Biomolecular condensates are characterized by interphase electric potentials.

Biomolecular condensates form via processes that combine phase separation and reversible associations of multivalent macromolecules. Condensates can be two- or multi-phase systems defined by coexisting dense and dilute phases. Here, we show that solution ions can partition asymmetrically across coexisting phases defined by condensates formed by intrinsically disordered proteins or homopolymeric RNA molecules. Our findings were enabled by direct measurements of the activities of cations and anions within coexisting phases of protein and RNA condensates. Asymmetries in ion partitioning between coexisting phases vary with protein sequence, condensate type, salt concentration, and ion type. The Donnan equilibrium set up by asymmetrical partitioning of solution ions generates interphase electric potentials known as Donnan and Nernst potentials. Our measurements show that the interphase potentials of condensates are of the same order of magnitude as membrane potentials of membrane-bound organelles. Interphase potentials quantify the degree to which microenvironments of coexisting phases are different from one another. Importantly, and based on condensate-specific interphase electric potentials, which are membrane-like potentials of membraneless bodies, we reason that condensates are mesoscale capacitors that store charge. Interphase potentials lead to electric double layers at condensate interfaces. This helps explain recent observations of condensate interfaces being electrochemically active.

Design of intrinsically disordered protein variants with diverse structural properties.

Intrinsically disordered proteins (IDPs) perform a wide range of functions in biology, suggesting that the ability to design IDPs could help expand the repertoire of proteins with novel functions. Designing IDPs with specific structural or functional properties has, however, been difficult, in part because determining accurate conformational ensembles of IDPs generally requires a combination of computational modelling and experiments. Motivated by recent advancements in efficient physics-based models for simulations of IDPs, we have developed a general algorithm for designing IDPs with specific structural properties. We demonstrate the power of the algorithm by generating variants of naturally occurring IDPs with different levels of compaction and that vary more than 100 fold in their propensity to undergo phase separation, even while keeping a fixed amino acid composition. We experimentally tested designs of variants of the low-complexity domain of hnRNPA1 and find high accuracy in our computational predictions, both in terms of single-chain compaction and propensity to undergo phase separation. We analyze the sequence features that determine changes in compaction and propensity to phase separate and find an overall good agreement with previous findings for naturally occurring sequences. Our general, physics-based method enables the design of disordered sequences with specified conformational properties. Our algorithm thus expands the toolbox for protein design to include also the most flexible proteins and will enable the design of proteins whose functions exploit the many properties afforded by protein disorder.

Workplace-based learning about health promotion in individual patient care: a scoping review.

OBJECTIVE: To outline current knowledge regarding workplace-based learning about health promotion in individual patient care. DESIGN: Scoping review. DATA SOURCES: PubMed, ERIC, CINAHL and Web of Science from January 2000 to August 2023. ELIGIBILITY CRITERIA: We included articles about learning (activities) for healthcare professionals (in training), about health promotion in individual patient care and in the context of workplace-based learning. DATA EXTRACTION AND SYNTHESIS: The studies were evaluated using a charting template and were analysed thematically using a template based on Designable Elements of Learning Environments model. RESULTS: From 7159 studies, we included 31 that described evaluations of workplace-based learning about health promotion, around a variety of health promotion topics, for different health professions. In the articles, health promotion was operationalised as knowledge, skills or attitudes related to specific lifestyle factors or more broadly, with concepts such as health literacy, advocacy and social determinants of health. We assembled an overview of spatial and instrumental, social, epistemic and temporal elements of learning environments in which health promotion is learnt. CONCLUSIONS: The studies included in our analysis varied greatly in their approach to health promotion topics and the evaluation of learning outcomes. Our findings suggest the importance of providing opportunities for health profession learners to engage in authentic practice situations and address potential challenges they may experience translating related theory into practice. Additionally, our results highlight the need for conscious and articulated integration of health promotion in curricula and assessment structures. We recommend the exploration of opportunities for health profession students, professionals and patients to learn about health promotion together. Additionally, we see potential in using participatory research methods to study future health promotion learning. STUDY REGISTRATION: Open Science Framework, https://doi.org/10.17605/OSF.IO/6QPTV.

