lifelong population of the native structure, as opposed to aggregation.

Slow formation of aggregation-resistant beta-sheet folding intermediates.

Protein folding has been studied extensively for decades, yet our ability to predict how proteins reach their native state from a mechanistic perspective is still rudimentary at best, limiting our understanding of folding-related processes in vivo and our ability to manipulate proteins in vitro. Here, we investigate the in vitro refolding mechanism of a large beta-helix protein, pertactin, which has an extended, elongated shape. At 55 kDa, this single domain, all-beta-sheet protein allows detailed analysis of the formation of beta-sheet structure in larger proteins. Using a combination of fluorescence and far-UV circular dichroism spectroscopy, we show that the pertactin beta-helix refolds remarkably slowly, with multiexponential kinetics. Surprisingly, despite the slow refolding rates, large size, and beta-sheet-rich topology, pertactin refolding is reversible and not complicated by off-pathway aggregation. The slow pertactin refolding rate is not limited by proline isomerization, and 30% of secondary structure formation occurs within the rate-limiting step. Furthermore, site-specific labeling experiments indicate that the beta-helix refolds in a multistep but concerted process involving the entire protein, rather than via initial formation of the stable core substructure observed in equilibrium titrations. Hence pertactin provides a valuable system for studying the refolding properties of larger, beta-sheet-rich proteins, and raises intriguing questions regarding the prevention of aggregation during the prolonged population of partially folded, beta-sheet-rich refolding intermediates. Proteins 2010. (c) 2009 Wiley-Liss, Inc.