Biochemistry, Biophysics, and Structural Biology Commons^™

Mechanisms Underlying The Exquisite Sensitivity Of Candida Albicans To Combinatorial Cationic And Oxidative Stress That Enhances The Potent Fungicidal Activity Of Phagocytes, Despoina Kaloriti, Mette Jacobsen, Zhikang Yin, Miranda Patterson, Anna Tillmann, Deborah A. Smith, Emily Cook, Tao You, Melissa J. Grimm, Iryna Bohovych, Celso Grebogi, Brahm H. Segal, Neil A.R. Gow, Ken Haynes, Janet Quinn, Alistair J.P. Brown

Department of Biochemistry: Faculty Publications

Immune cells exploit reactive oxygen species (ROS) and cationic fluxes to kill microbial pathogens, such as the fungus

Candida albicans. Yet, C. albicans is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity

of neutrophils? We show that simultaneous exposure to oxidative and cationic stresses is much more potent than the individual

stresses themselves and that this combinatorial stress kills C. albicans synergistically in vitro.We also show that the high

fungicidal activity of human neutrophils is dependent on the combinatorial effects of the oxidative burst and cationic fluxes, as

their pharmacological attenuation with …

New Clox Systems For Rapid And Efficient Gene Disruption In Candida Albicans, Shahida Shahana, Delma S. Childers, Elizabeth R. Ballou, Iryna Bohovych, Frank C. Odds, Neil A.R. Gow, Alistair J.P. Brown

Department of Biochemistry: Faculty Publications

Precise genome modification is essential for the molecular dissection of Candida albicans, and is yielding invaluable information about the roles of specific gene functions in this major fungal pathogen of humans. C. albicans is naturally diploid, unable to undergo meiosis, and utilizes a non-canonical genetic code. Hence, specialized tools have had to be developed for gene disruption in C. albicans that permit the deletion of both target alleles, and in some cases, the recycling of the Candida-specific selectable markers. Previously, we developed a tool based on the Cre recombinase, which recycles markers in C. albicans with 90–100% efficiency via …

Saturated Free Fatty Acids Induce Cholangiocyte Lipoapoptosis, Sathish Kumar Natarajan, Sally A. Ingham, Ashley M. Mohr, Cody J. Wehrkamp, Anuttoma Ray, Sohini Roy, Sophie C. Cazanave, Mary Anne Phillippi, Justin L. Mott

Department of Biochemistry: Faculty Publications

Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation …

Xiap Antagonist Embelin Inhibited Proliferation Of Cholangiocarcinoma Cells, Cody J. Wehrkamp, Ashley R. Gutwein, Sathish Kumar Natarajan, Mary Anne Phillippi, Justin L. Mott

Department of Biochemistry: Faculty Publications

Cholangiocarcinoma cells are dependent on antiapoptotic signaling for survival and resistance to death stimuli. Recent mechanistic studies have revealed that increased cellular expression of the E3 ubiquitin-protein ligase X-linked inhibitor of apoptosis (XIAP) impairs TRAIL- and chemotherapy-induced cytotoxicity, promoting survival of cholangiocarcinoma cells. This study was undertaken to determine if pharmacologic antagonism of XIAP protein was sufficient to sensitize cholangiocarcinoma cells to cell death. We employed malignant cholangiocarcinoma cell lines and used embelin to antagonize XIAP protein. Embelin treatment resulted in decreased XIAP protein levels by 8 hours of treatment with maximal effect at 16 hours in KMCH and Mz-ChA-1 …

Oxidative Stress, Redox Signaling, And Autophagy: Cell Death Versus Survival, Juliana Navarro-Yepes, Michaela Burns, Anandhan Annadurai, Oleh Khalimonchuk, Luz Maria Del Razo, Betzabet Quintanilla-Vega, Aglaia Pappa, Mihalis I. Panayiotidis, Rodrigo Franco