Crowder titrations enable the quantification of driving forces for macromolecular phase separation.

Macromolecular solubility is an important contributor to the driving forces for phase separation. Formally, the driving forces in a binary mixture comprising a macromolecule dissolved in a solvent can be quantified in terms of the saturation concentration, which is the threshold macromolecular concentration above which the mixture separates into coexisting dense and dilute phases. In addition, the second virial coefficient, which measures the effective strength of solvent-mediated intermolecular interactions provides direct assessments of solvent quality. The sign and magnitude of second virial coefficients will be governed by a combination of solution conditions and the nature of the macromolecule of interest. Here, we show, using a combination of theory, simulation, and in vitro experiments, that titrations of crowders, providing they are true depletants, can be used to extract the intrinsic driving forces for macromolecular phase separation. This refers to saturation concentrations in the absence of crowders and the second virial coefficients that quantify the magnitude of the incompatibility between macromolecules and the solvent. Our results show how the depletion-mediated attractions afforded by crowders can be leveraged to obtain comparative assessments of macromolecule-specific, intrinsic driving forces for phase separation.

Phase separation of protein mixtures is driven by the interplay of homotypic and heterotypic interactions.

Prion-like low-complexity domains (PLCDs) are involved in the formation and regulation of distinct biomolecular condensates that form via phase separation coupled to percolation. Intracellular condensates often encompass numerous distinct proteins with PLCDs. Here, we combine simulations and experiments to study mixtures of PLCDs from two RNA-binding proteins, hnRNPA1 and FUS. Using simulations and experiments, we find that 1:1 mixtures of A1-LCD and FUS-LCD undergo phase separation more readily than either of the PLCDs on their own due to complementary electrostatic interactions. Tie line analysis reveals that stoichiometric ratios of different components and their sequence-encoded interactions contribute jointly to the driving forces for condensate formation. Simulations also show that the spatial organization of PLCDs within condensates is governed by relative strengths of homotypic versus heterotypic interactions. We uncover rules for how interaction strengths and sequence lengths modulate conformational preferences of molecules at interfaces of condensates formed by mixtures of proteins.

Crowder titrations enable the quantification of driving forces for macromolecular phase separation.

Macromolecular solubility is an important contributor to the driving forces for phase separation. Formally, the driving forces in a binary mixture comprising a macromolecule dissolved in a solvent can be quantified in terms of the saturation concentration, which is the threshold macromolecular concentration above which the mixture separates into coexisting dense and dilute phases. Additionally, the second virial coefficient, which measures the effective strength of solvent-mediated intermolecular interactions provides direct assessments of solvent quality. The sign and magnitude of second virial coefficients will be governed by a combination of solution conditions and the nature of the macromolecule of interest. Here, we show, using a combination of theory, simulation, and in vitro experiments, that titrations of crowders, providing they are true depletants, can be used to extract the intrinsic driving forces for macromolecular phase separation. This refers to saturation concentrations in the absence of crowders and the second virial coefficients that quantify the magnitude of the incompatibility between macromolecules and the solvent. Our results show how the depletion-mediated attractions afforded by crowders can be leveraged to obtain comparative assessments of macromolecule-specific, intrinsic driving forces for phase separation. SIGNIFICANCE: Phase separation has emerged as a process of significant relevance to sorting macromolecules into distinct compartments, thereby enabling spatial and temporal control over cellular matter. Considerable effort is being invested into uncovering the driving forces that enable the separation of macromolecular solutions into coexisting phases. At its heart, this process is governed by the balance of macromolecule-solvent, inter-macromolecule, and solvent-solvent interactions. We show that the driving forces for phase separation, including the coefficients that measure interaction strengths between macromolecules, can be extracted by titrating the concentrations of crowders that enable macromolecules to phase separate at lower concentrations. Our work paves the way to leverage specific categories of measurements for quantitative characterizations of driving forces for phase separation.

