Biochemistry, Biophysics, and Structural Biology Commons^™

The Effects Of Tubulin Post-Translational Modifications On The Flagellar Motility Of Trypanosoma Brucei, Katherine Wentworth

All Theses

Trypanosoma brucei is a parasitic kinetoplastid that causes African trypanosomiasis and is transmitted to a mammalian host by the tsetse fly (Glossina spp.). T. brucei relies on its flagellar motility to carry out its morphological changes from the procyclic form (predominant in the fly vector) to the bloodstream form (infectious form in mammals) and navigate the bloodstream of its host. The driving structure within the flagellum is the axoneme, which is composed of microtubules and dynein motor proteins. The tubulin code hypothesis suggests that cells regulate microtubule motor protein activity through post-translational modifications (PTMs) of alpha and beta …

Proteasome Inhibitors Silence Oncogenes In Multiple Myeloma Through Localized Histone Deacetylase 3 (Hdac3) Stabilization And Chromatin Condensation, Laure Maneix, Polina Iakova, Shannon E Moree, Joanne I Hsu, Ragini M Mistry, Fabio Stossi, Premal Lulla, Zheng Sun, Ergun Sahin, Sarvari V Yellapragada, André Catic

Faculty, Staff and Students Publications

Proteasome inhibitors have become the standard of care for multiple myeloma (MM). Blocking protein degradation particularly perturbs the homeostasis of short-lived polypeptides such as transcription factors and epigenetic regulators. To determine how proteasome inhibitors directly impact gene regulation, we performed an integrative genomics study in MM cells. We discovered that proteasome inhibitors reduce the turnover of DNA-associated proteins and repress genes necessary for proliferation through epigenetic silencing. Specifically, proteasome inhibition results in the localized accumulation of histone deacetylase 3 (HDAC3) at defined genomic sites, which reduces H3K27 acetylation and increases chromatin condensation. The loss of active chromatin at super-enhancers critical …

Multicenter Randomized Clinical Trial Of A Mailed Outreach Strategy For Hepatocellular Carcinoma Surveillance, Amit G Singal, Sarah Reddy, Himani Radadiya Aka Patel, Deyaun Villarreal, Aisha Khan, Yan Liu, Vanessa Cerda, Nicole E Rich, Caitlin C Murphy, Jasmin A Tiro, Jennifer R Kramer, Ruben Hernaez

Faculty, Staff and Students Publications

BACKGROUND: The effectiveness of hepatocellular carcinoma (HCC) surveillance is mitigated by underuse in clinical practice, highlighting a need for interventions. We evaluated the effectiveness of mailed HCC surveillance outreach to promote HCC surveillance in patients with cirrhosis.

METHODS: We conducted a multicenter pragmatic randomized clinical trial comparing mailed outreach for surveillance ultrasound (n = 1436) and usual care with visit-based surveillance (n = 1436) among patients with cirrhosis at 3 health systems (tertiary care referral center, safety net health system, and Veterans Affairs medical center) from April 2018 to December 2019. The primary outcome of this interim analysis was guideline …

Distinctive Kidney Function Trajectories Following Left Ventricular Assist Device Implantation, Carl P Walther, Julia S Benoit, Harveen K Lamba, Andrew B Civitello, Kevin F Erickson, Nandan K Mondal, Kenneth K Liao, Sankar D Navaneethan

Faculty, Staff and Students Publications

BACKGROUND: The aim of this study was to assess for distinct kidney function trajectories following left ventricular assist device (LVAD) placement. Cohort studies of LVAD recipients demonstrate that kidney function tends to increase early after LVAD placement, followed by decline and limited sustained improvement. Inter-individual differences in kidney function response may be obscured.

METHODS: We identified continuous flow LVAD implantations in US adults (2016-2017) from INTERMACS (Interagency Registry for Mechanically Assisted Circulatory Support). Primary outcomes were estimated glomerular filtration rate (eGFR) trajectories pre-implantation to ∼12 months. Latent class mixed models were applied to primary and validation samples. Clinical differences among …

Pediatric Lung Transplantation For Covid-19: Unique Clinical And Psychosocial Barriers, Anupam Kumar, Gloria W Li, Justin M Segraves, Aladdein Mattar, Maheshwari Ramineni, Gabriel Loor, Puneet S Garcha

Faculty, Staff and Students Publications

BACKGROUND: SARS-CoV-2 infection in the age group of 0-17 years contributes to approximately 22% of all laboratory-confirmed SARS-CoV-2 infections. Fortunately, this age group has a lower death rate (0.5 per 100 000) that accounts for only 4% of the total deaths due to COVID-19. Despite the low mortality rate in the pediatric population, children of minority groups represented 78% of the deaths highlighting the existing disparities in access to health care.

