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Biology subjects

Michel, J.

Publications and source records attributed to Michel, J..

At least 19 recordsLinked to original sources

Teatime for Triticum: (how) can the presence of plants slow down decomposition?

Decomposition of organic matter is a key process in soils contributing to carbon and nutrient cycling. To identify management strategies for agroecosystems that reduce nutrient losses while maximizing plant growth, it is important to understand which parameters determine decomposition rates. This study therefore investigated how the presence of winter wheat (Triticum aestivum var. Asory) affects decomposition in a controlled Ecotron setup with two soil types with varying organic matter content across three simulated climates (2013, 2068, 2085). Using the tea bag index, interstitial soil pore water analyses, microbial biomass quantification, bacterial and fungal gene abundance, and soil respiration measurements, we tested the hypotheses that plant exudates would enhance decomposition rate and microbial biomass, while plant nitrogen uptake would deplete soil nitrate, potentially mitigated by fertilization. Contrary to expectations, decomposition rates were lower in planted than in unplanted soils, suggesting resource competition between plants and microbes. No significant differences were observed in microbial biomass or respiration due to plant presence, and fertilization effects on nitrate or microbial mineralization were undetectable, likely due to rapid turnover of organic molecules including uptake by plants and microbes. Mechanistically, fungi and soil humidity were more important for decomposition than bacteria or temperature. The findings corroborate climate impacts on decomposition but also indicate microbial resilience and highlight the potential of management strategies like cover crops, adjusted planting dates and crop residual management which can contribute to healthy soils by sustaining carbon and nutrient cycling.

ecology↗

3D, multi-omic imaging reveals molecular biomarkers of the pre-metastatic niche in lung cancer

The recurrence rate following complete surgical resection of primary non-small cell lung cancer is as high as 55%, yet no approach currently exists to evaluate the risk of local recurrence. The premetastatic paradigm is the recognition that metastasis is preceded by reprogramming naive tissues to prime a microenvironment for tumor cell survival and subsequent reactivation. Identification of biomarkers of the pre-metastatic niche would allow us to evaluate a patients risk of local relapse in the normal lung parenchyma surrounding the resected tumor. We designed a workflow incorporating in vivo modelling, radiology, and deep learning-guided three-dimensional (3D) imaging, spatial proteomics, and transcriptomics to identify previously unreported signals associated with the early transformation of the lung parenchyma announcing regional metastasis. We curated biorepository spanning timepoints before and after resection of primary Lewis Lung Carcinoma (LLC) tumors. Using radiology and cellular resolution 3D histology, we calculated the number and distribution of metastases in mouse lungs and developed an algorithm to guide placement of spatial proteomics and transcriptomics to regions containing early micro-metastases and the pre-metastatic microenvironment. Molecular and tissue features associated with presence, size, and location of metastases guided the identification of both myeloid (F4/80) and senescent (p16/p21) cell signatures in the premetastatic and metastatic environments. Finally, multiparametric flow cytometry of metastatic lungs in a senescence reporter GEMM (tdTomato-p16 INKA mice) resolved senescent cells including alveolar macrophages as the cellular phenotypes associated with these early premetastatic signatures. Altogether, this work highlights a novel AI-assisted approach for detection of biomarkers of tissue remodeling during lung cancer invasion.

bioengineering↗

Conserved programs and specificities of T cells targeting hematological malignancies

T cell-mediated immune surveillance is critical for cancer control, yet its endogenous effectiveness in hematological malignancies remains limited and poorly understood. Here, we integrate single-cell T cell receptor (TCR) profiling, HLA immunopeptidomics and functional antigen mapping to dissect the specificity landscape of bone marrow lymphocytes (BMLs) in multiple myeloma (MM) and acute myeloid leukemia (AML). We identify a rare subset of tumor-reactive T cells that exhibit a stereotyped transcriptional state distinct from bystander and virus-specific populations. Across both malignancies, immunopeptidomic profiling uncovers a partially conserved antigen repertoire enriched for noncanonical peptides, including products of novel or unannotated open reading frames (nuORFs), pseudogenes, and clonotypic immunoglobulin sequences. Several of these epitopes are recurrently presented and associated with convergent TCR responses across individuals. Based on this immune architecture, we develop a TCR-intrinsic fitness model that infers BML tumor specificity from transcriptional cues and stratifies immunotherapy response across three independent patient cohorts. Together, these findings map the latent potential of endogenous anti-tumor immunity in two biologically distinct diseases and provide a framework for decoding and restoring productive immune surveillance of hematological malignancies. HighlightsO_LISingle-cell resolved TCR profiling maps rare tumor-reactive T cells in the bone marrow of multiple myeloma (MM) and acute myeloid leukemia (AML) reveals conserved transcriptional programs C_LIO_LIA shared immunopeptidome across MM and AML includes noncanonical epitopes from nuORFs and idiotype sequences C_LIO_LIConserved tumor antigens elicit convergent T cell responses across patients C_LI O_LIA TCR fitness model predicts tumor specificity in bone marrow lymphocytes and stratifies immunotherapy response in both hematological malignancies C_LI

