Exploring GPCR conformational dynamics using single-molecule fluorescence.

G protein-coupled receptors (GPCRs) are membrane proteins that transmit specific external stimuli into cells by changing their conformation. This conformational change allows them to couple and activate G-proteins to initiate signal transduction. A critical challenge in studying and inferring these structural dynamics arises from the complexity of the cellular environment, including the presence of various endogenous factors. Due to the recent advances in cell-expression systems, membrane-protein purification techniques, and labeling approaches, it is now possible to study the structural dynamics of GPCRs at a single-molecule level both in vitro and in live cells. In this review, we discuss state-of-the-art techniques and strategies for expressing, purifying, and labeling GPCRs in the context of single-molecule research. We also highlight four recent studies that demonstrate the applications of single-molecule microscopy in revealing the dynamics of GPCRs. These techniques are also useful as complementary methods to verify the results obtained from other structural biology tools like cryo-electron microscopy and x-ray crystallography.

Single-molecule analysis reveals that a glucagon-bound extracellular domain of the glucagon receptor is dynamic.

Dynamic information is vital to understanding the activation mechanism of G protein-coupled receptors (GPCRs). Despite the availability of high-resolution structures of different conformational states, the dynamics of those states at the molecular level are poorly understood. Here, we used total internal reflection fluorescence microscopy to study the extracellular domain (ECD) of the glucagon receptor (GCGR), a class B family GPCR that controls glucose homeostasis. Single-molecule fluorescence resonance energy transfer was used to observe the ECD dynamics of GCGR molecules expressed and purified from mammalian cells. We observed that for apo-GCGR, the ECD is dynamic and spent time predominantly in a closed conformation. In the presence of glucagon, the ECD is wide open and also shows more dynamic behavior than apo-GCGR, a finding that was not previously reported. These results suggest that both apo-GCGR and glucagon-bound GCGRs show reversible opening and closing of the ECD with respect to the seven-transmembrane (7TM) domain. This work demonstrates a molecular approach to visualizing the dynamics of the GCGR ECD and provides a foundation for understanding the conformational changes underlying GPCR activation, which is critical in the development of new therapeutics.

Prenatal Perception of WIC Breastfeeding Recommendations Predicts Breastfeeding Exclusivity and Duration in the Infants' First Year.

BACKGROUND: Pregnant participants who perceived that the Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) recommends breastfeeding only were more likely to have better early breastfeeding outcomes. OBJECTIVES: Our objective was to examine the association between prenatal perception of WIC's breastfeeding recommendations and breastfeeding duration through the first year of infant life. METHODS: This observational study used a national longitudinal sample of 1594 pregnant participants in the WIC Infant and Toddler Feeding Practices Study-2 in 2013. Four measures of breastfeeding duration were used: 1) a discrete measure of exclusive breastfeeding through 5 mo; 2) a continuous measure of exclusive breastfeeding (in days up to 7 mo); 3) a discrete measure of any breastfeeding through 11 mo; and 4) a continuous measure of any breastfeeding (in days up to 13 mo). The primary explanatory variable was the participant's prenatal perception of whether WIC recommended breastfeeding only. The univariate analyses of time to breastfeeding cessation were performed using Kaplan-Meier curves. The Cox regression model was adopted to estimate the likelihood of breastfeeding outcomes over time. All analyses accounted for complex survey design effects. RESULTS: Compared with their peers who perceived WIC to recommend formula only or both breastfeeding and formula equally, participants who perceived WIC as recommending breastfeeding only were less likely to stop exclusive breastfeeding through 5 mo (HR = 0.83; 95% CI: 0.69, 0.99) or to stop any breastfeeding through 11 mo (HR = 0.80; 95% CI: 0.69, 0.92), without controlling for prenatal infant feeding intentions. Similar patterns were observed in the 2 continuous measures, as they were also less likely to stop exclusive breastfeeding by 7 mo (HR = 0.78; 95% CI: 0.69, 0.90) or to stop any breastfeeding by 13 mo (HR = 0.82; 95% CI: 0.71, 0.95). CONCLUSIONS: Prenatal perception of WIC's breastfeeding recommendation can be a useful predictor of breastfeeding duration in WIC participants.

PIP2 promotes conformation-specific dimerization of the EphA2 membrane region.