METHODS: With the emergence of the more contagious COVID-19 variants and the relatively slow pace of vaccination among the pediatric population, it is possible to see more cases of …

Patient And Clinician Perceptions Of Precision Cardiology Care: Findings From The Heartcare Study, Hadley Stevens Smith, Clarissa E Sanchez, Ronald Maag, Alexandria Buentello, David R Murdock, Ginger A Metcalf, Trevor D Hadley, Daniel L Riconda, Eric Boerwinkle, Xander H T Wehrens, Christie M Ballantyne, Richard A Gibbs, Amy L Mcguire, Stacey Pereira

Faculty, Staff and Students Publications

BACKGROUND: Routine genome-wide screening for cardiovascular disease risk may inform clinical decision-making. However, little is known about whether clinicians and patients would find such testing useful or acceptable within the context of a genomics-enabled learning health system.

METHODS: We conducted surveys with patients and their clinicians who were participating in the HeartCare Study, a precision cardiology care project that returned results from a next-generation sequencing panel of 158 genes associated with cardiovascular disease risk. Six weeks after return of results, we assessed patients' and clinicians' perceived utility and disutility of HeartCare, the effect of the test on clinical recommendations, and …

Assessment For Deeper Understanding Using Concept Maps: Lessons Learned From Flipped Teaching Of Pharmacology, Munder Zagaar, Weichao Chen

Faculty, Staff and Students Publications

To foster a deeper understanding of pharmacology concepts among physician assistant students, we integrated concept mapping into our flipped teaching to provide assessment for learning. Different mapping-based assessment strategies were adopted based on learner feedback, including in-person collaborative mapping, an individual computerized mapping-based quiz with automated feedback, and a collaborative computerized mapping-based quiz enhanced by the jigsaw strategy. Each mapping activity also leveraged the strength of a specific technology platform. Based on the findings from comparing learner ratings of these mapping activities and thematic analysis of learner feedback, we engaged in critical reflection and share our lessons learned.

Factors Affecting Need For Blood Transfusion In Paediatric Patients Undergoing Open Surgery For Hip Dysplasia, Adam C Adler, Lisa A H Hensch, Brittany E Bryant, Arvind Chandrakantan, Hai-Yen Nguyen, Brian H Nathanson, Scott B Rosenfeld

Faculty, Staff and Students Publications

BACKGROUND AND OBJECTIVES: The management of intraoperative blood loss in the surgical treatment of paediatric hip dysplasia is resource intensive. There are numerous clinical factors that impact the need for intraoperative transfusion. Identification of patient and surgical factors associated with increased blood loss may reduce the unnecessary use of resources. This study aimed to identify factors predictive of intraoperative transfusion in children undergoing hip dysplasia surgery.

MATERIALS AND METHODS: This is a single-centre retrospective review of patients undergoing surgery for hip dysplasia from 1 January 2012 to 15 April 2021. Patient demographic factors, anaesthetic, surgical and transfusion histories were reviewed. …

Metabolic Foundations Of Exercise-Induced Cardiac Growth., Kyle Fulghum

Electronic Theses and Dissertations

Regular aerobic exercise promotes physiological cardiac growth, which is an adaptive response thought to enable the heart to meet higher physical demands. Cardiac growth involves coordination of catabolic and anabolic activities to support ATP generation, macromolecule biosynthesis, and myocyte hypertrophy. Although previous studies suggest that exercise-induced reductions in cardiac glycolysis are critical for physiological myocyte hypertrophy, it remains unclear how exercise influences the many interlinked pathways of metabolism that support adaptive remodeling of the heart. In this thesis project, we tested the general hypothesis that aerobic exercise promotes physiological cardiac growth by coordinating myocardial metabolism to promote glucose-supported anabolic pathway …

Re-Engineering Cardio-Oncology Testing Using Biomimetic Heart Slice Cultures., Jessica Miller