Department of Biochemistry: Faculty Publications

Significance: The molecular machinery regulating autophagy has started becoming elucidated, and a number of studies have undertaken the task to determine the role of autophagy in cell fate determination within the context of human disease progression. Oxidative stress and redox signaling are also largely involved in the etiology of human diseases, where both survival and cell death signaling cascades have been reported to be modulated by reactive oxygen species (ROS) and reactive nitrogen species (RNS). Recent Advances: To date, there is a good understanding of the signaling events regulating autophagy, as well as the signaling processes by which alterations in …

Kinetic And Structural Characterization Of Tunnel-Perturbing Mutants In Bradyrhizobium Japonicum Proline Utilization A, Benjamin W. Arentson, Min Luo, Travis A. Pemberton, John J. Tanner, Donald F. Becker

Department of Biochemistry: Faculty Publications

Proline utilization A from Bradyrhizobium japonicum (BjPutA) is a bifunctional flavoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate Δ¹-pyrroline-5-carboxylate/glutamate-γ-semialdehyde (P5C/GSA). In this work, the structure of the channel was explored by inserting large side chain residues at four positions along the channel in BjPutA. Kinetic analysis of the different mutants revealed replacement of D779 with Tyr (D779Y) or Trp (D779W) significantly decreased the overall …

Evidence That The C‑Terminal Domain Of A Type B Puta Protein Contributes To Aldehyde Dehydrogenase Activity And Substrate Channeling, Min Luo, Shelbi Christgen, Nikhilesh Sanyal, Benjamin W. Arentson, Donald F. Becker, John J. Tanner

Department of Biochemistry: Faculty Publications

Proline utilization A (PutA) is a bifunctional enzyme that catalyzes the oxidation of proline to glutamate. Structures of type A PutAs have revealed the catalytic core consisting of proline dehydrogenase (PRODH) and Δ1- pyrroline-5-carboxylate dehydrogenase (P5CDH) modules connected by a substrate-channeling tunnel. Type B PutAs also have a C-terminal domain of unknown function (CTDUF) that is absent in type A PutAs. Small-angle X-ray scattering (SAXS), mutagenesis, and kinetics are used to determine the contributions of this domain to PutA structure and function. The 1127-residue Rhodobacter capsulatus PutA (RcPutA) is used as a representative CTDUF-containing type B PutA. The reaction progress …

Homogeneous Low-Molecular-Weight Heparins With Reversible Anticoagulant Activity, Yongmei Xu, Chao Cai, Kasemsiri Chandarajoti, Po-Hung Hsieh, Lingyun Li, Truong Q. Pham, Erica M. Sparkenbaugh, Juzheng Sheng, Nigel S. Key, Rafal Pawlinski, Edward N. Harris, Robert J. Linhardt, Jian Liu

Department of Biochemistry: Faculty Publications

Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.

Pharmacological Correction Of Obesity-Induced Autophagy Arrest Using Calcium Channel Blockers, Hwan-Woo Park, Haeli Park, Ian A. Semple, Insook Jang, Seung-Hyun Ro, Myungjin Kim, Victor A. Cazares, Edward L. Stuenkel, Jung-Jae Kim, Jeong Sig Kim, Jun Hee Lee

Department of Biochemistry: Faculty Publications

Autophagy deregulation during obesity contributes to the pathogenesis of diverse metabolic disorders. However, without understanding the molecular mechanism of obesity interference in autophagy, development of therapeutic strategies for correcting such defects in obese individuals is challenging. Here we show that a chronic increase of the cytosolic calcium concentration in hepatocytes during obesity and lipotoxicity attenuates autophagic flux by preventing the fusion between autophagosomes and lysosomes. As a pharmacological approach to restore cytosolic calcium homeostasis in vivo, we administered the clinically approved calcium channel blocker verapamil to obese mice. Such treatment successfully increases autophagosome–lysosome fusion in liver, preventing accumulation of …

Hepatoprotective Role Of Sestrin2 Against Chronic Er Stress, Hwan-Woo Park, Haeli Park, Seung-Hyun Ro, Insook Jang, Ian A. Semple, David N. Kim, Myungjin Kim, Myeongjin Nam, Deqiang Zhang, Lei Yin, Jun Hee Lee