immunology↗

Low and facultative mycorrhization of ferns in a low-montane tropical rainforest in Ecuador

Arbuscular mycorrhizal fungi (AMF) are amongst the most studied plant symbionts and regularly found in terrestrial plants. However, global estimates of AMF abundance amongst all land plants are difficult because i) the mycorrhizal status of many non-commercial, wild plant species is still unknown ii) numerous plant species engage in facultative symbiosis, meaning that they can, but do not always do, associate with mycorrhiza and iii) mycorrhizal status can vary amongst individuals of one same plant species at one location, as well as for different plant species within a given genus or family. To gain new insights to the pristine distribution of the plant-AMF symbiosis, we investigated the mycorrhizal status of one of the oldest lineages of extant vascular plants, Polypodiophyta (aka ferns), in one of the hotspots of natural plant diversification, the tropical rainforest. Providing a new data set of AMF abundance for 79 fern species, we hypothesized that (1) AMF would be found in 60-80% of the studied plants and (2) plant species with AMF symbionts would be more abundant than non-mycorrhizal species. Both hypotheses were rejected while unexpected observations were made: (1) AMF occurred in 33% of studied species, representing 56% of the studied fern families, (2) AMF colonisation was not correlated with species abundance, (3) a small but significant proportion of AMF-positive ferns was epiphytic (7%) and (4) as expected, mycorrhization was inconsistent among different populations of the same species. Together this empirical data supports recent reservations regarding global abundance of AMF, and further demonstrates that mycorrhization is not a taxonomic trait. In addition, the occurrence of AMF in epiphytic plants and no net benefits of AMF for plant abundance indicate that the mycorrhization observed here is on the commensalism, possibly parasitism, side of the symbiosis spectrum. HighlightsO_LISmall fraction of fern species mycorrhized (33% species level, 56% of families) C_LIO_LIMycorrhization of species can vary with location (facultative symbiosis) C_LIO_LIAMF colonisation does not increase plant species abundance C_LIO_LIAMF presence in epiphytic ferns suggests commensalism / parasitism C_LI

ecology↗

Tensions in tillage: Reduction in tillage intensity associates with lower wheat growth and nutritional grain quality despite enhanced soil biological indicators

Dryland ecosystems are particularly susceptible to the adverse effects of intensive agriculture, with intensive tillage exerting a major impact on soil health and its biotic components. The implementation of less disturbing soil management practices can be essential for preserving the soil environment and maintaining the diverse communities of microorganisms, micro- and mesofauna, which are essential contributors to soil fertility. In this study, we assessed soil chemical properties, soil biodiversity and functionality, and wheat crop growth across a tillage gradient encompassing no-tillage (NT), minimum tillage (MT), and standard tillage (ST). Results showed that NT resulted in increased soil macronutrient levels compared to MT and ST. In general, reduced tillage increased the abundance of soil biota, with significantly higher levels of bacterial and fungal marker genes observed in MT and NT compared to ST. Nematode abundance increased by 25% in MT and 50% in NT, compared to ST and predatory acari were significantly more abundant in NT, while numbers of total acari were higher in both NT and ST compared to MT. Community structure analysis revealed that tillage strongly influenced bacterial, fungal and acari community composition, reflecting a gradient of soil disturbance intensity. Corresponding to the increased abundance of soil biota, reduced tillage increased microbial activity and soil functionality along the disturbance gradient. This was evident in the potential activity of carbon, nitrogen and phosphorus cycling enzymes, as well as the microbial capacity for carbon utilisation. In addition, evidence of the formation of biocrust as a possible source of carbon input was found. Furthermore, we observed important wheat pathogens to decrease and fungal antagonists to increase in NT compared to ST. Despite enhanced soil biological indicators under reduced tillage, wheat growth, nitrogen uptake and grain B vitamin contents were higher in ST compared to NT. In addition, we observed a shift in technological grain properties across tillage practices. The higher root:shoot ratio (an indicator of nitrogen deficiency) and median root diameter (hormone-driven lateral expansion) in NT suggest that soil compaction could be a potential cause of reduced wheat performance. These results suggest that despite improved soil biological indicators, other factors such as a low rates of N mineralization potential and prevalence of soil compaction may be limiting wheat performance in NT systems. HighlightsO_LIEnhanced microbial activity and functionality under reduced tillage C_LIO_LITillage intensity shaped community structure of microbes, nematodes and acari C_LIO_LISoil biocrust development under NT may increase soil organic carbon C_LIO_LIRoot traits revealed soil compaction and nutrient limitation in NT systems C_LIO_LIReduced tillage impaired wheat quality and changed technological grain properties C_LI