The impact of the EphA2 receptor on cancer malignancy hinges on the two different ways it can be activated. EphA2 induces antioncogenic signaling after ligand binding, but ligand-independent activation of EphA2 is pro-oncogenic. It is believed that the transmembrane (TM) domain of EphA2 adopts two alternate conformations in the ligand-dependent and the ligand-independent states. However, it is poorly understood how the difference in TM helical crossing angles found in the two conformations impacts the activity and regulation of EphA2. We devised a method that uses hydrophobic matching to stabilize two conformations of a peptide comprising the EphA2 TM domain and a portion of the intracellular juxtamembrane (JM) segment. The two conformations exhibit different TM crossing angles, resembling the ligand-dependent and ligand-independent states. We developed a single-molecule technique using styrene maleic acid lipid particles to measure dimerization in membranes. We observed that the signaling lipid PIP2 promotes TM dimerization, but only in the small crossing angle state, which we propose corresponds to the ligand-independent conformation. In this state the two TMs are almost parallel, and the positively charged JM segments are expected to be close to each other, causing electrostatic repulsion. The mechanism PIP2 uses to promote dimerization might involve alleviating this repulsion due to its high density of negative charges. Our data reveal a conformational coupling between the TM and JM regions and suggest that PIP2 might directly exert a regulatory effect on EphA2 activation in cells that is specific to the ligand-independent conformation of the receptor.

Intake assessments of salivary cortisol, survey responses, and adverse childhood experiences are associated with recovery success in an abstinence-based treatment program for substance use disorders.

BACKGROUND: Connecting patients to treatment for a substance use disorder (SUD) that satisfies their needs is often complicated by confounding factors. A reliable measurement of patients' underlying stress level may be helpful because it often reflects many of the same confounders as their SUD. Whereas cortisol levels reflect physiological responses to stress, patients' cortisol levels during recovery from an SUD may serve as biomarkers for stressors that result in poor treatment outcomes, including early discontinuation of treatment. However, further exploration of the relationship between patients' cortisol levels and their treatment outcomes is needed for this approach to be clinically useful. METHODS: We enrolled participants from an abstinence-based, male-only, residential alcohol and drug recovery program to examine the relationship between salivary cortisol, stress exposure, ACEs, and treatment retention. RESULTS: Participants who remained in the program <90 days had significantly higher initial cortisol levels than those who remained ≥90 days (0.62 ± 0.074 µg/dl vs. 0.36 ± 0.037 µg/dl). Kaplan-Meier curves differed significantly when we grouped participants according to whether their cortisol level was below or above the overall average of 0.49 ± 0.044 µg/dl, with the median numbers of days before discontinuing being 110 and 60, respectively. A Cox proportional hazards model indicated that elevated salivary cortisol (with increases in µg/dl), marital/relationship status, and adverse childhood experiences (ACEs) score correlated significantly with hazards of discontinuing the program (hazard ratios for the three factors were 3.49, 2.39, and 1.50, respectively). DISCUSSION: Cortisol level may predict, at least partially, SUD treatment program retention regardless of individuals' numerous confounding factors or the substance used. If this approach is validated, it could enable providers to consider patients' cortisol levels at the time of admission to treatment to facilitate their retention in treatment and thereby enhance their recovery.

Novel variants of engineered water soluble mu opioid receptors with extensive mutations and removal of cysteines.

We have shown that water-soluble variants of the human mu opioid receptor (wsMOR) containing a reduced number of hydrophobic residues at the lipid-facing residues of the transmembrane (TM) helices can be expressed in E. coli. In this study, we tested the consequences of increasing the number of mutations on the surface of the transmembrane domain on the receptor's aqueous solubility and ligand binding properties, along with mutation of 11 cysteine residues regardless of their solvent exposure value and location in the protein. We computationally engineered 10 different variants of MOR, and tested four of them for expression in E. coli. We found that all four variants were successfully expressed and could be purified in high quantities. The variants have alpha helical structural content similar to that of the native MOR, and they also display binding affinities for the MOR antagonist (naltrexone) similar to the wsMOR variants we engineered previously that contained many fewer mutations. Furthermore, for these full-length variants, the helical content remains unchanged over a wide range of pH values (pH 6 ~ 9). This study demonstrates the flexibility and robustness of the water-soluble MOR variants with respect to additional designed mutations in the TM domain and changes in pH, whereupon the protein's structural integrity and its ligand binding affinity are maintained. These variants of the full-length MOR with less hydrophobic surface residues and less cysteines can be obtained in large amounts from expression in E. coli and can serve as novel tools to investigate structure-function relationships of the receptor.

