bluuebunny/crossref_metadata_2025_split huggingface dataset:
Exploring the effect of conserved motifs on the structure and activity of the RNA‐binding protein LARP6c from Arabidopsis thaliana
Authors: Foster Jessica Sara , Foster Corina L. , Otte‐Petrill Courtney , Betancourt Francisco C. , Lewis Karen A. , Antonelli Cecile , Billey Elodie 
Published: June 2021

La Related Protein 6 (LARP6) is an RNA binding protein that belongs to the La‐Related Protein superfamily (LARPS). LARP6 contains structural motifs that are conserved across vertebrates and invertebrates. In vertebrates, LARP6 regulates the translation of collagen mRNA and thereby the synthesis of type I collagen. However, the biological function of LARP6 in plants is not well understood. Plants express three LARP6 paralogs: LARP6a, LARP6b, and LARP6c. Our study focuses on Arabidopsis thaliana LARP6c (AtLARP6c), which contains three structural motifs: a poly‐A binding protein (PABP)‐interacting motif 2 (PAM2), an RNA‐binding domain consisting of the La module comprised of the La motif and RNA recognition motif (RRM), and a C‐terminal La‐and SUZ‐C associated (LSA) motif. We hypothesize that the PAM2 motif may affect the RNA binding activity of AtLARP6c. To test this hypothesis, we recombinantly expressed and purified three forms of AtLARP6c: full length AtLARP6c, AtLARP6c‐ΔPAM2, and the isolated La module. Limited proteolysis was used to probe the structural stability of the purified proteins, demonstrating that the three constructs were stably folded. Our current work is now focused on establishing electrophoretic mobility shift assays with biotinylated RNA ligands to test the role of the deleted protein domains on the RNA binding activity of our AtLARP6c variants. Previous published studies found that full length AtLARP6c binds to polyA, polyU, and polyC oligonucleotides. These same ligands are being used to directly test how the PAM2 and auxiliary domains of AtLARP6c affect the RNA binding activity of the La module. This investigation provides insight into potential regulatory mechanisms of RNA binding in the plant LARP6 proteins.Support or Funding InformationSupport provided by Texas State University and the National Institutes of Health ‐ National Institute of General Medical Studies (GM119096) to K.A.L.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Thermodynamic Analysis of ATP Dissociation in a DEAD‐Box Protein”
Authors: Yoder Lisa , Garcia Ivelitza , Patterson Jacob , Bardine Conner 
Published: June 2021

RNA plays an essential role in many cellular functions. Similar to proteins, RNA function is strictly determined by the overall structure and dynamics of the RNA macromolecule. Thus, many cellular proteins or accessory factors are associated with RNA folding. One example of these RNA binding proteins are DEAD‐Box proteins. DEAD‐Box proteins utilize ATP hydrolysis to unwind and reanneal RNA duplexes to promote correct folding during RNA processing. DEAD‐Box proteins possess two conserved RecA‐like domains that bind ATP and RNA. In addition, these proteins also contain N‐ and/or C‐ terminal domains (NTD and CTD, respectively). Peripheral domains in DEAD‐Box proteins are proposed to regulate ATP hydrolysis as well as confer RNA specificity. Thus, the NTD and/or CTD can alter the ATP binding pocket and subsequently alter ATP association and/or dissociation. For example, Rok1p, a yeast DEAD‐Box protein, contains both a NTD and a CTD. The NTD in Rok1p is suggested to affect the structure and stability of the protein. Therefore, the dissociation rate of a fluorescence ATP analog was determined through transient kinetics and stopped‐flow spectroscopy under various thermal conditions. Resulting trends conform to double exponential fits suggesting a two‐step mechanism. The observed slow rate was consistent with a conformation change in the protein that allows rapid dissociation. The rate in which the conformation change occurs was observed to be temperature dependent. The current hypothesis centers on the ability, of the Rok1p peripheral domains, to affect the slow structural change needed for ATP dissociation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Assessing Cysteine Residue Thiol Status of t‐DARPP, A Protein Involved in Chemoresistance
Authors: Aldana‐Mendoza Jesus Antonio , Momand Jamil Antonio 
Published: June 2021

