Homologous Recombination Shapes the Architecture and Evolution of Bacterial Genomes.

Homologous recombination is a key evolutionary force that varies considerably across bacterial species. However, how the landscape of homologous recombination varies across genes and within individual genomes has only been studied in a few species. Here, we used Approximate Bayesian Computation to estimate the recombination rate along the genomes of 145 bacterial species. Our results show that homologous recombination varies greatly along bacterial genomes and shapes many aspects of genome architecture and evolution. The genomic landscape of recombination presents several key signatures: rates are highest near the origin of replication in most species, patterns of recombination generally appear symmetrical in both replichores (i.e. replicational halves of circular chromosomes) and most species have genomic hotpots of recombination. Furthermore, many closely related species share conserved landscapes of recombination across orthologs indicating that recombination landscapes are conserved over significant evolutionary distances. We show evidence that recombination drives the evolution of GC-content through increasing the effectiveness of selection and not through biased gene conversion, thereby contributing to an ongoing debate. Finally, we demonstrate that the rate of recombination varies across gene function and that many hotspots of recombination are associated with adaptive and mobile regions often encoding genes involved in pathogenicity.

Evolution of homologous recombination rates across bacteria.

Bacteria are nonsexual organisms but are capable of exchanging DNA at diverse degrees through homologous recombination. Intriguingly, the rates of recombination vary immensely across lineages where some species have been described as purely clonal and others as "quasi-sexual." However, estimating recombination rates has proven a difficult endeavor and estimates often vary substantially across studies. It is unclear whether these variations reflect natural variations across populations or are due to differences in methodologies. Consequently, the impact of recombination on bacterial evolution has not been extensively evaluated and the evolution of recombination rate-as a trait-remains to be accurately described. Here, we developed an approach based on Approximate Bayesian Computation that integrates multiple signals of recombination to estimate recombination rates. We inferred the rate of recombination of 162 bacterial species and one archaeon and tested the robustness of our approach. Our results confirm that recombination rates vary drastically across bacteria; however, we found that recombination rate-as a trait-is conserved in several lineages but evolves rapidly in others. Although some traits are thought to be associated with recombination rate (e.g., GC-content), we found no clear association between genomic or phenotypic traits and recombination rate. Overall, our results provide an overview of recombination rate, its evolution, and its impact on bacterial evolution.

The Mechanism of Folding of Human Frataxin in Comparison to the Yeast Homologue - Broad Energy Barriers and the General Properties of the Transition State.

The funneled energy landscape theory suggests that the folding pathway of homologous proteins should converge at the late stages of folding. In this respect, proteins displaying a broad energy landscape for folding are particularly instructive, allowing inferring both the early, intermediate and late stages of folding. In this paper we explore the folding mechanisms of human frataxin, an essential mitochondrial protein linked to the neurodegenerative disorder Friedreich's ataxia. Building upon previous studies on the yeast homologue, the folding pathway of human frataxin is thoroughly examined, revealing a mechanism implying the presence of a broad energy barrier, reminiscent of the yeast counterpart. Through an extensive site-directed mutagenesis, we employed a Φ -value analysis to map native-like contacts in the folding transition state. The presence of a broad energy barrier facilitated the exploration of such contacts in both early and late folding events. We compared results from yeast and human frataxin providing insights into the impact of native topology on the folding mechanism and elucidating the properties of the underlying free energy landscape. The findings are discussed in the context of the funneled energy landscape theory of protein folding.

The binding selectivity of the C-terminal SH3 domain of Grb2, but not its folding pathway, is dictated by its contiguous SH2 domain.

The adaptor protein Grb2, or growth factor receptor-bound protein 2, possesses a pivotal role in the transmission of fundamental molecular signals in the cell. Despite lacking enzymatic activity, Grb2 functions as a dynamic assembly platform, orchestrating intracellular signals through its modular structure. This study delves into the energetic communication of Grb2 domains, focusing on the folding and binding properties of the C-SH3 domain linked to its neighboring SH2 domain. Surprisingly, while the folding and stability of C-SH3 remain robust and unaffected by SH2 presence, significant differences emerge in the binding properties when considered within the tandem context compared with isolated C-SH3. Through a double mutant cycle analysis, we highlighted a subset of residues, located at the interface with the SH2 domain and far from the binding site, finely regulating the binding of a peptide mimicking a physiological ligand of the C-SH3 domain. Our results have mechanistic implications about the mechanisms of specificity of the C-SH3 domain, indicating that the presence of the SH2 domain optimizes binding to its physiological target, and emphasizing the general importance of considering supramodular multidomain protein structures to understand the functional intricacies of protein-protein interaction domains.