Variations in utilization and clinical outcomes for endoscopic sphenopalatine artery ligation and endovascular arterial embolization in a single multi-hospital network.

PURPOSE: Endoscopic sphenopalatine artery ligation (ESPAL) and endovascular arterial embolization (EAE) are increasingly common treatment options for patients with refractory epistaxis. The objective of this study was to compare the utilization pattern and clinical outcomes between these interventions within our single multi-hospital network. MATERIALS AND METHODS: A retrospective study of all patients undergoing ESPAL and/or EAE within any of the hospitals in a single healthcare network between 2008 and 2017 was conducted. We compared differences in procedure utilization with various hospital characteristics. Secondarily, we evaluated clinical outcomes and costs associated with each procedure. RESULTS: Forty-three ESPAL and 33 EAE procedures were performed across 7 hospitals, with the majority of procedures being performed at teaching institutions (65% and 91%, p = .013). The majority of both interventions were performed in larger hospitals and EAE patients were more likely to undergo inter-hospital transfer compared to ESPAL patients (48.5% and 16.3%, p = .02). Success rates for ESPAL and EAE were comparable (95% and 93%); however, the median direct cost of treatment for EAE was significantly higher than the cost for ESPAL ($12984.89 and $5002.02, p < .0001). CONCLUSIONS: The majority of both ESPAL and EAE interventions were performed at teaching and larger hospitals. Transfers occurring prior to EAE may have been due to the limited availability of interventional radiology services, and likely contributed to the increased cost of treatment. ESPAL is a known cost-effective management strategy and should be considered early in treatment algorithms of refractory epistaxis.

Growth charts for individuals with Coffin-Siris syndrome.

Coffin-Siris syndrome (CSS; OMIM #135900) is a rare, multisystem syndrome caused by pathogenic variants in genes encoding the BRG-1 associated factors complex (BAF). Individuals with CSS often present with feeding difficulties and failure to thrive during infancy, in addition to a number of variable congenital anomalies. Nutritional interventions are used to support growth in this population, and growth hormone therapy has been reported in a limited number of cases. The purpose of this study was to construct CSS-specific growth charts to better characterize the growth in this population. Anthropometric data were collected from 99 individuals enrolled in the CSS/BAF pathway international registry via a retrospective chart review. All measurements obtained after the first exposure to growth hormone therapy were excluded from this analysis. Sex-specific centiles (5th, 50th, and 95th) were estimated for height, weight, and head circumference from birth to age 10. Cubic smoothing splines were then fit to the centile estimates and superimposed on normative male and female growth curves for comparison. The CSS patients in this cohort exhibited normal growth parameters at birth. By age 10, the weight and head circumference of the CSS cohort began to approach normal parameters. Stature, however, remained shortened at 10 years of age.

A DEAD-box protein acts through RNA to promote HIV-1 Rev-RRE assembly.

The HIV-1 Rev protein activates nuclear export of unspliced and partially spliced viral RNA transcripts, which encode the viral genome and the genes encoding viral structural proteins, by binding to and oligomerizing on the Rev Response Element (RRE). The human DEAD-box protein 1 (DDX1) enhances the RNA export activity of Rev through an unknown mechanism. Using a single-molecule assembly assay and various DDX1 mutants, we show that DDX1 acts through the RRE RNA to specifically accelerate the nucleation step of the Rev-RRE assembly process. Single-molecule Förster resonance energy transfer (smFRET) experiments using donor-labeled Rev and acceptor-labeled DDX1 show that both proteins can associate with a single RRE molecule. However, simultaneous interaction is only observed in a subset of binding events and does not explain the extent to which DDX1 promotes the nucleation step of Rev-RRE assembly. Together, these results are consistent with a model wherein DDX1 acts as an RNA chaperone, remodeling the RRE into a conformation that is pre-organized to bind the first Rev monomer, thereby promoting the overall Rev-RRE assembly process.

Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor ß2AR.