A Qualitative Study on How Entrustable Professional Activities Support Medical Students in Their Transitions across Clerkships.

Introduction: Medical students regularly transition between clerkships. These transitions can lead to discontinuity in their development because of the need to adapt to a new environment. The use of entrustable professional activities (EPAs) might facilitate less disruptive transitions across clerkships, as they could provide support at the start of a clerkship. This study aims to shed light on how an EPA-based curriculum contributes to medical students' learning processes during transitions. Methods: The authors used a constructivist rapid ethnographic design. They conducted observations and interviews with 11 medical students in their Pediatrics clerkship; six of them were in clerkships not utilizing EPAs, and five were using EPAs. Data collection was followed by template analysis such that all data were coded with a template that was continually updated until the authors all agreed upon a definitive template. Results: Four themes proved important when considering the impact of EPAs during transitions between clerkships: transitions as a learning opportunity, building relationships in context, taking leadership in the landscape of practice and feedback-seeking behavior. Discussion: EPAs smooth clerkship transitions, as they establish continuity in the student's development and facilitate navigating discontinuity in transitions. Students build skills and confidence in order to grow and work with increasing independence within the clerkships. Transitions offer important learning opportunities for students, which can be fully exploited by using EPA guidance.

Radiotherapy in Combination with Systemic Therapy for Multiple Myeloma-A Critical Toxicity Evaluation in the Modern Treatment Era.

Radiotherapy (RT) is an established treatment modality in the management of patients with multiple myeloma (MM), aiming at analgesia and stabilization of osteolytic lesions. As a multifocal disease, the combined use of RT, systemic chemotherapy, and targeted therapy (ST) is pivotal to achieve better disease control. However, adding RT to ST may lead to increased toxicity. The aim of this study was to evaluate the tolerability of ST given concurrently with RT. Overall, 82 patients treated at our hematological center with a median follow-up of 60 months from initial diagnosis and 46.5 months from the start of RT were evaluated retrospectively. Toxicities were recorded from 30 days before RT up to 90 days after RT. 54 patients (65.9%) developed at least one non-hematological toxicity, with 50 patients (61.0%) showing low-grade (grade I or II) and 14 patients (17.1%) revealing high-grade (grade III and IV) toxicities. Hematological toxicities were documented in 50 patients (61.0%) before RT, 60 patients (73.2%) during RT, and 67 patients (81.7%) following RT. After RT, patients who had received ST during RT showed a significant increase in high-grade hematological toxicities (p = 0.018). In summary, RT can be safely implemented into modern treatment regimens for MM, but stringent monitoring of potential toxicities even after completion of RT has to be ensured.

An Innovative Undergraduate Medical Curriculum Using Entrustable Professional Activities.

The need to educate medical professionals in changing medical organizations has led to a revision of the Radboudumc's undergraduate medical curriculum. Entrustable professional activities (EPAs) were used as a learning tool to support participation and encourage feedback-seeking behavior, in order to offer students the best opportunities for growth. This paper describes the development of the Radboudumc's EPA-based Master's curriculum and how EPAs can facilitate continuity in learning in the clerkships. Four guiding principles were used to create a curriculum that offers possibilities for the students' development: (1) working with EPAs, (2) establishing entrustment, (3) providing continuity in learning, and (4) organizing smooth transitions. The new curriculum was designed with the implementation of EPAs and an e-portfolio, based on these 4 principles. The authors found that the revised curriculum corresponds to daily practice in clerkships. Students used their e-portfolios throughout all clerkships, which stimulates feedback-seeking behavior. Moreover, EPAs promote continuity in learning while rotating clerkships every 1 to 2 months. This might encourage curriculum developers to use EPAs when aiming for greater continuity in the development of students. Future research needs to focus on the effect of EPAs on transitions across clerkships in order to further improve the undergraduate medical curriculum.