Electronic Theses and Dissertations

28% of drug withdrawal from the market are due to unforeseen disruptions in cardiomyocyte contractility and electrophysiology. The most commonly used platforms for drug testing are in vivo animal models and in vitro cell culture models. While both have been of paramount importance for the discovery and detection of many cardiotoxicities and mechanisms of action, they lack the ability to model an intact human myocardium. This work aims to establish cardiac tissue slices, which are 300-micron thin tissue sections taken from the left ventricular myocardium, as an alternative platform for cardio-oncology studies, specifically cardiotoxicity testing. Additionally, this work aims to …

Structure, Function, And Immunogenic Applications Of Ab5-Type Adp-Ribosylating Bacterial Toxins, Elise Marie Overgaard

Boise State University Theses and Dissertations

Bacterial mono-ADP-ribosyltransferases (ARTs) catalyze the singular transfer of an ADP-ribose moiety from an NAD+ molecule onto a target molecule. ARTs contain an ancient and highly conserved tertiary structure and have a wide variety of intracellular targets and effects. Some, but not all, bacterial ARTs have an AB₅-type multimeric structure consisting of an enzymatically active subunit non-covalently situated atop of a non-toxic pentamer. The active, or A, subunit of AB₅-type toxins has a catalytic action that contributes to bacterial pathogenicity, and it is sometimes, but not always, an ART. ArtAB is an ART with AB₅-type …

Characterization Of The Isothermal Titration Calorimetry Single Injection Method Using T4 Polynucleotide Kinase As A Model System For Kinases, Rebecca Chaehee Lim

UNLV Theses, Dissertations, Professional Papers, and Capstones

Kinases are an important class of enzymes involved in the regulation of different cellular processes. The dysfunctional activity, either hyperactivity or inactivity, of kinases has been associated with many types of diseases, making kinases a major therapeutic target. As of 2020, more than 80 kinase inhibitors have been FDA-approved and have revolutionized the treatment for progressive disorders such as cancers and Alzheimer's diseases. However, there is always the possibility of developing severe side effects or resistance to drugs so the search for new therapeutics must continue with efficiency and accuracy.Isothermal titration calorimetry (ITC) is a state-of-the-art technique specialized in detecting …

Ankyrin Dependent Mitochondrial Function And Bioenergetics In The Heart, Janani Subramaniam, Janani Subramaniam

Dissertations and Theses (Open Access)

ANK2 mutations in patients are associated with numerous arrhythmias, cardiomyopathies, and other heart defects. In the heart, AnkB, the protein encoded by ANK2, clusters relevant ion channels and cell adhesion molecules in several important domains; however, its role at Mitochondria Associated ER/SR Membranes (MAMs) has yet to be investigated. MAMs are crucial to mitochondrial function and metabolism and are signaling hubs implicated in various cardiac pathologies. Among several functions, these sites mediate the direct transfer of calcium from the ER/SR to the mitochondria to modulate ATP synthesis. Given that mitochondrial function and energy production are paramount to cardiovascular heath, …

The Metabolic Change Of Lipid Species In Stam Mice With Hepatocellular Carcinoma, Devin J. Peart

UNLV Theses, Dissertations, Professional Papers, and Capstones

Primary liver cancer hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. In the US, primary liver cancer is the most rapidly increasing type of cancer due to increases in hepatitis B and C and the continued trend of greater and greater percentages of the population suffering from obesity, diabetes mellitus and metabolic syndrome. The prognosis for this disease is poor, with a 5-year survival rate of 26% as compared to only 2% when the cancer is metastatic. Unfortunately, current strategies for early detection of HCC, including routine CT imaging or alpha-fetoprotein biomarker, are suboptimal and tend …

The Role Of Fatty Acid Metabolism In The Pathogenesis Of Trypanosoma Brucei, Nava Poudyal

All Dissertations

Trypanosoma brucei is the protozoan parasite that causes African Sleeping Sickness in humans and nagana, a wasting disease in cattle. T. brucei completes its life cycle in two hosts, mammals and the tsetse fly insect vector. Due to the geographical restriction of the tsetse fly, the disease is endemic in sub-Saharan Africa. Both the insect and mammalian forms of the parasite need fatty acids to anchor their surface proteins. We worked on three projects on fatty acid metabolism and its role in immune evasion strategies of T. brucei. First, we assessed the role of T. brucei surface proteins in …