Binding of extracellular ligands to G protein-coupled receptors (GPCRs) initiates transmembrane signaling by inducing conformational changes on the cytoplasmic receptor surface. Knowledge of this process provides a platform for the development of GPCR-targeting drugs. Here, using a site-specific Cy3 fluorescence probe in the human ß2-adrenergic receptor (ß2AR), we observed that individual receptor molecules in the native-like environment of phospholipid nanodiscs undergo spontaneous transitions between two distinct conformational states. These states are assigned to inactive and active-like receptor conformations. Individual receptor molecules in the apo form repeatedly sample both conformations, with a bias toward the inactive conformation. Experiments in the presence of drug ligands show that binding of the full agonist formoterol shifts the conformational distribution in favor of the active-like conformation, whereas binding of the inverse agonist ICI-118,551 favors the inactive conformation. Analysis of single-molecule dwell-time distributions for each state reveals that formoterol increases the frequency of activation transitions, while also reducing the frequency of deactivation events. In contrast, the inverse agonist increases the frequency of deactivation transitions. Our observations account for the high level of basal activity of this receptor and provide insights that help to rationalize, on the molecular level, the widely documented variability of the pharmacological efficacies among GPCR-targeting drugs.

Disparities in emergency department visits in American children with asthma: 2006-2010.

OBJECTIVE: This article was to examine the trends in emergency department (ED) visits for asthma among American children in 2006-2010 across sociodemographic factors, parental smoking status, and children's body weight status. METHODS: We analyzed 5,535 children aged 2-17 years with current asthma in the Asthma Call-Back Survey in 2006-2010. Multivariate log binomial regression was used to examine the disparities of ED visits by demographics, socioeconomic status, parental smoking status, children's body weight status, and the level of asthma control. We controlled for average state-level air pollutants. Prevalence ratios (PRs) and 95% confidence intervals (CIs) were reported. RESULTS: Minority children with current asthma had higher risks of ED visits compared with white children in 2009 and 2010, e.g., the PR (95% CI) for black children in 2009 was 3.64 (1.79, 7.41). Children who had current asthma and more highly educated parents experienced a higher risk of ED visits in 2007 (PRs [95% CI] = 2.15 [1.02, 4.53] and 2.97 [1.29, 6.83] for children with some college or college-graduated parents), but not significant in other years. Children with uncontrolled asthma were significantly more likely to visit the ED in 2008 (PRs [95% CI] = 2.79 [1.44, 5.41] and 6.96 [3.55, 13.64] for not-well-controlled and very poorly controlled children with asthma). CONCLUSIONS: Minority children with current asthma or children with uncontrolled asthma were more likely to visit EDs for asthma treatment. However, the disparities in ED visits across sociodemographics, health status, or asthma control vary in scale and significance across time. More research is needed to explain these differences.

Risk of Colorectal Cancer among Patients with One or Multiple Metabolic Syndrome Components.

Background/Objectives: Dysfunctions of metabolic syndrome (MetS) have been identified as a significant risk factor for colorectal cancer (CRC). However, current colon cancer guidelines do not classify patients with MetS as high risk, thereby leaving these individuals vulnerable. Consequently, we explored the relationship between MetS, its individual components, and the development of CRC in a cohort of patients with MetS to assess the necessity for CRC screening in these individuals. Methods: This study included patients ages 18 and older that received a service from the Marshall-Health (MH) practice plan, Cabell-Huntington Hospital (CHH), MU/JCESOM's Edwards Comprehensive Cancer Center (ECCC), or the University of Kentucky HealthCare (UKHC) system between 2010 and 2018. We implemented log-binomial regression models to assess the individual and collective effects of MetS components after adjusting other CRC risk factors. Results: Given that CRC prevalence increases in the older population (aged 65 years and above), and that multiple components of MetS are observed within the same population, we analyzed the concurrent impact of all MetS components on CRC. Log-binomial regression models were implemented to assess the risk of CRC due to MetS components after adjusting other risk factors. Conclusions: We identified specific components that markedly increased CRC risk, suggesting that individuals with these components should be prioritized for early screening. These findings could significantly influence early CRC screening protocols, with the ultimate aim to reduce mortality associated with the disease.

Cholesterol inhibits assembly and activation of the EphA2 receptor.