Assessing the performance of group-based trajectory modeling method to discover different patterns of medication adherence.

It is well known that medication adherence is critical to patient outcomes and can decrease patient mortality. The Pharmacy Quality Alliance (PQA) has recognized and identified medication adherence as an important indicator of medication-use quality. Hence, there is a need to use the right methods to assess medication adherence. The PQA has endorsed the proportion of days covered (PDC) as the primary method of measuring adherence. Although easy to calculate, the PDC has however several drawbacks as a method of measuring adherence. PDC is a deterministic approach that cannot capture the complexity of a dynamic phenomenon. Group-based trajectory modeling (GBTM) is increasingly proposed as an alternative to capture heterogeneity in medication adherence. The main goal of this paper is to demonstrate, through a simulation study, the ability of GBTM to capture treatment adherence when compared to its deterministic PDC analogue and to the nonparametric longitudinal K-means. A time-varying treatment was generated as a quadratic function of time, baseline, and time-varying covariates. Three trajectory models are considered combining a cat's cradle effect, and a rainbow effect. The performance of GBTM was compared to the PDC and longitudinal K-means using the absolute bias, the variance, the c-statistics, the relative bias, and the relative variance. For all explored scenarios, we find that GBTM performed better in capturing different patterns of medication adherence with lower relative bias and variance even under model misspecification than PDC and longitudinal K-means.

Effect of statin use for the primary prevention of cardiovascular disease among older adults: a cautionary tale concerning target trials emulation.

OBJECTIVES: Evidence concerning the effect of statins in primary prevention of cardiovascular disease (CVD) among older adults is lacking. Using Quebec population-wide administrative data, we emulated a hypothetical randomized trial including older adults >65 years on April 1, 2013, with no CVD history and no statin use in the previous year. STUDY DESIGN AND SETTING: We included individuals who initiated statins and classified them as exposed if they were using statin at least 3 months after initiation and nonexposed otherwise. We followed them until March 31, 2018. The primary outcome was the composite endpoint of coronary events (myocardial infarction, coronary bypass, and percutaneous coronary intervention), stroke, and all-cause mortality. The intention-to-treat (ITT) effect was estimated with adjusted Cox models and per-protocol effect with inverse probability of censoring weighting. RESULTS: A total of 65,096 individuals were included (mean age = 71.0 ± 5.5, female = 55.0%) and 93.7% were exposed. Whereas we observed a reduction in the composite outcome (ITT-hazard ratio (HR) = 0.75; 95% CI: 0.68-0.83) and mortality (ITT-HR = 0.69; 95% CI: 0.61-0.77) among exposed, coronary events increased (ITT-HR = 1.46; 95% CI: 1.09-1.94). All multibias E-values were low indicating that the results were not robust to unmeasured confounding, selection, and misclassification biases simultaneously. CONCLUSION: We cannot conclude on the effectiveness of statins in primary prevention of CVD among older adults. We caution that an in-depth reflection on sources of biases and careful interpretation of results are always required in observational studies.

Addressing the Binding Mechanism of the Meprin and TRAF-C Homology Domain of the Speckle-Type POZ Protein Using Protein Engineering.

Protein-protein interactions play crucial roles in a wide range of biological processes, including metabolic pathways, cell cycle progression, signal transduction, and the proteasomal system. For PPIs to fulfill their biological functions, they require the specific recognition of a multitude of interacting partners. In many cases, however, protein-protein interaction domains are capable of binding different partners in the intracellular environment, but they require precise regulation of the binding events in order to exert their function properly and avoid misregulation of important molecular pathways. In this work, we focused on the MATH domain of the E3 Ligase adaptor protein SPOP in order to decipher the molecular features underlying its interaction with two different peptides that mimic its physiological partners: Puc and MacroH2A. By employing stopped-flow kinetic binding experiments, together with extensive site-directed mutagenesis, we addressed the roles of specific residues, some of which, although far from the binding site, govern these transient interactions. Our findings are compatible with a scenario in which the binding of the MATH domain with its substrate is characterized by a fine energetic network that regulates its interactions with different ligands. Results are briefly discussed in the context of previously existing work regarding the MATH domain.

Longitudinal plasmode algorithms to evaluate statistical methods in realistic scenarios: an illustration applied to occupational epidemiology.