ecology↗

Low-input soil management increases yield and decreases CO2-emissions but aggravates risk of nitrate leaching and diseases in winter wheat cropping systems under climate change

Empirical data is key to anticipate the impact of climate change on cropping systems and develop land management strategies that are sustainable while ensuring food security. Here, the combined effects of projected increases in temperature, atmospheric CO2-concentrations, solar irradiation and altered precipitation patterns on winter wheat cropping systems were investigated using an Ecotron. Experimental plant-soil systems were subjected to three different climatic conditions representing a gradient of ongoing climate change implementing the weather patterns of the years 2013, 2068, and 2085 respectively. The wheat plants were grown in two differentially manged agricultural soil types: one with long-term low organic matter (OM) inputs and the other one with long-term high OM inputs. In the low OM system, the risk for plant diseases and nitrate leaching was increased, but it outperformed the high OM system with higher yields and lower CO2-emissions. Developing high-yielding cropping systems leveraging the CO2-fertilisation effect without sacrificing environmental health will therefore require further refined of management practices to improve nutrient cycling and reduce greenhouse gas emissions. One possibility is adapting crop rotations and cover crops to the shorter wheat cycle observed in the future climates to replenish soil nutrients and break disease cycles. Further, in both here studied soil types the wheat plants developed natural coping mechanisms against environmental stressors, such as enhanced root growth and increased levels of proline and silicon. Unravelling the molecular mechanisms that trigger such inherent plant defences is a further interesting target for breeding future crops. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=177 HEIGHT=200 SRC="FIGDIR/small/626142v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@fefc32org.highwire.dtl.DTLVardef@5f6757org.highwire.dtl.DTLVardef@1799ca4org.highwire.dtl.DTLVardef@11aec5c_HPS_FORMAT_FIGEXP M_FIG C_FIG

ecology↗

Molecular recording of cellular protein kinase activity with chemical labeling

Protein kinases control most cellular processes and aberrant kinase activity is involved in numerous diseases. To investigate the link between specific kinase activities and cellular phenotypes in heterogeneous cell populations and in vivo, we introduce molecular recorders of kinase activities for later analysis. Based on split-HaloTag and a phosphorylation-dependent molecular switch, our recorders become rapidly labeled in the presence of a specific kinase activity and a fluorescent HaloTag substrate. The kinase activity in a given cell controls the degree of fluorescent labeling whereas the recording window is set by the presence of the fluorescent substrate. We have designed specific recorders for four protein kinases, including protein kinase A. We apply our protein kinase A recorder for the sorting of heterogeneous cell populations and subsequent transcriptome analysis, in genome-wide CRISPR screens to discover regulators of PKA activity and for the tracking of neuromodulation in freely moving mice.

biochemistry↗

Aging and viral evolution impair immunity against dominant pan-coronavirus-reactive T cell epitope