The Antimicrobial Resistance Plasmid Mobilome Of Salmonella Enterica And Related Enteric Species And Factors That Influence The Transfer Efficiency., Suad Algarni

Graduate Theses and Dissertations

The dynamic distribution of antimicrobial resistance genes in Salmonella enterica is considered a public health risk. S. enterica is one of the most important etiological agents of foodborne illness and has a critical impact on global human health. In S. enterica and related species, mobile genetic elements (MGEs) serve as primary vehicles for the dissemination of antibiotic resistance genes in the bacterial evolution. This dissemination can be impacted by different selective pressures that leads to diverse antibiotic response phenotypes. This project focusses on the dynamics of antimicrobial resistance genes, particularly exploring the transfer efficiency of multidrug resistance plasmids in S. …

Using Molecular Dynamics Simulations To Decipher Mechanistic Details Of Biomolecular Processes Of Biology And Biotechnology Oriented Applications, Adithya Polasa

Graduate Theses and Dissertations

Researchers in chemistry and biology often utilize computer simulations, in conjunction with experimental data, to model and predict the structures, energies, kinetics, processes, and functions of the systems that are their focus of study, ranging from single molecules to whole viruses. Here, we use molecular dynamics (MD) techniques to gain a deeper understanding of biomolecular processes in biology and biotechnology-oriented applications. Using a mixture of equilibrium and non-equilibrium MD simulations, this work describes the insertion process of YidC at the atomic level. In order to better comprehend the insertion process, several docking models of YidC-Pf3 in the lipid bilayer were …

Molecular Characterization Of Nitrogenase Regulation In Methanosarcina Acetivorans, Melissa Chanderban

Graduate Theses and Dissertations

Nitrogenase is the metalloenzyme only found in bacteria and archaea that is essential for biological nitrogen fixation (diazotrophy), but it can also serve as a catalyst in biofuel production. All diazotrophs contain a molybdenum (Mo) nitrogenase, while some species contain additional alternative nitrogenases where either vanadium (V) or iron (Fe) replace Mo in the active site cofactor. Nitrogen fixation by bacteria has been extensively studied. The limited investigation of nitrogen fixation in methanogenic archaea (methanogens) indicates production of nitrogenase is simpler than in bacteria and methanogen nitrogenase has different biochemical properties. Thus, methanogen nitrogenases provide a promising alternative for genetic …

The Binding Of The Micronutrient Transition Metals To The Alkylation Products Of Chemical Warfare Agent, Sulfur Mustard, And Thiols, Potentially Giving New Understanding To Physiological Effects Of Exposure And Increased Toxicity, Colin O'Donnell

Graduate Theses and Dissertations

Model compounds, 3,6,9-trithaiundecane-1,11-dicarboxylic acid (TTDPA), 2,5,8-trithianonane-1,9-dicarboxylic acid (TTDAA), and 1,11-diamide-3,6,9-trithiaundecane (TTDAce), closely related to the adducts formed by cysteine alkylation of the chemical weapon, sulfur mustard, were synthesized. It is shown that TTDPA forms complexes with key metal micronutrients: copper, nickel, cobalt, manganese, and zinc. Though the strength of binding to TTDPA varies, the complexes in many cases precipitate from solution. All metals produced a visible precipitate upon interaction with TTDPA under the conditions tested, however only Cu2+, Mn2+, and Zn2+ produced enough to be measured. The mass of formed precipitate seemed to peak at an equimolar ratio of TTDPA …

Comparative Analysis Of Conformational Transition Pathways In Homologous Proteins, Dylan Sebastien Ogden

Graduate Theses and Dissertations

Molecular dynamics (MD) simulations are routinely used to study the dynamics of proteins. However, conventional MD limited to the sampling of local conformational changes as the functionally important conformational transitions of proteins often extend beyond the timescales of the simulations employed, for example, membrane transport proteins. We have determined the combination of multiple MD based techniques that allows for a rigorous characterization of energetics and kinetics of large-scale conformational changes in membrane proteins. The methodology is based on biased, nonequilibrium collective variable based simulations including nonequilibrium pulling, string method with swarms of trajectories, bias-exchange umbrella sampling, and rate estimation techniques. …

