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Since the Force Concept Inventory in 1992, many concept inventories have been developed to cover classical scientific fields. However, there is a lack of concept inventories for interdisciplinary fields, such as biophysics. We introduce a Biophysics Concept Inventory Survey (BCIS), a 20-question, multiple-choice survey to measure student gains in biophysical concepts. The BCIS contains 5 question classifications: remember, understand, apply, analyze, and create, as well as question concepts divided into primarily physics or primarily biology questions. We administered the BCIS to 3 cohorts of students over 4 years. Each cohort participated in a 10-week summer Research Experience for Undergraduates (REU) in biophysics. We compared the presurvey (before REU) and postsurvey (after REU) scores to determine the fraction of the maximum possible gain or loss realized. Our analysis of the results suggests that the BCIS shows no biases based on sex or ethnicity. Further, we used the BCIS to show that 69% of the REU participants showed gains in biophysics concepts, with most of the total participant mean of gain occurring at higher levels of Bloom’s taxonomy: create and analyze. Overall, participants obtain slightly higher scores in physics (8% increase) than biology (5% increase) when comparing the pre- and postscores. The coronavirus disease 2019 pandemic allows a splitting of prepandemic and postpandemic cohorts, with the postpandemic cohort showing significantly larger gains than the prepandemic students. These results show that the BCIS, with question classifications and concepts, probes the students’ ability to apply knowledge to various biophysical science topics without underlying biases and enables instructors to obtain answers to important questions about the effectiveness of the educational programs. The BCIS fills a gap for interdisciplinary concept inventories. 

Transcription factors (TFs) regulate gene expression by binding to specific DNA sequences and gating access to genes. Even when the binding of TFs and their cofactors to DNA is reversible, indicating a reversible control of gene expression, there is little knowledge about the molecular effect DNA has on TFs. Using single-molecule multiparameter fluorescence spectroscopy, molecular dynamics simulations, and biochemical assays, we find that the monomeric form of the forkhead (FKH) domain of the human FoxP1 behaves as a disordered protein and increases its folded population when it dimerizes. Notably, DNA binding promotes a disordered FKH dimer bound to DNA, negatively controlling the stability of the dimeric FoxP1:DNA complex. The DNA-mediated reversible regulation on FKH dimers suggests that FoxP1-dependent gene suppression is unstable, and it must require the presence of other dimerization domains or cofactors to revert the negative impact exerted by the DNA. 

Transcription factors regulate gene expression by binding to DNA. They have disordered regions and specific DNA-binding domains. Binding to DNA causes structural changes, including folding and interactions with other molecules. The FoxP subfamily of transcription factors in humans is unique because they can form heterotypic interactions without DNA. However, it is unclear how they form heterodimers and how DNA binding affects their function. We used computational and experimental methods to study the structural changes in FoxP1's DNA-binding domain when it forms a heterodimer with FoxP2. We found that FoxP1 has complex and diverse conformational dynamics, transitioning between compact and extended states. Surprisingly, DNA binding increases the flexibility of FoxP1, contrary to the typical folding-upon-binding mechanism. In addition, we observed a 3-fold increase in the rate of heterodimerization after FoxP1 binds to DNA. These findings emphasize the importance of structural flexibility in promoting heterodimerization to form transcriptional complexes. 

Transcription factors are multidomain proteins with specific DNA binding and regulatory domains. In the human FoxP subfamily (FoxP1, FoxP2, FoxP3, and FoxP4) of transcription factors, a 90 residue-long disordered region links a Leucine Zipper (ZIP)—known to form coiled-coil dimers—and a Forkhead (FKH) domain—known to form domain swapping dimers. We used replica exchange discrete molecular dynamics simulations, single-molecule fluorescence experiments, and other biophysical tools to understand how domain tethering in FoxP1 impacts dimerization at ZIP and FKH domains and how DNA binding allosterically regulates their dimerization. We found that domain tethering promotes FoxP1 dimerization but inhibits a FKH domain-swapped structure. Furthermore, our findings indicate that the linker mediates the mutual organization and dynamics of ZIP and FKH domains, forming closed and open states with and without interdomain contacts, thus highlighting the role of the linkers in multidomain proteins. Finally, we found that DNA allosterically promotes structural changes that decrease the dimerization propensity of FoxP1. We postulate that, upon DNA binding, the interdomain linker plays a crucial role in the gene regulatory function of FoxP1. 

Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule studies of Förster Resonance Energy Transfer (smFRET) between a tethered donor and acceptor dye pair are a powerful tool to investigate the structure and dynamics of labeled molecules. However, capturing and quantifying conformational dynamics in intensity-based smFRET experiments remains challenging when the dynamics occur on the sub-millisecond timescale. The method of multiparameter fluorescence detection addresses this challenge by simultaneously registering fluorescence intensities and lifetimes of the donor and acceptor. Together, two FRET observables, the donor fluorescence lifetime τ D and the intensity-based FRET efficiency E, inform on the width of the FRET efficiency distribution as a characteristic fingerprint for conformational dynamics. We present a general framework for analyzing dynamics that relates average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms. We present parametric relations of these observables for interpreting the location of FRET populations in E–τ D diagrams, called FRET-lines. To facilitate the analysis of complex exchange equilibria, FRET-lines serve as reference curves for a graphical interpretation of experimental data to (i) identify conformational states, (ii) resolve their dynamic connectivity, (iii) compare different kinetic models, and (iv) infer polymer properties of unfolded or intrinsically disordered proteins. For a simplified graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data that decouples the motion of the fluorescence labels from the conformational dynamics of the biomolecule. Importantly, FRET-lines facilitate exploring complex dynamic models via easily computed experimental observables. We provide extensive computational tools to facilitate applying FRET-lines. 

Single-molecule Förster Resonance Energy Transfer (smFRET) experiments are ideally suited to resolve the structural dynamics of biomolecules. A significant challenge to date is capturing and quantifying the exchange between multiple conformational states, mainly when these dynamics occur on the sub-millisecond timescale. Many methods for quantitative analysis are challenged if more than two states are involved, and the appropriate choice of the number of states in the kinetic network is difficult. An additional complication arises if dynamically active molecules coexist with pseudo-static molecules in similar conformational states with undistinguishable Förster Resonance Energy Transfer (FRET) efficiencies. To address these problems, we developed a quantitative integrative analysis framework that combines the information from FRET-lines that relate average fluorescence lifetimes and intensities in two-dimensional burst frequency histograms, fluorescence decays obtained by time-correlated single-photon-counting, photon distribution analysis of the intensities, and fluorescence correlation spectroscopy. Individually, these methodologies provide ambiguous results for the characterization of dynamics in complex kinetic networks. However, the global analysis approach enables accurate determination of the number of states, their kinetic connectivity, the transition rate constants, and species fractions. To challenge the potential of smFRET experiments for studying multi-state kinetic networks, we apply our integrative framework using a set of synthetic data for three-state systems with different kinetic connectivity and exchange rates. Our methodology paves the way toward an integrated analysis of multiparameter smFRET experiments that spans all dimensions of the experimental data. Finally, we propose a workflow for the analysis and show examples that demonstrate the usefulness of this toolkit for dynamic structural biology. 

The scaffold protein PSD-95 links postsynaptic receptors to sites of presynaptic neurotransmitter release. Flexible linkers between folded domains in PSD-95 enable a dynamic supertertiary structure. Interdomain interactions within the PSG supramodule, formed by P DZ3, S H3, and G uanylate Kinase domains, regulate PSD-95 activity. Here we combined discrete molecular dynamics and single molecule Förster resonance energy transfer (FRET) to characterize the PSG supramodule, with time resolution spanning picoseconds to seconds. We used a FRET network to measure distances in full-length PSD-95 and model the conformational ensemble. We found that PDZ3 samples two conformational basins, which we confirmed with disulfide mapping. To understand effects on activity, we measured binding of the synaptic adhesion protein neuroligin. We found that PSD-95 bound neuroligin well at physiological pH while truncated PDZ3 bound poorly. Our hybrid structural models reveal how the supertertiary context of PDZ3 enables recognition of this critical synaptic ligand. 

Thiamine pyrophosphate (TPP) riboswitches regulate thiamine metabolism by inhibiting the translation of enzymes essential to thiamine synthesis pathways upon binding to thiamine pyrophosphate in cells across all domains of life. Recent work on the Arabidopsis thaliana TPP riboswitch suggests a multistep TPP binding process involving multiple riboswitch configurational ensembles and Mg 2+ dependence underlies the mechanism of TPP recognition and subsequent transition to the expression-inhibiting state of the aptamer domain followed by changes in the expression platform. However, details of the relationship between TPP riboswitch conformational changes and interactions with TPP and Mg 2+ in the aptamer domain constituting this mechanism are unknown. Therefore, we integrated single-molecule multiparameter fluorescence and force spectroscopy with atomistic molecular dynamics simulations and found that conformational transitions within the aptamer domain's sensor helices associated with TPP and Mg 2+ ligand binding occurred between at least five different ensembles on timescales ranging from µs to ms. These dynamics are orders of magnitude faster than the 10 sec-timescale folding kinetics associated with expression-state switching in the switch helix. Together, our results show that a TPP and Mg 2+ dependent mechanism determines dynamic configurational state ensemble switching of the aptamer domain's sensor helices that regulate the switch helix's stability, which ultimately may lead to the expression-inhibiting state of the riboswitch. Additionally, we propose that two pathways exist for ligand recognition and that this mechanism underlies a kinetic rheostat-like behavior of the Arabidopsis thaliana TPP riboswitch.