Department of Biochemistry: Faculty Publications

Upon prolonged endoplasmic reticulum (ER) stress, cells attenuate protein translation to prevent accumulation of unfolded proteins. Here we show that Sestrin2 is critical for this process. Sestrin2 expression is induced by an ER stress-activated transcription factor CCAATenhancer- binding protein beta (c/EBPβ). Once induced, Sestrin2 halts protein synthesis by inhibiting mammalian target of rapamycin complex 1 (mTORC1). As Sestrin2-deficient cells continue to translate a large amount of proteins during ER stress, they are highly susceptible to ER stress-associated cell death. Accordingly, dietary or genetically induced obesity, which does not lead to any pathological indication other than simple fat accumulation in the …

Analysis Of Biomolecular Interactions Using Affinity Microcolumns: A Review, Xiwei Zheng, Zhao Li, Sandya Beeram, Maria Podariu, Ryan Matsuda, Erika L. Pfaunmiller, Christopher J. White Ii, Natasha Carter, David S. Hage

David Hage Publications

Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and …

Further Characterization Of A Secreted Lipase From The Human Pathogen Leishmania Donovani By Determining The Effect Of Various Metal Ions On Its Enzymatic Activity., Lana Hoertz

Pell Scholars and Senior Theses

Leishmania donovani, a protozoan parasite, is the causative agent of the often fatal disease visceral leishmaniasis. The current treatments available are minimal and toxic to the patient. It has been shown that these organisms exhibit lipolytic activity during their growth in vitro. Lipases are enzymes that are known to aid in the development and virulence of several pathogenic organisms such as Candida albicans and Staphylococcus warneri. Little information is known, however, about the role of lipases in Leishmania species. We hypothesize that lipase may play a part in Leishmania's ability to survive within the human host as well as its …

Culturable Aerobic And Facultative Anaerobic Intestinal Bacterial Flora Of Black Cobra (Naja Naja Karachiensis) In Southern Pakistan, Junaid Iqbal, Mehwish Sagheer, Nazneen Tabassum, Ruqaiyyah Siddiqui, Naveed Ahmed Khan

Department of Biological & Biomedical Sciences

Using morphological analysis and biochemical testing, here for the first time, we determined the culturable gut bacterial flora (aerobes and facultative anaerobes) in the venomous Black Cobra (Naja naja karachiensis) from South Asia. The findings revealed that these snakes inhabit potentially pathogenic bacteria including Serratia marcescens, Pseudomonas aeruginosa, Shewanella putrefaciens, Aeromonas hydrophila, Salmonella sp., Moraxella sp., Bacillus sp., Ochrobactrum anthropi, and Providencia rettgeri. These findings are of concern, as injury from snake bite can result in wound infections and tissue necrosis leading to sepsis/necrotizing fasciitis and/or expose consumers of snake meat/medicine in the community to infections.

Synthesis And Characterization Of Pt(Ii) Complexes For Anticancer Therapy, Mihaela A. Ciulei, Pradip K. Bhowmik

McNair Poster Presentations

The first platinum-based drug was discovered and approved by Food and Drug Administration (FDA) in 1978 is cis-diamminedichloroplatinum (II) (cisplatin or CDDP). Cisplatin is used for about 50% of the chemotherapeutic cancer treatments along with its two analogues carboplatin and oxaliplatin. So far these drugs have been used extensively as treatment for ovarian, bladder, head and neck, and lung cancers. Although cisplatin has been used so often, it has toxic side effects and drug resistance.1-4 Due to these limitations other compounds have been synthesized. Specifically, our lab in conjunction with a biochemistry lab has recently published one article …

A Loose Domain Swapping Organization Confers A Remarkable Stability To The Dimeric Structure Of The Arginine Binding Protein From Thermotoga Maritima, Alessia Ruggiero, Jonathan D. Dattelbaum, Maria Staiano, Rita Berisio, Sabato D'Auria, Luigi Vitagliano