INTRODUCTION: Plasmode simulations are a type of simulations that use real data to determine the synthetic data-generating equations. Such simulations thus allow evaluating statistical methods under realistic conditions. As far as we know, no plasmode algorithm has been proposed for simulating longitudinal data. In this paper, we propose a longitudinal plasmode framework to generate realistic data with both a time-varying exposure and time-varying covariates. This work was motivated by the objective of comparing different methods for estimating the causal effect of a cumulative exposure to psychosocial stressors at work over time. METHODS: We developed two longitudinal plasmode algorithms: a parametric and a nonparametric algorithms. Data from the PROspective Québec (PROQ) Study on Work and Health were used as an input to generate data with the proposed plasmode algorithms. We evaluated the performance of multiple estimators of the parameters of marginal structural models (MSMs): inverse probability of treatment weighting, g-computation and targeted maximum likelihood estimation. These estimators were also compared to standard regression approaches with either adjustment for baseline covariates only or with adjustment for both baseline and time-varying covariates. RESULTS: Standard regression methods were susceptible to yield biased estimates with confidence intervals having coverage probability lower than their nominal level. The bias was much lower and coverage of confidence intervals was much closer to the nominal level when considering MSMs. Among MSM estimators, g-computation overall produced the best results relative to bias, root mean squared error and coverage of confidence intervals. No method produced unbiased estimates with adequate coverage for all parameters in the more realistic nonparametric plasmode simulation. CONCLUSION: The proposed longitudinal plasmode algorithms can be important methodological tools for evaluating and comparing analytical methods in realistic simulation scenarios. To facilitate the use of these algorithms, we provide R functions on GitHub. We also recommend using MSMs when estimating the effect of cumulative exposure to psychosocial stressors at work.

Characterization and description of Gabonibacter chumensis sp. nov., isolated from feces of a patient with non-small cell lung cancer treated with immunotherapy.

A polyphasic taxonomic approach, incorporating analysis of phenotypic features, cellular fatty acid profiles, 16S rRNA gene sequences, and determination of average nucleotide identity (ANI) plus digital DNA-DNA hybridization (dDDH), was applied to characterize an anaerobic bacterial strain designated KD22T isolated from human feces. 16S rRNA gene-based phylogenetic analysis showed that strain KD22T was found to be most closely related to species of the genus Gabonibacter. At the 16S rRNA gene level, the closest species from the strain KD22T corresponded with Gabonibacter massiliensis GM7T, with a similarity of 97.58%. Cells of strain KD22T were Gram-negative coccobacillus, positive for indole and negative for catalase, nitrate reduction, oxidase, and urease activities. The fatty acid analysis demonstrated the presence of a high concentration of iso-C15: 0 (51.65%). Next, the complete whole-genome sequence of strain KD22T was 3,368,578 bp long with 42 mol% of DNA G + C contents. The DDH and ANI values between KD22T and type strains of phylogenetically related species were 67.40% and 95.43%, respectively. These phylogenetic, phenotypic, and genomic results supported the affiliation of strain KD22T as a novel bacterial species within the genus Gabonibacter. The proposed name is Gabonibacter chumensis and the type strain is KD22T (= CSUR Q8104T = DSM 115208 T).

Marginal structural models with latent class growth analysis of treatment trajectories: Statins for primary prevention among older adults.

Latent class growth analysis is increasingly proposed as a solution to summarize the observed longitudinal treatment into a few distinct groups. When latent class growth analysis is combined with standard approaches like Cox proportional hazards models, confounding bias is not properly addressed because of time-varying covariates that have a double role of confounders and mediators. We propose to use latent class growth analysis to classify individuals into a few latent classes based on their medication adherence pattern, then choose a working marginal structural model that relates the outcome to these groups. The parameter of interest is defined as a projection of the true marginal structural model onto the chosen working model. Simulation studies are used to illustrate our approach and compare it with unadjusted, baseline covariates adjusted, time-varying covariates adjusted, and inverse probability of trajectory groups weighted adjusted models. Our proposed approach yielded estimators with little or no bias and appropriate coverage of confidence intervals in these simulations. We applied our latent class growth analysis and marginal structural model approach to a database comprising information on 52,790 individuals from the province of Quebec, Canada, aged more than 65 and who were statin initiators to estimate the effect of statin-usage trajectories on a first cardiovascular event.

Characterization of the folding and binding properties of the PTB domain of FRS2 with phosphorylated and unphosphorylated ligands.