The gene PPP1R1B expresses t‐Darpp, a protein that activates protein kinase A and the AKT pathway in breast cancer cells. t‐Darpp, when overexpressed in breast cancer cells, also confers resistance to trastuzumab, a therapeutic that binds to the Her2 receptor. Although t‐Darpp expression is relatively high in gastric, breast and colon cancers, its structure is unknown. Human t‐Darpp has two cysteine residues that can be exploited to explore t‐Darpp structure, Cys36 and Cys119. Methoxypolyethylene glycol‐maleimide (Mal‐PEG) was used to determine whether recombinant t‐Darpp cysteine thiols are on the surface of the protein and to find out if they are reversibly oxidized. Later, a quantitative analysis was conducted using Ellman's test. 5,5′‐dithiobis‐(2‐nitrobenzoic acid) (Ellman's reagent) was used to determine whether recombinant t‐Darpp cysteine thiols are free or not. Both studies suggest that the two cysteine thiols are either buried or oxidized. Further work will be conducted to distinguish between these two possibilities.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein lysine acetylation is a regulatory mechanism for Bacillus subtilis multicellularity
Authors: Reverdy Alicyn , Chen Yun , Chai Yunrong 
Published: June 2021

Lysine acetylation is a post‐translational modification that alters the charge, conformation, and stability of proteins. Published lists of acetylated proteins, or acetylomes, in bacteria have demonstrated that lysine acetylation occurs on proteins with a wide diversity of function, including central metabolism, DNA transcription and binding, chemotaxis, and cell size. Bacillus subtilis is a non‐pathogenic soil bacterium, and model organism for sporulation, motility, biofilm formation, and general multicellular development. The genetic regulation of these processes has been extensively studied, thus, we aimed to investigate the role of lysine acetylation in multicellular development (or biofilm formation) in B. subtilis. To further investigate acetylation, we generated an acetylome under biofilm‐inducing conditions and identified acetylated proteins involved in multicellularity; specifically, swarming and biofilm formation. Upon deletion of known genes for acetylation, cells had significantly slower swarming kinetics and a significantly smaller final swarming radius compared to wildtype. This suggests that acetylation impacts bacterial swarming. Biofilm is a mixture of cells and secreted matrix consisting of exopolysaccharides, proteins, and nucleotides that assists bacterial communities in colonization and survival. Genetic analysis of acetylation in biofilm formation demonstrated that lack of acetylation impacts biofilm development as shown by a decrease in matrix production. Targeting the acetylated biofilm proteins, we performed site‐directed mutagenesis of known acetylated lysine residues and assayed for biofilm formation. Two biofilm proteins, YmcA, a biofilm regulatory protein, and GtaB, an enzyme for biosynthesis of a precursor sugar for exopolysaccharides, demonstrated severe biofilm defects upon mutation of lysine residues. This suggests the importance of YmcA Lys64 and GtaB Lys89 and Lys191 for function in biofilm formation. In summary, we believe that protein lysine acetylation is a novel regulatory mechanism linking cell metabolism to bacterial multicellular development.Support or Funding InformationNational Science Foundation and Northeastern UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Towards Visualizing Citrullinated Proteins in SDS‐PAGE Gels Using Phenylglyoxal‐Based Chemistries
Authors: Thompson Kourtland A. , Grant Jennifer E. 
Published: June 2021

Citrullination is the deimination of arginine, a post‐translational modification that is the root cause of rheumatoid arthritis and multiple sclerosis. The result of this modification in these diseases is an aberrant immune response to the citrullinated proteins which results in the disease phenotype. Citrullination is notoriously difficult to visualize due to the minimal mass upon modification, and the limited options for chemical derivatization of citrulline over arginine. Furthermore, amino acid specificity patterns defining protein citrullination remain obscure, although protein secondary structure maintain an influence (Olson et al., 2017, Comp. Biol. Chem., 70:107–115). The goal of this project is to design an assay system that allows visualization of citrulline‐modified proteins on an SDS‐PAGE gel, to aid in understanding of citrullination patterns within proteins and the discovery of citrullinated proteins.A model system has been designed to detect citrullinated peptides using SDS‐PAGE and related technologies. Peptides likely to be highly citrullinated by PAD4, like histone H3, were synthesized; citrullinated versions of peptides were also prepared to serve as positive controls. We also examined the feasibility of actively citrullinating full‐length proteins as part of the methods development process. The basis of the assay is to use PAD4 to citrullinate peptides and proteins in an in vitro system, and then employ phenylglyoxal‐based chemistry to detect citrullination events. The design of peptides and proteins, optimization of conditions for citrullination proteins in vitro by PAD4, and use of SDS‐PAGE gels in visualizing citrullinated proteins, are presented.In sum, developing more direct methods for visualization of citrulline, like the SDS‐PAGE method, will facilitate identification of proteins susceptible to citrullination events.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Studying the Dimerization of the FNR Transcription Factor by Mutant Analysis at the 130 and 140 Residue Positions
Authors: Kazmouz Sobhi , Moore Laura J. 
Published: June 2021