Chemistry Faculty Publications

The arginine binding protein from Thermatoga maritima (TmArgBP), a substrate binding protein (SBP) involved in the ABC system of solute transport, presents a number of remarkable properties. These include an extraordinary stability to temperature and chemical denaturants and the tendency to form multimeric structures, an uncommon feature among SBPs involved in solute transport. Here we report a biophysical and structural characterization of the TmArgBP dimer. Our data indicate that the dimer of the protein is endowed with a remarkable stability since its full dissociation requires high temperature as well as SDS and urea at high concentrations. In order to elucidate …

Bipolar Nanosecond Electric Pulses Are Less Efficient At Electropermeabilization And Killing Cells Than Monopolar Pulses, Bennett L. Ibey, Olga N. Pakhomova, Caleb C. Roth, Shu Xiao, Karl Schoenbach, Andrei G. Pakhomov

Bioelectrics Publications

Multiple studies have shown that bipolar (BP) electric pulses in the microsecond range are more effective at permeabilizing cells while maintaining similar cell survival rates as compared to monopolar (MP) pulse equivalents. In this paper, we investigated whether the same advantage existed for BP nanosecond-pulsed electric fields (nsPEF) as compared to MP nsPEF. To study permeabilization effectiveness, MP or BP pulses were delivered to single Chinese hamster ovary (CHO) cells and the response of three dyes, Calcium Green-1, propidium iodide (PI), and FM1-43, was measured by confocal microscopy. Results show that BP pulses were less effective at increasing intracellular calcium …

Enzyme Optimization For Lignocellulose Hydrolysis Using Mechanistic Modeling, Adam S. Marlowe

Dissertations, Master's Theses and Master's Reports - Open

A novel mechanistic model for the saccharification of cellulose and hemicellulose is utilized to predict the products of hydrolysis over a range of enzyme loadings and times. The mechanistic model considers the morphology of the substrate and the kinetics of enzymes to optimize enzyme concentrations for the enzymatic hydrolysis of cellulose and hemicellulose simultaneously. Substrates are modeled based on their fraction of accessible sites, glucan content, xylan content, and degree of polymerizations. This enzyme optimization model takes into account the kinetics of six core enzymes for lignocellulose hydrolysis: endoglucanase I (EG1), cellobiohydrolase I (CBH1), cellobiohydrolase II (CBH2), and endo-xylanase (EX) …

Evaluations Of A Mechanistic Hypothesis For The Influence Of Extracellular Ions On Electroporation Due To High-Intensity, Nanosecond Pulsing, V. Sridhara, R. P. Joshi

Electrical & Computer Engineering Faculty Publications

The effect of ions present in the extracellular medium on electroporation by high-intensity, short-duration pulsing is studied through molecular dynamic simulations. Our simulation results indicate that mobile ions in the medium might play a role in creating stronger local electric fields across membranes that then reinforce and strengthen electroporation. Much faster pore formation is predicted in higher conductivity media. However, the impact of extracellular conductivity on cellular inflows, which depend on transport processes such as electrophoresis, could be different as discussed here. Our simulation results also show that interactions between cations (Na+ in this case) and the carbonyl oxygen of …

Evolution And Divergence Of The Structural And Physical Properties Of Dna Binding By Methyl-Cytosine Binding Domain Family Members 2 And 3, Jason Cramer

Theses and Dissertations

The studies presented in this dissertation, Evolution And Divergence Of The Structural And Physical Properties Of DNA Binding By Methyl-Cytosine Binding Domain Family Members 2 And 3, pertain primarily to two key epigenetic regulators involved with the biological interpretation of methylated DNA marks. We provide insights into the emergence and evolution of the MBD2 and MBD3 and how those molecular entities influence heritable changes in gene activity. We further provide details regarding the mystery surrounding MBD3 function and the MBD2-mediated capacity of primitive animals to carry out methylation-specific epigenetic mechanisms. In chapter two, we describe the DNA binding properties of …

A Summary Of Three Antibiotic Resistant Bacteria Studies Of Plaster Creek, Yeaeun Lee, David P. Warners

Faculty and Professional Research

No abstract provided.