Phase Separation in Mixtures of Prion-Like Low Complexity Domains is Driven by the Interplay of Homotypic and Heterotypic Interactions.

Prion-like low-complexity domains (PLCDs) are involved in the formation and regulation of distinct biomolecular condensates that form via coupled associative and segregative phase transitions. We previously deciphered how evolutionarily conserved sequence features drive phase separation of PLCDs through homotypic interactions. However, condensates typically encompass a diverse mixture of proteins with PLCDs. Here, we combine simulations and experiments to study mixtures of PLCDs from two RNA binding proteins namely, hnRNPA1 and FUS. We find that 1:1 mixtures of the A1-LCD and FUS-LCD undergo phase separation more readily than either of the PLCDs on their own. The enhanced driving forces for phase separation of mixtures of A1-LCD and FUS-LCD arise partly from complementary electrostatic interactions between the two proteins. This complex coacervation-like mechanism adds to complementary interactions among aromatic residues. Further, tie line analysis shows that stoichiometric ratios of different components and their sequence-encoded interactions jointly contribute to the driving forces for condensate formation. These results highlight how expression levels might be tuned to regulate the driving forces for condensate formation in vivo . Simulations also show that the organization of PLCDs within condensates deviates from expectations based on random mixture models. Instead, spatial organization within condensates will reflect the relative strengths of homotypic versus heterotypic interactions. We also uncover rules for how interaction strengths and sequence lengths modulate conformational preferences of molecules at interfaces of condensates formed by mixtures of proteins. Overall, our findings emphasize the network-like organization of molecules within multicomponent condensates, and the distinctive, composition-specific conformational features of condensate interfaces.

Sequence-specific interactions determine viscoelasticity and aging dynamics of protein condensates.

Biomolecular condensates are viscoelastic materials. Here, we report results from investigations into molecular-scale determinants of sequence-encoded and age-dependent viscoelasticity of condensates formed by prion-like low-complexity domains (PLCDs). The terminally viscous forms of PLCD condensates are Maxwell fluids. Measured viscoelastic moduli of these condensates are reproducible using a Rouse-Zimm model that accounts for the network-like organization engendered by reversible physical crosslinks among PLCDs in the dense phase. Measurements and computations show that the strengths of aromatic inter-sticker interactions determine the sequence-specific amplitudes of elastic and viscous moduli as well as the timescales over which elastic properties dominate. PLCD condensates also undergo physical aging on sequence-specific timescales. This is driven by mutations to spacer residues that weaken the metastability of terminally viscous phases. The aging of PLCD condensates is accompanied by disorder-to-order transitions, leading to the formation of non-fibrillar, beta-sheet-containing, semi-crystalline, terminally elastic, Kelvin-Voigt solids. Our results suggest that sequence grammars, which refer to the identities of stickers versus spacers in PLCDs, have evolved to afford control over the metastabilities of terminally viscous fluid phases of condensates. This selection can, in some cases, render barriers for conversion from metastable fluids to globally stable solids to be insurmountable on functionally relevant timescales.

Phase Separation in Mixtures of Prion-Like Low Complexity Domains is Driven by the Interplay of Homotypic and Heterotypic Interactions.