Immune evasion by escape mutations subverts immunity against SARS-CoV-2. A role of pan-coronavirus immunity for more durable protection is being discussed but has remained understudied. We here investigated the effects of age, mutations, and homo-/heterologous vaccination regimens on the dominant pan-coronavirus-specific cellular and humoral epitope iCope after SARS-CoV-2 infection and vaccination in detail. In the older, quantitatively, and qualitatively reduced iCope-reactive CD4+ T cell responses with narrow TCR repertoires could not be enhanced by vaccination and were further compromised by emerging spike mutations. In contrast pan-coronavirus-reactive humoral immunity was affected only by mutations and not by age. Our results reveal a distinct deficiency of the dichotomous layer of pan-coronavirus immunity in the older, critical for long-term protection against SARS-CoV-2 variants. One-Sentence SummaryAging and viral evolution impair dominant pan-coronavirus immunity, a hallmark of efficient and broad immune competence against SARS-CoV-2

immunology↗

Comparison of Methodologies for Absolute Binding Free Energy Calculations of Ligands to Intrinsically Disordered Proteins

Modulating the function of Intrinsically Disordered Proteins (IDPs) with small molecules is of considerable importance given the crucial roles of IDPs in the patho-physiology of numerous diseases. Reported binding affinities for ligands to diverse IDPs vary broadly and little is known about the detailed molecular mechanisms that underpin ligand efficacy. Molecular simulations of IDP-ligand binding mechanisms can help us understand the mode of action of small molecule inhibitors of IDP function, but it is still unclear how binding energies can be modeled rigorously for such a flexible class of proteins. Here we compare alchemical absolute binding free energy calculation (ABFE) and Markov-State Modelling (MSM) protocols to model the binding of the small molecule 10058-F4 to a disordered peptide extracted from a segment of the oncoprotein c-Myc. The ABFE results produce binding energy estimates that are sensitive to the choice of reference structure. In contrast, the MSM results produce more reproducible binding energy estimates consistent with weak mM binding affinities and transient intermolecular contacts reported in the literature.

biophysics↗

Vegetative stage and soil horizon respectively determine direction and magnitude of rhizosphere priming effects in contrasting treeline soils

O_LITreelines in high latitudes and high altitudes are considered sentinels of global change. This manifests in accelerated encroachment of trees and shrubs and enhanced plant productivity, with currently unknown implications for the carbon balance of these biomes. Given the large soil organic carbon stocks in many treeline soils, we here wondered whether introducing highly productive plants would accelerate carbon cycling through rhizosphere priming effects and if certain soils would be more vulnerable to carbon loss from positive priming than others. C_LIO_LITo test this, organic and mineral soils were sampled above and below treelines in the Swedish sub-arctic and the Peruvian Andes. A greenhouse experiment was then performed to quantify plant-induced changes in soil mineralisation rates (rhizosphere priming effect) and new C formation using natural abundance labelling and the C4-species Cynodon dactylon. Several environmental, plant, soil and microbial parameter were monitored during the experiment to complement the observations on soil C cycling. C_LIO_LIPriming was predominantly positive at the beginning of the experiment, then systematically decreased in all soils during the plant growth season to be mostly negative at the end of the experiment at plant senescence. Independent of direction of priming, the magnitude of priming was always greater in organic than in corresponding mineral soils, which was best explained by the higher C contents of these soils. Integrated over the entire study period, the overall impact of priming (positive and negative) on the soil C balance was mostly negligible. Though, net soil C loss was observed in organic soils from the sub-arctic tundra in Sweden. C_LIO_LIMost notably, positive and negative priming effects were not mutually exclusive, rather omnipresent across ecosystems, depending on sampling time. The direction of priming seems to be fluctuating with plant productivity, rhizosphere carbon inputs and nutrient uptake. This highlights the need for integrative long-term studies if we aim to understand priming effects at ecosystem scale and greenhouse and laboratory studies must be validated in situ to enable reliable ecological upscaling. C_LI

ecology↗

Possible adaption of the 2022 Monkeypox virus to the human host through gene duplication and loss

Poxviruses are known to evolve slower than RNA viruses, with rearrangements such as gene gain and loss as the main driver for host adaption. In 2022 the world is being challenged by the largest global outbreak so far of Monkeypox virus, and the virus seems to have established itself in the human community. Here we report five MPXV genomes with extensive gene duplication and loss, including duplications of up to 18,000 bp to the opposed genome end, and deletions at the site of insertion of up to 16,000 bp, as a possible adaption to the human host.

molecular biology↗

Culture of human nasal olfactory stem cells and their extracellular vesicles as advanced therapy medicinal products