Nine Year Assessment Of Soil Treatment On Aguacatillo (Nectandontisii And Ocotea Austinii, Lauraceae) Seedling Success In The Restoration Of A Costa Rican Cloud Forestra Cufo, Sheila Devries, David Warners

Faculty and Professional Research

We tracked seedling growth of two species of aguacatillo (Nectandra cufodontisii (O. C. Schmidt) C. K. Allen and Ocotea austinii C. K. Allen) that were planted into an early successional cloud forest hillside in San Gerardo de Dota, Costa Rica over nine years. Seedlings were collected from underneath nearby mother trees and transplanted into one of three soil treatments: indigenous soil; a 50:50 mixture of horse manure and indigenous soil; and a 50:50 mixture of kitchen compost and indigenous soil. After seedlings were transplanted they were measured and each specific location was evaluated for light exposure. Two months later seedlings …

Single-Molecule Analysis Of Alzheimer's Β-Peptide Oligomer Disassembly At Physiological Concentration, Chen Chen

Theses and Dissertations--Chemistry

The diffusible soluble oligomeric amyloid β-peptide (Aβ) has been identified as a toxic agent in Alzheimer’s disease that can cause synaptic dysfunction and memory loss, indicating its role as potential therapeutic targets for AD treatment. Recently an oligomer-specific sandwich biotin-avidin interaction based assay identified the Aβ oligomer dissociation potency of a series of dihydroxybenzoic acid (DHBA) isomers. Because the sandwich assay is an ensemble method providing limited size information, fluorescence correlation spectroscopy (FCS) was employed to provide single molecule resolution of the disassembly mechanism.

Using FCS coupled with atomic force microscopy, we investigated the size distribution of fluorescein labeled synthetic …

Stability Studies Of Membrane Proteins, Cui Ye

Theses and Dissertations--Chemistry

The World Health Organization has identified antimicrobial resistance as one of the top three threats to human health. Gram-negative bacteria such as Escherichia coli are intrinsically more resistant to antimicrobials. There are very few drugs either on the market or in the pharmaceutical pipeline targeting Gram-negative pathogens. Two mechanisms, the protection of the outer membrane and the active efflux by the multidrug transporters, play important roles in conferring multidrug resistance to Gram-negative bacteria. My work focuses on two main directions, each aligning with one of the known multidrug resistance mechanisms.

The first direction of my research is in the area …

Increase Of Basal Oxidative Stress Levels And Impairment Of Heme Oxygenase-1/Biliverdin Reductase Post-Translational Modification By The Defect Of Parkinson-Related Gene Of Pink1, Zhaoshu Zhang

Theses and Dissertations--Chemistry

Parkinson disease (PD) is the most common movement disorder and the second most common neurodegenerative disease. PINK1, PTEN-induced kinase 1, functions as a serine/threonine kinase as well as a protector of mitochondrial function. Mutations in PINK1 gene result in either mitochondria dysfunction or disruption of kinase signaling pathways involved in the pathogenesis of PD.

In this thesis, oxidative stress levels were examined in the brain of PINK1 knockout mice, and also how heme oxygenase-1 and biliverdin reductase are affected in brain of PINK1 knockout mice. In addition, posttranslational modifications are a way to control the behavior of proteins, so posttranslational …

The Differences Between Iron And Iron-Substituted Manganese Superoxide Dismutase With Respect To Hydrogen Peroxide Treatment, Jianing Wang

Theses and Dissertations--Chemistry

Iron-substituted manganese superoxide dismutase (Fe(Mn)SOD) was produced using an in vivo preparation method. It’s an inactive enzyme in catalyzing superoxide radical dismutation owing to the mis-incorporation of Fe in the active site evolved to use Mn. To investigate the possible toxicity of human Fe(Mn)SOD proposed by Yamakura, we studied the properties of Fe(Mn)SOD upon H₂O₂ treatment and compared to that of FeSOD. It’s found that the responses to H₂O₂ treatment were different, including the changes of optical spectra, variations of active site coordination and secondary structures. Fe³⁺ reduction was not observed in Fe(Mn)SOD …