Prion-like low-complexity domains (PLCDs) are involved in the formation and regulation of distinct biomolecular condensates that form via coupled associative and segregative phase transitions. We previously deciphered how evolutionarily conserved sequence features drive phase separation of PLCDs through homotypic interactions. However, condensates typically encompass a diverse mixture of proteins with PLCDs. Here, we combine simulations and experiments to study mixtures of PLCDs from two RNA binding proteins namely, hnRNPA1 and FUS. We find that 1:1 mixtures of the A1-LCD and FUS-LCD undergo phase separation more readily than either of the PLCDs on their own. The enhanced driving forces for phase separation of mixtures of A1-LCD and FUS-LCD arise partly from complementary electrostatic interactions between the two proteins. This complex coacervation-like mechanism adds to complementary interactions among aromatic residues. Further, tie line analysis shows that stoichiometric ratios of different components and their sequence-encoded interactions jointly contribute to the driving forces for condensate formation. These results highlight how expression levels might be tuned to regulate the driving forces for condensate formation in vivo . Simulations also show that the organization of PLCDs within condensates deviates from expectations based on random mixture models. Instead, spatial organization within condensates will reflect the relative strengths of homotypic versus heterotypic interactions. We also uncover rules for how interaction strengths and sequence lengths modulate conformational preferences of molecules at interfaces of condensates formed by mixtures of proteins. Overall, our findings emphasize the network-like organization of molecules within multicomponent condensates, and the distinctive, composition-specific conformational features of condensate interfaces. Significance Statement: Biomolecular condensates are mixtures of different protein and nucleic acid molecules that organize biochemical reactions in cells. Much of what we know about how condensates form comes from studies of phase transitions of individual components of condensates. Here, we report results from studies of phase transitions of mixtures of archetypal protein domains that feature in distinct condensates. Our investigations, aided by a blend of computations and experiments, show that the phase transitions of mixtures are governed by a complex interplay of homotypic and heterotypic interactions. The results point to how expression levels of different protein components can be tuned in cells to modulate internal structures, compositions, and interfaces of condensates, thus affording distinct ways to control the functions of condensates.

Condensates formed by prion-like low-complexity domains have small-world network structures and interfaces defined by expanded conformations.

Biomolecular condensates form via coupled associative and segregative phase transitions of multivalent associative macromolecules. Phase separation coupled to percolation is one example of such transitions. Here, we characterize molecular and mesoscale structural descriptions of condensates formed by intrinsically disordered prion-like low complexity domains (PLCDs). These systems conform to sticker-and-spacers architectures. Stickers are cohesive motifs that drive associative interactions through reversible crosslinking and spacers affect the cooperativity of crosslinking and overall macromolecular solubility. Our computations reproduce experimentally measured sequence-specific phase behaviors of PLCDs. Within simulated condensates, networks of reversible inter-sticker crosslinks organize PLCDs into small-world topologies. The overall dimensions of PLCDs vary with spatial location, being most expanded at and preferring to be oriented perpendicular to the interface. Our results demonstrate that even simple condensates with one type of macromolecule feature inhomogeneous spatial organizations of molecules and interfacial features that likely prime them for biochemical activity.

Quantifying Coexistence Concentrations in Multi-Component Phase-Separating Systems Using Analytical HPLC.

Over the last decade, evidence has accumulated to suggest that numerous instances of cellular compartmentalization can be explained by the phenomenon of phase separation. This is a process by which a macromolecular solution separates spontaneously into dense and dilute coexisting phases. Semi-quantitative, in vitro approaches for measuring phase boundaries have proven very useful in determining some key features of biomolecular condensates, but these methods often lack the precision necessary for generating quantitative models. Therefore, there is a clear need for techniques that allow quantitation of coexisting dilute and dense phase concentrations of phase-separating biomolecules, especially in systems with more than one type of macromolecule. Here, we report the design and deployment of analytical High-Performance Liquid Chromatography (HPLC) for in vitro separation and quantification of distinct biomolecules that allows us to measure dilute and dense phase concentrations needed to reconstruct coexistence curves in multicomponent mixtures. This approach is label-free, detects lower amounts of material than is accessible with classic UV-spectrophotometers, is applicable to a broad range of macromolecules of interest, is a semi-high-throughput technique, and if needed, the macromolecules can be recovered for further use. The approach promises to provide quantitative insights into the balance of homotypic and heterotypic interactions in multicomponent phase-separating systems.