PTB (PhosphoTyrosine Binding) domains are protein domains that exert their function by binding phosphotyrosine residues on other proteins. They are commonly found in a variety of signaling proteins and are important for mediating protein-protein interactions in numerous cellular processes. PTB domains can also exhibit binding to unphosphorylated ligands, suggesting that they have additional binding specificities beyond phosphotyrosine recognition. Structural studies have reported that the PTB domain from FRS2 possesses this peculiar feature, allowing it to interact with both phosphorylated and unphosphorylated ligands, such as TrkB and FGFR1, through different topologies and orientations. In an effort to elucidate the dynamic and functional properties of these protein-protein interactions, we provide a complete characterization of the folding mechanism of the PTB domain of FRS2 and the binding process to peptides mimicking specific regions of TrkB and FGFR1. By analyzing the equilibrium and kinetics of PTB folding, we propose a mechanism implying the presence of an intermediate along the folding pathway. Kinetic binding experiments performed at different ionic strengths highlighted the electrostatic nature of the interaction with both peptides. The specific role of single amino acids in early and late events of binding was pinpointed by site-directed mutagenesis. These results are discussed in light of previous experimental works on these protein systems.

An intramolecular energetic network regulates ligand recognition in a SH2 domain.

In an effort to investigate the molecular determinants of ligand recognition of the C-terminal SH2 domain of the SHP2 protein, we conducted extensive site-directed mutagenesis and kinetic binding experiments with a peptide mimicking a specific portion of a physiological ligand (the scaffold protein Gab2). Obtained data provided an in-depth characterization of the binding reaction, allowing us to pinpoint residues topologically far from the binding pocket of the SH2 domain to have a role in the recognition and binding of the peptide. The presence of a sparse energetic network regulating the interaction with Gab2 was identified and characterized through double mutant cycle analysis, performed by challenging all the designed site-directed variants of C-SH2 with a Gab2 peptide mutated at +3 position relative to its phosphorylated tyrosine, a key residue for C-SH2 binding specificity. Results highlighted non-optimized residues involved in the energetic network regulating the binding with Gab2, which may be at the basis of the ability of this SH2 domain to interact with different partners in the intracellular environment. Moreover, a detailed analysis of kinetic and thermodynamic parameters revealed the role of the residue at +3 position on Gab2 in the early and late events of the binding reaction with the C-SH2 domain.

Biophysical Characterization of the Binding Mechanism between the MATH Domain of SPOP and Its Physiological Partners.

SPOP (Speckle-type POZ protein) is an E3 ubiquitin ligase adaptor protein that mediates the ubiquitination of several substrates. Furthermore, SPOP is responsible for the regulation of both degradable and nondegradable polyubiquitination of a number of substrates with diverse biological functions. The recognition of SPOP and its physiological partners is mediated by two protein-protein interaction domains. Among them, the MATH domain recognizes different substrates, and it is critical for orchestrating diverse cellular pathways, being mutated in several human diseases. Despite its importance, the mechanism by which the MATH domain recognizes its physiological partners has escaped a detailed experimental characterization. In this work, we present a characterization of the binding mechanism of the MATH domain of SPOP with three peptides mimicking the phosphatase Puc, the chromatin component MacroH2A, and the dual-specificity phosphatase PTEN. Furthermore, by taking advantage of site-directed mutagenesis, we address the role of some key residues of MATH in the binding process. Our findings are briefly discussed in the context of previously existing data on the MATH domain.

Tractidigestivibacter montrealensis sp. nov., a new member of human gut microbiota isolated from a healthy volunteer.

Strain KD21T, isolated from the fecal sample of a healthy female volunteer, is a strictly anaerobic, non-motile, Gram-staining-positive, saccharolytic small rod that does not produce spores. Strain KD21T was able to grow in the range of temperature 28°C-37°C (optimum, 37 °C), pH 6.0-8.0 (optimum, pH 7.0), and with 0-5.0 g/l NaCl (optimum, 0 g/l NaCl). Bacteria cells reduced nitrates to nitrites. Its major fatty acids were C18:1ω9c, C16:0, C18:0, and summed in feature 8 (C18:1ω7c and/or C18:1ω6c). 16S rRNA gene phylogenetic analysis revealed that KD21T is a member of the genus Tractidigestivibacter and is distinct from any species with validly published names. The sequence showed 98.48% similarity with T. scatoligenes SK9K4T. The DNA G + C content of strain KD21T was 62.6 mol%. The DNA-DNA hybridization and OrthoANI values between strain KD21T and T. scatoligenes SK9K4T were 40.2% and 90.2%, respectively. Differences in phenotypic, phylogenetic, chemotaxonomic, and genomic characteristics indicated that strain KD21T represents a novel species within the genus Tractidigestivibacter. The name T. montrealensis sp. nov. is proposed and the type strain is KD21T (= CSUR Q8103T =  DSM 115111T).