The olfactory ecto-mesenchymal stem cell (OE-MSC) are mesenchymal stem cells originating from the lamina propria of the nasal mucosa. They have neurogenic and immune-modulatory properties and showed therapeutic potential in animal models of spinal cord trauma, hearing loss, Parkinsonss disease, amnesia, and peripheral nerve injury. In this paper we designed a protocol that meet the requirements set by human health agencies to manufacture these stem cells for clinical applications. Once purified, OE-MSCs can be used per se or expanded in order to get the extracellular vesicles (EV) they secrete. A protocol for the extraction of these vesicles was validated and the EV from the OE-MSC were functionally tested on an in vitro model. Nasal mucosa biopsies from three donors were used to validate the manufacturing process of clinical grade OE-MSC. All stages were performed by expert staff of the cell therapy laboratory according to aseptic handling manipulations, requiring grade A laminar airflow. Enzymatic digestion provides more rapidly a high number of cells and is less likely to be contaminated. Foetal calf serum was replaced with human platelet lysate and allowed stronger cell proliferation, with the optimal percentage of platelet lysate being 10%. Cultivated OE-MSCs are sterile, highly proliferative (percentage of CFU-F progenitors was 15,5%) and their maintenance does not induce chromosomal rearrangement (karyotyping and chromosomal microarray analysis were normal). These cells express the usual phenotypic markers of OE-MSC. Purification of the EVs was performed with ultracentrifugation and size exclusion chromatography. Purified vesicles expressed the recognized markers of EVs (Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines) and promoted cell differentiation and neurite elongation in a model of neuroblastoma Neuro2a cell line. We developed a safer and more efficient manufacturing process for clinical-grade olfactory stem cells, these cells can now be used in humans. A phase I clinical trial will begin soon.An efficient protocol for the purification of the OE-MSC EVs have been validated. These EVs exert neurogenic properties in vitro. More studies are needed to understand the exact mechanisms of action of these EVs and prove their efficacy and safety in animal models.

neuroscience↗

Energetics of a protein disorder-order transition in small molecule recognition

Many proteins recognise other proteins via mechanisms that involve the folding of intrinsically disordered regions upon complex formation. Here we investigate how the selectivity of a drug-like small molecule arises from its modulation of a protein disorder-to-order transition. Binding of the compound AM-7209 has been reported to confer order upon an intrinsically disordered lid region of the oncoprotein MDM2. Calorimetric measurements revealed that truncation of the lid region of MDM2 increases the apparent dissociation constant of AM-7209 250-fold. By contrast, lid truncation has little effect on the binding of the ligand Nutlin-3a. Insights into these differential binding energetics were obtained via a complete thermodynamic analysis that featured adaptive absolute alchemical free energy of binding calculations with enhanced-sampling molecular dynamics simulations. The simulations reveal that in apo MDM2 the ordered lid state is energetically disfavoured. AM-7209, but not Nutlin-3a, shows a significant energetic preference for ordered lid conformations, thus shifting the balance towards ordering of the lid in the AM-7209/MDM2 complex. The methodology reported herein should facilitate broader targeting of intrinsically disordered regions in medicinal chemistry.

biophysics↗

Active Cytoskeletal Composites Display Emergent Tunable Contractility and Restructuring

The cytoskeleton is a model active matter system that controls diverse cellular processes from division to motility. While both active actomyosin dynamics and actin-microtubule interactions are key to the cytoskeletons versatility and adaptability, an understanding of their interplay is lacking. Here, we couple microscale experiments with mechanistic modeling to elucidate how connectivity, rigidity, and force-generation affect emergent material properties in in vitro composites of actin, tubulin, and myosin. We use time-resolved differential dynamic microscopy and spatial image autocorrelation to show that ballistic contraction occurs in composites with sufficient flexibility and motor density, but that a critical fraction of microtubules is necessary to sustain controlled dynamics. Our active double-network models reveal that percolated actomyosin networks are essential for contraction, but that networks with comparable actin and microtubule densities can uniquely resist mechanical stresses while simultaneously supporting substantial restructuring. Our findings provide a much-needed blueprint for designing cytoskeleton-inspired materials that couple tunability with resilience and adaptability.

biophysics↗

Morphometry of SARS-CoV and SARS-CoV-2 particles in ultrathin sections of infected Vero cell cultures.