The Fumarate Nitrate Reductase (FNR) transcription factor is the main anaerobic switch in Escherichia coli bacteria. In the absence of oxygen, the FNR protein exists as a dimer with a [4Fe‐4S] cluster and binds to DNA to regulate transcription of genes needed for anaerobic respiration. When oxygen is present, however, the [4Fe‐4S] cluster degrades causing FNR to return to its inactive monomeric form. Examination of the crystal structure of Alivibrio fischeri FNR, a close variant of E. coli FNR, suggested that the dimerization and, thus, activation of FNR is dependent upon a salt bridge interaction between the Asp 130 residue and the Arg 140 residue.[1] Site‐directed mutagenesis was used to analyze how various mutations at those two particular residues can affect FNR activity. Analysis of double mutants where the 130 and 140 residues were swapped showed that the salt bridge interaction may not be playing a role in FNR dimerization since the double mutants had no FNR activity. Therefore, additional mutations were constructed to study the importance of the 130 and 140 residues in promoting dimerization. Data from β – galactosidase assays carried out on an E. coli reporter strain to measure FNR activity suggest that only Arg 140 is essential for regulating dimerization. Single mutants in which Asp 130 was replaced by hydrophobic and polar amino acids display FNR activity almost the same as that of the wild type FNR while the same mutations that replaced Arg 140 resulted in almost no FNR activity. Furthermore, alanine and leucine double mutants at positions 130 and 140 displayed FNR activity even higher than the wild type FNR activity, further suggesting a salt bridge is not required for dimerization.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural analysis of the active site and DNA binding of human cytidine deaminase APOBEC3B
Authors: Hou Shurong , Silvas Tania V. , Leidner Florian , Nalivaika Ellen , Yilmaz Nese Kurt , Schiffer Celia A. 
Published: June 2021

APOBEC3s proteins (A3s), a family of human cytidine deaminases, protect the host cell from endogenous retro‐elements and exogenous viral infections by introducing hypermutations. However, their ability to mutate genomic DNA makes them a potential cancer source. Of the 7 human A3s, A3A, A3B and A3H have been implicated as an endogenous cause for multiple cancers. Among all A3s, A3B and A3A share highest sequence identity, but have distinct deamination activities with A3B about 15‐fold lower activity compared to A3A. Over the past few years, our lab along with other groups has determined several structures of apo as well as DNA‐bound A3A and A3B complexes. These structures revealed the molecular determinants of nucleotide specificity and importance of the loops around the active site, including the critical loop 1, in DNA binding. However, the current A3B‐DNA co‐crystal structure is that of a chimera with loop 1 from A3A. Hence, the molecular mechanism for how A3B binds to DNA within the context of native loop 1 and how A3B regulates its deamination activity resulting in much lower activity compared to A3A still remain unknown. Here we identified an auto‐inhibited mode of A3B that restricts its activity, revealed the molecular mechanism for DNA binding to A3B, as well as the sequence specificity upstream of substrate cytidine. Using structural analysis and molecular dynamics, we revealed a unique hydrogen bonding network in A3B loop 1. This network auto‐inhibits A3B's activity by locking the active site in a DNA‐binding incompatible mode. In addition, the critical residues for DNA binding to A3B have been identified through molecular modeling and simulations. The results were verified experimentally by binding assays with different engineered A3B mutants. Overall, structural analysis of the active site and DNA binding of A3B demonstrate how A3B structurally regulates its deamination activity and will potentially lead to discovering A3B‐specific inhibitors that may be developed as anti‐cancer drugs to benefit cancer therapeutics.Support or Funding InformationNIH R01GM118474This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FRET‐based Measurements of Protein Multimerization in Pyruvate Carboxylase
Authors: Koza Anya Lei , Hakala Joshua , St. Maurice Martin 
Published: June 2021