Time trends in infectious and chronic disease consultations in Dakar, Senegal: Impact of COVID-19 sanitary measures.

Background: The impact of COVID-19 sanitary measures on the time trends in infectious and chronic disease consultations in Sub-Saharan Africa remains unknown. Methods: We conducted a cohort study on all emergency medical consultations from January 2016 to July 2020, from SOS Medecins in Dakar, Senegal. The consultation records provided basic demographic information such as age, ethnicity (Senegalese or Caucasian), and sex as well as the principal diagnosis using an ICD-10 classification ("infectious", "chronic", and "other"). We first investigated how the pattern in emergency consultation differed from March to July 2020 compared to previous years. Then, we examined any potential racial/ethnic disparities in COVID-19 consultation. Results: We obtained data on emergency medical consultations from 53 583 patients of all ethnic origins. The mean age of patients was 37.0 (standard deviation (SD) = 25.2) and 30.3 (SD = 21.7) in 2016-2019 and 45.5 (SD = 24.7) and 39.5 (SD = 23.3) in 2020 for Senegalese and Caucasian patients, respectively. The type of consultations between January and July were similar from 2016 to 2019; however, in 2020, there was a drop in the number of infectious disease consultations, particularly from April to May 2020, when sanitary measures for COVID-19 were applied (average of 366.5 and 358.2 in 2016-1019 and 133.0 and 125.0 in 2020). The prevalence of chronic conditions remained steady during the same period (average of 381.0 and 394.7 in 2016-2019 and 373.0 and 367.0 in 2020). In a multivariate analysis adjusted for age and sex, infectious disease consultations were significantly more likely to occur in 2016-2019 compared to 2020 (2016 odds ratio (OR) = 2.39, 2017 OR = 2.74, 2018 OR = 2.39, 2019 OR = 2.01). Furthermore, the trend in the number of infectious and chronic consultations was similar among Senegalese and Caucasian groups, indicating no disparities among those seeking treatment. Conclusions: During the implementation of COVID-19 sanitary measures, infectious disease rates dropped as chronic disease rates remained stagnant in Dakar. We observed no racial/ethnic disparities among the infectious and chronic consultations.

Understanding the molecular basis of folding cooperativity through a comparative analysis of a multidomain protein and its isolated domains.

Although cooperativity is a well-established and general property of folding, our current understanding of this feature in multidomain folding is still relatively limited. In fact, there are contrasting results indicating that the constituent domains of a multidomain protein may either fold independently on each other or exhibit interdependent supradomain phenomena. To address this issue, here we present the comparative analysis of the folding of a tandem repeat protein, comprising two contiguous PDZ domains, in comparison to that of its isolated constituent domains. By analyzing in detail the equilibrium and kinetics of folding at different experimental conditions, we demonstrate that despite each of the PDZ domains in isolation being capable of independent folding, at variance with previously characterized PDZ tandem repeats, the full-length construct folds and unfolds as a single cooperative unit. By exploiting quantitatively, the comparison of the folding of the tandem repeat to those observed for its constituent domains, as well as by characterizing a truncated variant lacking a short autoinhibitory segment, we successfully rationalize the molecular basis of the observed cooperativity and attempt to infer some general conclusions for multidomain systems.

Pangenomic analysis of Coxiella burnetii unveils new traits in genome architecture.

Coxiella burnetii is the etiological agent of Q fever, a worldwide zoonosis able to cause large outbreaks. The disease is polymorphic. Symptomatic primary infection is named acute Q fever and is associated with hepatitis, pneumonia, fever, and auto-immune complications while persistent focalized infections, mainly endocarditis, and vascular infections, occur in a minority of patients but are potentially lethal. In order to evaluate the genomic features, genetic diversity, evolution, as well as genetic determinants of antibiotic resistance, pathogenicity, and ability to cause outbreaks of Q fever, we performed a pangenomic analysis and genomic comparison of 75 C. burnetii strains including 63 newly sequenced genomes. Our analysis demonstrated that C. burnetii has an open pangenome, unique genes being found in many strains. In addition, pathogenicity islands were detected in all genomes. In consequence C. burnetii has a high genomic plasticity, higher than that of other intracellular bacteria. The core- and pan-genomes are made of 1,211 and 4,501 genes, respectively (ratio 0.27). The core gene-based phylogenetic analysis matched that obtained from multi-spacer typing and the distribution of plasmid types. Genomic characteristics were associated to clinical and epidemiological features. Some genotypes were associated to specific clinical forms and countries. MST1 genotype strains were associated to acute Q fever. A significant association was also found between clinical forms and plasmids. Strains harboring the QpRS plasmid were never found in acute Q fever and were only associated to persistent focalized infections. The QpDV and QpH1 plasmids were associated to acute Q fever. In addition, the Guyanese strain CB175, the most virulent strain to date, exhibited a unique MST genotype, a distinct COG profile and an important variation in gene number that may explain its unique pathogenesis. Therefore, strain-specific factors play an important role in determining the epidemiological and clinical manifestations of Q fever alongside with host-specific factors (valvular and vascular defects notably).