The receptor tyrosine kinase EphA2 drives cancer malignancy by facilitating metastasis. EphA2 can be found in different self-assembly states: as a monomer, dimer, and oligomer. However, our understanding remains limited regarding which EphA2 state is responsible for driving pro-metastatic signaling. To address this limitation, we have developed SiMPull-POP, a single-molecule method for accurate quantification of membrane protein self-assembly. Our experiments revealed that a reduction of plasma membrane cholesterol strongly promoted EphA2 self-assembly. Indeed, low cholesterol caused a similar effect to the EphA2 ligand ephrinA1-Fc. These results indicate that cholesterol inhibits EphA2 assembly. Phosphorylation studies in different cell lines revealed that low cholesterol increased phospho-serine levels, the signature of oncogenic signaling. Investigation of the mechanism that cholesterol uses to inhibit the assembly and activity of EphA2 indicate an in-trans effect, where EphA2 is phosphorylated by protein kinase A downstream of beta-adrenergic receptor activity, which cholesterol also inhibits. Our study not only provides new mechanistic insights on EphA2 oncogenic function, but also suggests that cholesterol acts as a molecular safeguard mechanism that prevents uncontrolled self-assembly and activation of EphA2.

Dynamics of site switching in DNA polymerase.

DNA polymerases replicate DNA by catalyzing the template-directed polymerization of deoxynucleoside triphosphate (dNTP) substrates onto the 3' end of a growing DNA primer strand. Many DNA polymerases also possess a separate 3'-5' exonuclease activity that is used to remove misincorporated nucleotides from the nascent DNA (proofreading). The polymerase (pol) and exonuclease (exo) activities are spatially separated in different enzyme domains, indicating that a mechanism must exist to transfer the growing primer terminus from one site to the other. Here we report a single-molecule Förster resonance energy transfer (smFRET) system that directly monitors the movement of a DNA substrate between the pol and exo sites of DNA polymerase I Klenow fragment (KF). FRET trajectories recorded during the encounter between single polymerase and DNA molecules reveal that DNA can channel between the pol and exo sites in both directions while remaining closely associated with the enzyme (intramolecular transfer). In addition, it is evident from the trajectories that DNA can also dissociate from one site and subsequently rebind at the other (intermolecular transfer). Rate constants for each pathway have been determined by dwell-time analysis, revealing that intramolecular transfer is the faster of the two pathways. Unexpectedly, a mispaired primer terminus accesses the exo site more frequently when dNTP substrates are also present in solution, which is expected to enhance proofreading. Together, these results explain how the separate pol and exo activities of KF are physically coordinated to achieve efficient proofreading.

RNA looping by PTB: Evidence using FRET and NMR spectroscopy for a role in splicing repression.

Alternative splicing plays an important role in generating proteome diversity. The polypyrimidine tract-binding protein (PTB) is a key alternative splicing factor involved in exon repression. It has been proposed that PTB acts by looping out exons flanked by pyrimidine tracts. We present fluorescence, NMR, and in vivo splicing data in support of a role of PTB in inducing RNA loops. We show that the RNA recognition motifs (RRMs) 3 and 4 of PTB can bind two distant pyrimidine tracts and bring their 5' and 3' ends in close proximity, thus looping the RNA. Efficient looping requires an intervening sequence of 15 nucleotides or longer between the pyrimidine tracts. RRM3 and RRM4 bind the 5' and the 3' pyrimidine tracts, respectively, in a specific directionality and work synergistically for efficient splicing repression in vivo.

Maternal and Neonatal Outcomes in Women with Metabolic Syndrome and Substance Use Disorder.

INTRODUCTION: Metabolic syndrome amplifies the risk of gestational diabetes, preeclampsia, and preterm labor in pregnant women. Similarly, women with substance use disorder have worsened obstetric and birth outcomes. Despite these two conditions being major healthcare disparities in Appalachia, the health outcomes of this cohort have not been studied thus far. This study looks at the health outcomes of this cohort. METHOD AND RESULTS: In this retrospective cohort study, we analyzed 27,955 mothers who delivered at Cabell Huntington Hospital between January 2010 and November 2021. We implemented Chi-square tests to determine the associations and multiple logistic regression methods for comparison after controlling for other factors, and found that MetS, together with SUD, significantly increases the risk as well as the number of pregnancy complications such as gestational diabetes (p-value < 0.001), preeclampsia (p-value < 0.001), premature rupture (p-value < 0.001), preterm labor (p-value < 0.001), and newborn disorder (p-value < 0.001) compared to the women who had none or had either MetS or SUD alone. CONCLUSION: Women with both metabolic syndrome and substance abuse had worsened pregnancy and neonatal outcomes compared to women with metabolic syndrome or SUD alone. In conclusion, analysis of all the variables is crucial to strategically planning and implementing health interventions that will positively influence the health outcome of the pregnant woman as well as the child.