How an EPA-based curriculum supports professional identity formation.

BACKGROUND: Entrustable professional activities (EPAs) are widely used in medical education, and they might be an important incentive to stimulate professional identity formation (PIF) of medical students, by actively encouraging participation in the workplace. The goal of this study was to explore the effects of an EPA-based curriculum on the PIF of medical students in undergraduate curricula. METHODS: In this study at the Radboud University Medical Center in Nijmegen, the Netherlands, the authors interviewed twenty-one medical students in three focus group interviews (November 2019), and conducted a thematic analysis based on both the synthesizing concepts PIF, communities of practice and EPAs, and newly defined themes. RESULTS: Four central themes proved crucial for understanding the influence of EPAs on PIF: creating learning opportunities, managing feedback, dealing with supervision in context and developing confidence. EPAs helped students to create learning opportunities and to choose activities purposefully, and the use of EPAs stimulated their feedback-seeking behavior. The context and way of supervision had a great impact on their development, where some contexts offer better learning opportunities than others. EPAs helped them develop trust and self-confidence, but trust from supervisors hardly appears to result from using EPAs. CONCLUSIONS: An EPA-based curriculum does stimulate PIF in the complex context of working and learning by supporting participation in the workplace and by encouraging feedback-seeking behavior. Striking the right balance between participation, feedback-seeking behavior and choosing learning activities is essential. TRIAL REGISTRATION: This study was approved by the ethics committee of the Netherlands Association of Medical Education (NVMO, case number 2019.5.12).

Deciphering how naturally occurring sequence features impact the phase behaviours of disordered prion-like domains.

Prion-like low-complexity domains (PLCDs) have distinctive sequence grammars that determine their driving forces for phase separation. Here we uncover the physicochemical underpinnings of how evolutionarily conserved compositional biases influence the phase behaviour of PLCDs. We interpret our results in the context of the stickers-and-spacers model for the phase separation of associative polymers. We find that tyrosine is a stronger sticker than phenylalanine, whereas arginine is a context-dependent auxiliary sticker. In contrast, lysine weakens sticker-sticker interactions. Increasing the net charge per residue destabilizes phase separation while also weakening the strong coupling between single-chain contraction in dilute phases and multichain interactions that give rise to phase separation. Finally, glycine and serine residues act as non-equivalent spacers, and thus make the glycine versus serine contents an important determinant of the driving forces for phase separation. The totality of our results leads to a set of rules that enable comparative estimates of composition-specific driving forces for PLCD phase separation.

How do intrinsically disordered protein regions encode a driving force for liquid-liquid phase separation?

Liquid-liquid phase separation is the mechanism underlying the formation of biomolecular condensates. Disordered protein regions often drive phase separation, but the molecular interactions mediating this phenomenon are not well understood, sometimes leading to the conflation that all disordered protein regions drive phase separation. Given the critical role of phase separation in many cellular processes, and that dysfunction of phase separation can lead to debilitating diseases, it is important that we understand the interactions and sequence properties underlying phase behavior. A conceptual framework that divides IDRs into interacting and solvating regions has proven particularly useful, and analytical instantiations and coarse-grained models can test our understanding of the driving forces against experimental phase behavior. Validated simulation paradigms enable the exploration of sequence space to help our understanding of how disordered protein regions can encode phase behavior, which IDRs may mediate phase separation in cells, and which IDRs are highly soluble.

Microfluidic characterization of macromolecular liquid-liquid phase separation.