SH2 Domains: Folding, Binding and Therapeutical Approaches.

SH2 (Src Homology 2) domains are among the best characterized and most studied protein-protein interaction (PPIs) modules able to bind and recognize sequences presenting a phosphorylated tyrosine. This post-translational modification is a key regulator of a plethora of physiological and molecular pathways in the eukaryotic cell, so SH2 domains possess a fundamental role in cell signaling. Consequently, several pathologies arise from the dysregulation of such SH2-domains mediated PPIs. In this review, we recapitulate the current knowledge about the structural, folding stability, and binding properties of SH2 domains and their roles in molecular pathways and pathogenesis. Moreover, we focus attention on the different strategies employed to modulate/inhibit SH2 domains binding. Altogether, the information gathered points to evidence that pharmacological interest in SH2 domains is highly strategic to developing new therapeutics. Moreover, a deeper understanding of the molecular determinants of the thermodynamic stability as well as of the binding properties of SH2 domains appears to be fundamental in order to improve the possibility of preventing their dysregulated interactions.

Gene flow and introgression are pervasive forces shaping the evolution of bacterial species.

BACKGROUND: Although originally thought to evolve clonally, studies have revealed that most bacteria exchange DNA. However, it remains unclear to what extent gene flow shapes the evolution of bacterial genomes and maintains the cohesion of species. RESULTS: Here, we analyze the patterns of gene flow within and between >2600 bacterial species. Our results show that fewer than 10% of bacterial species are truly clonal, indicating that purely asexual species are rare in nature. We further demonstrate that the taxonomic criterion of ~95% genome sequence identity routinely used to define bacterial species does not accurately represent a level of divergence that imposes an effective barrier to gene flow across bacterial species. Interruption of gene flow can occur at various sequence identities across lineages, generally from 90 to 98% genome identity. This likely explains why a ~95% genome sequence identity threshold has empirically been judged as a good approximation to define bacterial species. Our results support a universal mechanism where the availability of identical genomic DNA segments required to initiate homologous recombination is the primary determinant of gene flow and species boundaries in bacteria. We show that these barriers of gene flow remain porous since many distinct species maintain some level of gene flow, similar to introgression in sexual organisms. CONCLUSIONS: Overall, bacterial evolution and speciation are likely shaped by similar forces driving the evolution of sexual organisms. Our findings support a model where the interruption of gene flow-although not necessarily the initial cause of speciation-leads to the establishment of permanent and irreversible species borders.

Exploring the effect of tethered domains on the folding of Grb2 protein.

Two thirds of eukaryotic proteins have evolved as multidomain constructs, and in vivo, domains fold within a polypeptide chain, with inter-domain interactions possibly crucial for correct folding. However, to date, most of the experimental folding studies are based on domains in isolation. In an effort to better understand multidomain folding, in this work we analyzed, through equilibrium and kinetic folding experiments, the folding properties of the Growth factor receptor-bound protein 2 (Grb2), composed by one SRC homology 2 domain flanked by two SRC homology 3 domains. In particular we compared the kinetic features of the multidomain construct with the domains expressed in isolation. By performing single and double mixing folding experiments, we demonstrated that the folding of the SH2 domain is kinetically trapped in a misfolded intermediate when tethered to the C-SH3. Importantly, within the multidomain construct, misfolding occurred independently if refolding is started with C-SH3 in its unfolded or native state. Interestingly, our data reported a peculiar scenario, in which SH2 and C-SH3 domain reciprocally and transiently interact during folding. Altogether, the analysis of kinetic folding data provided a quantitative description of the multidomain folding of Grb2 protein, discussed under the light of previous works on multidomain folding.