SARS-CoV-2 is the causative of the COVID-19 disease, which has spread pandemically around the globe within a few months. It is therefore necessary to collect fundamental information about the disease, its epidemiology and treatment, as well as about the virus itself. While the virus has been identified rapidly, detailed ultrastructural analysis of virus cell biology and architecture is still in its infancy. We therefore studied the virus morphology and morphometry of SARS-CoV-2 in comparison to SARS-CoV as it appears in Vero cell cultures by using conventional thin section electron microscopy and electron tomography. Both virus isolates, SARS-CoV Frankfurt 1 and SARS-CoV-2 Italy-INMI1, were virtually identical at the ultrastructural level and revealed a very similar particle size distribution with a median of about 100 nm without spikes. Maximal spike length of both viruses was 23 nm. The number of spikes per virus particle was about 30% higher in the SARS-CoV than in the SARS-CoV-2 isolate. This result complements a previous qualitative finding, which was related to a lower productivity of SARS-CoV-2 in cell culture in comparison to SARS-CoV.

microbiology↗

The SAMPL6 SAMPLing challenge: Assessing the reliability and efficiency of binding free energy calculations

Approaches for computing small molecule binding free energies based on molecular simulations are now regularly being employed by academic and industry practitioners to study receptor-ligand systems and prioritize the synthesis of small molecules for ligand design. Given the variety of methods and implementations available, it is natural to ask how the convergence rates and final predictions of these methods compare. In this study, we describe the concept and results for the SAMPL6 SAMPLing challenge, the first challenge from the SAMPL series focusing on the assessment of convergence properties and reproducibility of binding free energy methodologies. We provided parameter files, partial charges, and multiple initial geometries for two octa-acid (OA) and one cucurbit[8]uril (CB8) host-guest systems. Participants submitted binding free energy predictions as a function of the number of force and energy evaluations for seven different alchemical and physical-pathway (i.e., potential of mean force and weighted ensemble of trajectories) methodologies implemented with the GROMACS, AMBER, NAMD, or OpenMM simulation engines. To rank the methods, we developed an efficiency statistic based on bias and variance of the free energy estimates. For the two small OA binders, the free energy estimates computed with alchemical and potential of mean force approaches show relatively similar variance and bias as a function of the number of energy/force evaluations, with the attach-pull-release (APR), GROMACS expanded ensemble, and NAMD double decoupling submissions obtaining the greatest efficiency. The differences between the methods increase when analyzing the CB8-quinine system, where both the guest size and correlation times for system dynamics are greater. For this system, nonequilibrium switching (GROMACS/NS-DS/SB) obtained the overall highest efficiency. Surprisingly, the results suggest that specifying force field parameters and partial charges is insufficient to generally ensure reproducibility, and we observe differences between seemingly converged predictions ranging approximately from 0.3 to 1.0 kcal/mol, even with almost identical simulations parameters and system setup (e.g., Lennard-Jones cutoff, ionic composition). Further work will be required to completely identify the exact source of these discrepancies. Among the conclusions emerging from the data, we found that Hamiltonian replica exchange--while displaying very small variance--can be affected by a slowly-decaying bias that depends on the initial population of the replicas, that bidirectional estimators are significantly more efficient than unidirectional estimators for nonequilibrium free energy calculations for systems considered, and that the Berendsen barostat introduces non-negligible artifacts in expanded ensemble simulations.

biophysics↗

Atomic Level Characterisation of Millisecond-Time Scale Protein Motions through a Combined Molecular Simulations and NMR Approach

Proteins need to interconvert between many conformations in order to function, many of which are formed transiently, and sparsely populated. Particularly when the lifetimes of these states approach the millisecond timescale, identifying the relevant structures and the mechanism by which they inter-convert remains a tremendous challenge. Here we introduce a novel combination of accelerated MD (aMD) simulations and Markov State modelling (MSM) to explore these excited conformational states. Applying this to the highly dynamic protein CypA, a protein involved in immune response and associated with HIV infection, we identify five principally populated conformational states and the atomistic mechanism by which they interconvert. A rational design strategy predicted that the mutant D66A should stabilise the minor conformations and substantially alter the dynamics whereas the similar mutant H70A should leave the landscape broadly unchanged. These predictions are confirmed using CPMG and R1{rho} solution state NMR measurements. By accurately and reliably exploring functionally relevant, but sparsely populated conformations with milli-second lifetimes in silico, our aMD/MSM method has tremendous promise for the design of dynamic protein free energy landscapes for both protein engineering and drug discovery.

biophysics↗