Liquid-liquid phase separation plays important roles in the compartmentalization of cells. Developing an understanding of how phase separation is encoded in biomacromolecules requires quantitative mapping of their phase behavior. Given that such experiments require large quantities of the biomolecule of interest, these efforts have been lagging behind the recent breadth of biological insights. Herein, we present a microfluidic phase chip that enables the measurement of saturation concentrations over at least three orders of magnitude for a broad spectrum of biomolecules and solution conditions. The phase chip consists of five units, each made of twenty individual sample chambers to allow the measurement of five sample conditions simultaneously. The analytes are slowly concentrated via evaporation of water, which is replaced by oil, until the sample undergoes phase separation into a dilute and dense phase. We show that the phase chip lowers the required sample quantity by 98% while offering six-fold better statistics in comparison to standard manual experiments that involve centrifugal separation of dilute and dense phases. We further show that the saturation concentrations measured in chips are in agreement with previously reported data for a variety of biomolecules. Concomitantly, time-dependent changes of the dense phase morphology and potential off-pathway processes, including aggregation, can be monitored microscopically. In summary, the phase chip is suited to exploring sequence-to-binodal relationships by enabling the determination of a large number of saturation concentrations at low protein cost.

Impact of Adjuvant Radiation Therapy in Patients With Male Breast Cancer: A Multicenter International Analysis.

PURPOSE: Breast cancer in men accounts for approximately 1% of all breast cancers. Breast cancer trials have routinely excluded men. The aim of this analysis was to determine the effect of different treatment factors, in particular, postoperative radiation therapy (RT) on long-term outcomes. METHODS AND MATERIALS: Seventy-one patients with male breast cancer treated in 5 closely cooperating institutions between 2003 and 2019 were analyzed. RESULTS: Almost all patients (95%) underwent surgical resection. Forty-two patients (59%) received chemotherapy, and 59 (83%) received adjuvant hormonal therapy. Of the 71 patients, 52 (73%) were treated with RT. The rate of recurrence was 20% in the whole cohort, with a locoregional recurrence rate of 3%. In the entire group, the 5-year local control (LC) was 95%, whereas 5-year progression-free survival (PFS) and 5-year overall survival (OS) were 62% and 96%, respectively. There was a lower rate of relapses after adjuvant RT (19% vs 32%, P = .05) without in-field relapse after postoperative RT (0%) versus 10% in patients without RT (P = .02). In the multivariate analysis performed, hormonal therapy administration was found to have a possible significant effect on LC and PFS. Administration of adjuvant RT and stage affect PFS. In patients who received RT, there were no grade 3 or 4 acute toxicities. CONCLUSIONS: Adjuvant RT is an effective and safe treatment for male breast cancer patients with no infield relapses and better PFS. Hormonal therapy administration was found to have a possible effect on LC and PFS.

Similar Yet Different-Structural and Functional Diversity among Arabidopsis thaliana LEA_4 Proteins.

The importance of intrinsically disordered late embryogenesis abundant (LEA) proteins in the tolerance to abiotic stresses involving cellular dehydration is undisputed. While structural transitions of LEA proteins in response to changes in water availability are commonly observed and several molecular functions have been suggested, a systematic, comprehensive and comparative study of possible underlying sequence-structure-function relationships is still lacking. We performed molecular dynamics (MD) simulations as well as spectroscopic and light scattering experiments to characterize six members of two distinct, lowly homologous clades of LEA_4 family proteins from Arabidopsis thaliana. We compared structural and functional characteristics to elucidate to what degree structure and function are encoded in LEA protein sequences and complemented these findings with physicochemical properties identified in a systematic bioinformatics study of the entire Arabidopsis thaliana LEA_4 family. Our results demonstrate that although the six experimentally characterized LEA_4 proteins have similar structural and functional characteristics, differences concerning their folding propensity and membrane stabilization capacity during a freeze/thaw cycle are obvious. These differences cannot be easily attributed to sequence conservation, simple physicochemical characteristics or the abundance of sequence motifs. Moreover, the folding propensity does not appear to be correlated with membrane stabilization capacity. Therefore, the refinement of LEA_4 structural and functional properties is likely encoded in specific patterns of their physicochemical characteristics.