Bioc 435: Advanced Topics In Biochemistry—A Peer Review Of Teaching Project Benchmark Portfolio, Edward N. Harris

UNL Faculty Course Portfolios

BIOC 435 or Advanced Topics in Biochemistry is an ACE 10 (Achievement centered education)course for students in their senior year. This is a capstone course for Biochemistry majors and is the last of several biochemistry courses that they will take in their undergraduate enrollment. The course is also open to non-majors in a related science such as Biology and Biological Sciences, although these students typically do not do as well as the majors. Students who take this course should be well-prepared in the basic concepts of biochemistry with regards to nucleic acids and proteins as well as some familiarity with …

High Fat Diet Increased Serum Glutamate Dehydrogenase More Than Chronic Acetaminophen Dosing In Female Mice, Elizabeth Ruth Behmer

All Graduate Theses, Dissertations, and Other Capstone Projects

This laboratory study examined the effects of acetaminophen overdose in normal fed and high-fat fed female mice. Forty female mice were placed on normal and high-fat diets at 4 weeks old. When the mice were significantly different in weight (between 6 and 9 months old), half the mice were dosed with acetaminophen. These mice were daily given an overdose of acetaminophen for 14 days. The dose used was 300 mg/kg mouse (LD50 = 338 mg/kg). The control group was given 10 µl water/g mouse. Levels of serum glutamate dehydrogenase (GDH) were measured to indicate liver damage. GDH is released from …

Catalytic Mechanism And Maturation Of The Metalloenzyme Nitrile Hydratase, Natalie I. Gumataotao

Dissertations

Nitrile hydratases are metalloenzymes that catalyze the hydration of nitriles to their corresponding amides in a specific manner at ambient pressures and temperatures at neutral pH. Traditional industrial methods require high temperature and pressure, extreme pH, and heavy metals. NHases are used as biocatalysts in the large scale industrial production of amide precursors to textiles, animal feedstock, and polymers. Notably, NHase is used in the production of ~100,000 tons of acrylamide annually by the Mitsubishi Corporation.

Despite being used extensively in industry, questions remain about NHase. The catalytic mechanism is not defined. Understanding the way in which the nitrile is …

Numerical Study Of Lipid Translocation Driven By Nanoporation Due To Multiple High-Intensity, Ultrashort Electrical Pulses, Viswanadham Sridhara, Ravindra P. Joshi

Electrical & Computer Engineering Faculty Publications

The dynamical translocation of lipids from one leaflet to another due to membrane permeabilization driven by nanosecond, high-intensity (>100 kV/cm) electrical pulses has been probed. Our simulations show that lipid molecules can translocate by diffusion through water-filled nanopores which form following high voltage application. Our focus is on multiple pulsing, and such simulations are relevant to gauge the time duration over which nanopores might remain open, and facilitate continued lipid translocations and membrane transport. Our results are indicative of a N^1/2 scaling with pulse number for the pore radius. These results bode well for the use of pulse …

Mutations In Phospholamban Alter The Structure And Function Of The Calcium Atpase Regulatory Complex, Neha Abrol

Dissertations

Phospholamban (PLB) is an integral membrane protein that plays an important role in regulation of cardiac calcium handling and contractility. PLB exists as a homopentamer in the membrane, which upon deoligomerization into active monomers reversibly inhibits sarco/endoplasmic reticulum calcium ATPase (SERCA). Mutations in PLB that change the PLB monomer-pentamer equilibrium result in dysregulation of SERCA. To determine the structural and regulatory role of the C-terminal residues of PLB in the membranes of living cells, we fused fluorescent protein tags to PLB and SERCA. We then studied the effect of C-terminal alanine substitutions and truncation mutations on PLB oligomerization and SERCA …