Aspergillus Coinfection in a Hydatid Cyst Cavity of Lung in an Immunocompetent Host: A Case Report and Review of Literature.

Aspergilloma (a saprophytic infection) typically colonizes lung cavities due to underlying diseases such as tuberculosis, bronchiectasis, cavitary lung cancer, sarcoidosis, and pulmonary infarctions. Rarely, aspergilloma has been noted within a hydatid cyst. Even if this was the case, it is more common to find the coexistence of aspergilloma and pulmonary echinococcal cysts in immunocompromised individuals. It is, however, very uncommon to find this coinfection in normal immune status individuals. Here, we report on the successfully treated case of a 30-year-old immunocompetent female from Western Nepal with histologically proven coinfection by these two pathogens. She had a prolonged history of exposure to domesticated dogs. She suffered from hemoptysis from time to time for 3 years with increased frequency in the last 30 days. She was misdiagnosed clinically during a past medical visit at a local health center. Her computed tomography (CT) scans showed well-defined nonenhancing cystic lesions in the anterior basal segment of the right lower lobe adjacent to the major fissure. She underwent enucleation of the cyst via right posterolateral thoracotomy. On further histopathological evaluation, laminated membranes of the ectocyst along with fungal elements were found, and periodic acid-Schiff (PAS) staining revealed Aspergillus in the form of septate hyphae and acute angle branching. Owing to patient's economic constraints and unavailability in our center, DNA testing and molecular characterization could not be performed which further highlights the essence of diagnosing and managing such cases in resource poor settings. Eventually, we reviewed 12 confirmed cases of this coinfection in immunocompetent individuals during a period of 7 years (2015-2022) comparing them to a systematic review of 22 confirmed cases of the same coinfection from 1995 to 2014.

Combined personalized therapy for the treatment of multiple giant keloids: a case report and literature review.

Keloids are the result of an abnormal wound-healing process and are associated with various risk factors. The majority of diagnoses are clinical. Successful treatment of keloid is challenging due to its nonregressing and recurring nature. Case presentation: We discuss the case of a 30-year-old mongoloid male who had multiple swellings over his body for the past 10 years. More striking are the giant keloids that are present over his bilateral scapulae. Diagnosis of keloid was made clinically. Smaller sessile lesions over his shoulder and upper limbs were subjected to intralesional 5-fluorouracil and triamcinolone injections, whereas the giant bilateral scapular keloids underwent excision and split skin grafting. Clinical discussion: Keloids usually present with firm and rubbery masses that extend beyond the site of the previous wound/injury. Keloids are diagnosed and evaluated clinically. Its differentiation from the hypertrophic scar is done based on the presence of multiple lesions beyond the site of the previous wound/injury. Conclusion: Treatment of keloids is difficult due to their nonregressing and recurring nature. Hence, the main goal of treatment is to tailor the therapy to the patient's needs such that the benefits outweigh the risks.

Single-molecule visualization of human A2A adenosine receptor activation by a G protein and constitutively activating mutations.

Mutations that constitutively activate G protein-coupled receptors (GPCRs), known as constitutively activating mutations (CAMs), modify cell signaling and interfere with drugs, resulting in diseases with limited treatment options. We utilize fluorescence imaging at the single-molecule level to visualize the dynamic process of CAM-mediated activation of the human A2A adenosine receptor (A2AAR) in real time. We observe an active-state population for all CAMs without agonist stimulation. Importantly, activating mutations significantly increase the population of an intermediate state crucial for receptor activation, notably distinct from the addition of a partner G protein. Activation kinetics show that while CAMs increase the frequency of transitions to the intermediate state, mutations altering sodium sensitivity increase transitions away from it. These findings indicate changes in GPCR function caused by mutations may be predicted based on whether they favor or disfavor formation of an intermediate state, providing a framework for designing receptors with altered functions or therapies that target intermediate states.