T-Cell Antigen Receptors In Multiple Sclerosis, Lisa Lanée Keyes Jones

Biomedical Sciences Theses & Dissertations

Multiple Sclerosis (MS) is T-cell mediated autoimmune disease characterized by inflammation, demyelination, and degeneration of axons in the brain and spinal cord. A T cell-mediated immune response in MS is directed against myelin components and possibly other antigens in genetically susceptible individuals and is triggered by a viral infection. The T-cell antigen receptor (TCR) on T cells is responsible for antigen recognition and determines specificity. Our overall hypothesis is to determine whether clonally expanded T cells in patients with MS recognize viral or self-antigens and to determine whether molecular mimicry is involved in the development of the disease. To study …

Functional Characterization Of The Newly Discovered Type V Crispr-Cas Protein Cas12a2, Dylan J. Keiser

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Similarly to people, bacteria are under the treat of infection by viruses. To circumvent these threats, bacteria evolve complex immune systems. Our understanding of some of these immune systems has led to many advancements in the field of Biotechnology including tools that made expressing proteins for study in a lab easier, tools that revolutionized the feasibility of gene editing, and tools that could change the way we think about viral diagnostics and cancer therapeutics. A certain type of immune system that bacteria use to fight virus is called a CRISPR system. Presented here is work to understand the function of …

A Pipeline To Generate Deep Learning Surrogates Of Genome-Scale Metabolic Models, Achilles Rasquinha

School of Computing: Dissertations, Theses, and Student Research

Genome-Scale Metabolic Models (GEMMs) are powerful reconstructions of biological systems that help metabolic engineers understand and predict growth conditions subjected to various environmental factors around the cellular metabolism of an organism in observation, purely in silico. Applications of metabolic engineering range from perturbation analysis and drug-target discovery to predicting growth rates of biotechnologically important metabolites and reaction objectives within dierent single-cell and multi-cellular organism types. GEMMs use mathematical frameworks for quantitative estimations of flux distributions within metabolic networks. The reasons behind why an organism activates, stuns, or fluctuates between alternative pathways for growth and survival, however, remain relatively unknown. GEMMs …

Analyzing Interactions Of Calmodulin With Hiv-1 Matrix Protein, Andrea Sandoval, D. Mau, N. Karimi, K. Sakamaki, C. Owens, Jerry Larue

Student Scholar Symposium Abstracts and Posters

Human immunodeficiency virus (HIV) attacks the immune system and if left untreated, could cause acquired immunodeficiency syndrome (AIDS). The HIV matrix protein (HIV-MA) is involved in replication and regulation of the HIV virus. Calmodulin (CaM), a calcium-binding protein found in all eukaryotes, has a potential role in the viral replication of HIV-MA which plays a key role in the replication of HIV. In order to investigate the interactions between calmodulin and the HIV-MA, a series of titrations with CaM are performed using circular dichroism. Circular dichroism (CD) uses circularly polarized light to observe the secondary structure of a molecule. The …

Association Of Bradycardia And Asystole Episodes With Dialytic Parameters: An Analysis Of The Monitoring In Dialysis (Mid) Study, Qandeel H Soomro, Nisha Bansal, Wolfgang C Winkelmayer, Bruce A Koplan, Alexandru I Costea, Prabir Roy-Chaudhury, James A Tumlin, Vijay Kher, Don E Williamson, Saurabh Pokhariyal, Candace K Mcclure, David M Charytan, Mid Investigators

Faculty, Staff and Students Publications

BACKGROUND: Bradycardia and asystole events are common among patients treated with maintenance hemodialysis. However, triggers of these events in patients on maintenance hemodialysis (HD), particularly during the long interdialytic period when these events cluster, are uncertain.

METHODS: The Monitoring in Dialysis Study (MiD) enrolled 66 patients on maintenance HD who were implanted with loop recorders and followed for 6 months. We analyzed associations of predialysis laboratory values with clinically significant bradyarrhythmia or asystole (CSBA) during the 12 hours before an HD session. Associations with CSBA were analyzed with mixed-effect models. Adjusted negative binomial mixed-effect regression was used to estimate incidence …

Monitoring The Recruitment And Fusion Of Autophagosomes To Phagosomes During The Clearance Of Apoptotic Cells In The Nematode Caenorhabditis Elegans, Omar Peña-Ramos, Zheng Zhou

Faculty, Staff and Students Publications

During an animal's development, a large number of cells undergo apoptosis, a suicidal form of death. These cells are promptly phagocytosed by other cells and degraded inside phagosomes. The recognition, engulfment, and degradation of apoptotic cells is an evolutionarily conserved process occurring in all metazoans. Recently, we discovered a novel event in the nematode Caenorhabditis elegans: the double-membrane autophagosomes are recruited to the surface of phagosomes; subsequently, the outer membrane of an autophagosome fuses with the phagosomal membrane, allowing the inner vesicle to enter the phagosomal lumen and accumulate there over time. This event facilitates the degradation of the apoptotic …

Spatial Definition Of The Human Progesterone Receptor-B Transcriptional Complex, Xinzhe Yu, Ping Yi, Anil K Panigrahi, Lance Edward V Lumahan, John P Lydon, David M Lonard, Steven J Lutdke, Zhao Wang, Bert W O'Malley

Faculty, Staff and Students Publications

We report the quaternary structure of core transcriptional complex for the full-length human progesterone receptor-B (PR-B) homodimer with primary coactivator steroid receptor coactivator-2 (SRC-2) and the secondary coactivator p300/CREB-binding protein (CBP). The PR-B homodimer engages one SRC-2 mainly through its activation function 1 (AF1) in N-terminus. SRC-2 is positioned between PR-B and p300 leaving space for direct interaction between PR-B and p300 through PR-B's C-terminal AF2 and its unique AF3. Direct AF3/p300 interaction provides long-desired structural insights into the known functional differences between PR-B and the PR-A isoform lacking AF3. We reveal the contributions of each AF and demonstrate their …

Polyphenolics, Glucosinolates And Isothiocyanates Profiling Of Aerial Parts Of Nasturtium Officinale (Watercress), Sotiris Kyriakou, Kyriaki Michailidou, Tom Amery, Kyle Stewart, Paul G. Winyard, Dimitrios T. Trafalis, Rodrigo Franco, Aglaia Pappa, Mihalis I. Panayiotidis

School of Veterinary and Biomedical Sciences: Faculty Publications

Watercress (Nasturtium officinale) is a rich source of secondary metabolites with disease-preventing and/or health-promoting properties. Herein, we have utilized extraction procedures to isolate fractions of polyphenols, glucosinolates and isothiocyanates to determine their identification, and quantification. In doing so, we have utilized reproducible analytical methodologies based on liquid chromatography with tandem mass spectrometry by either positive or negative ion mode. Due to the instability and volatility of isothiocyanates, we followed an ammonia derivatization protocol which converts them into respective ionizable thiourea derivatives. The analytes’ content distribution map was created on watercress flowers, leaves and stems. We have demonstrated that …

Applications Of Covalent Chemistry In Targeted Protein Degradation, Dong Lu, Xin Yu, Hanfeng Lin, Ran Cheng, Erika Y Monroy, Xiaoli Qi, Meng C Wang, Jin Wang

Faculty, Staff and Students Publications

Proteolysis-targeting chimeras (PROTACs) and targeted covalent inhibitors (TCIs) are currently two exciting strategies in the fields of chemical biology and drug discovery. Extensive research in these two fields has been conducted, and significant progress in these fields has resulted in many clinical candidates, some of which have been approved by FDA. Recently, a novel concept termed covalent PROTACs that combine these two strategies has emerged and gained an increasing interest in the past several years. Herein, we briefly review and highlight the mechanism and advantages of TCIs and PROTACs, respectively, and the recent development of covalent PROTACs using irreversible and …

Gating Intermediates Reveal Inhibitory Role Of The Voltage Sensor In A Cyclic Nucleotide-Modulated Ion Channel, Xiaolong Gao, Philipp A M Schmidpeter, Vladimir Berka, Ryan J Durham, Chen Fan, Vasanthi Jayaraman, Crina M Nimigean

Faculty, Staff and Student Publications

Understanding how ion channels gate is important for elucidating their physiological roles and targeting them in pathophysiological states. Here, we used SthK, a cyclic nucleotide-modulated channel from Spirochaeta thermophila, to define a ligand-gating trajectory that includes multiple on-pathway intermediates. cAMP is a poor partial agonist for SthK and depolarization increases SthK activity. Tuning the energy landscape by gain-of-function mutations in the voltage sensor domain (VSD) allowed us to capture multiple intermediates along the ligand-activation pathway, highlighting the allosteric linkage between VSD, cyclic nucleotide-binding (CNBD) and pore domains. Small, lateral displacements of the VSD S4 segment were necessary to open the …