Changes in environmental conditions can affect protein multimerization which, in turn, may alter an enzyme's activity. The multi‐domain homotetrameric enzyme pyruvate carboxylase (PC) serves as a model to study protein multimerization. Past studies on PC have shown that it equilibrates between different oligomeric forms, but that it is active exclusively as a tetramer. When diluted to low concentrations, the PC equilibrium shifts to favor inactive monomeric and dimeric oligomerization states[1],[2]. Since the in vitro activity of PC depends on its oligomerization state, it stands to reason that regulation of oligomerization states offers a way to control the enzyme activity in vivo.In a heterogeneous population, smaller PC oligomers can reassociate into a population containing mixed heterotetramers. Controlled manipulation of PC multimerization will aid in developing hybrid tetramers that would serve as a tool to reveal key mechanistic insights into inter‐subunit coordination during catalysis. Past remixing studies have failed to precisely control the assembly of heterogeneous tetramers. This research will contribute to a more complete description of the factors influencing PC multimerization by using a FRET‐based approach to measure the assembly of PC tetramers under a wide array of remixing conditions. A non‐conserved residue (Leu 339) on the surface of the biotin carboxylase (BC) domain of Staphylococcus aureus PC (SaPC) was mutated to a Cys for site‐specific maleimide‐based labeling with either a Cy3 or Cy5 fluorophore. These site‐specifically incorporated fluorophores will form a FRET pair when the BC domain of one PC population dimerizes with a labeled BC domain of another PC population en route to re‐associating into a heterogeneous tetramer. Preliminary characterization revealed no significant difference in the maleimide‐based labeling of WT SaPC and L339C in SaPC. However, the addition of ADP along with the BC domain transition state analogues, phosphonoacetate (PPA), dramatically reduced the labeling of WT SaPC without affecting labeling of SaPC L339C. The site‐specifically labeled FRET pair of Cy3 and Cy5 offers a method to measure PC multimerization, which enables the investigation of conditions that alter the formation of the heterogeneous PC tetramer. These studies represent the first efforts to carefully control remixing of PC through manipulation of PC oligomerization states and may provide insight into ways to control the oligomerization states in other oligomeric proteins.Support or Funding InformationThis work was supported by the National Institutes of Health grant GM117540.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reconstitution of a Tail‐anchored Mitochondrial Membrane Protein
Authors: Bakkum Amber L. , Hill Blake 
Published: June 2021

Tail‐anchored (TA) proteins have a single transmembrane domain (TMD) located on the C‐terminus with the bulk of the protein facing the cytosol; in contrast with the conventional membrane protein that is translated directly into the membrane of interest. TA proteins are involved in organelle division, vesicle trafficking, and apoptosis. TA proteins lack an N‐terminal signal sequence and exhibit wide sequence variation in the TMD. Thus, TA proteins are likely inserted into the membrane post translationally. We asked if we can reconstitute Fis1, a mitochondrial outer membrane TA protein, in a detergent micelle to determine its structure. We were able to purify soluble recombinant Fis1 in the absence of detergent. Preliminary results suggest that in the absence of detergent, Fis1 exists in a soluble aggregate that can be disrupted by addition of detergent. This soluble aggregate is likely facilitated by the TMD of Fis1. We further show that in the detergent micelle Fis1 is oriented such that the cytoplasmic domain faces the solvent, reminiscent of the orientation on the mitochondrial surface. Using properly reconstituted Fis1 in detergent micelles, we collected CD, DLS, NMR, and EPR data with the goal of determining a high‐resolution structure of Fis1 in the membrane, which is an outstanding question in the mitochondrial fission field. More generally, this work has many implications for TA membrane protein reconstitution.Support or Funding InformationNIH R01‐GM067180This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NMR structural studies of membrane proteins in bilayer environments
Authors: Radoicic Jasmina , Park Sang Ho , Opella Stanley J. 
Published: June 2021

Membrane protein research focused on studying the structure, function, and interactions of proteins in membrane environments by NMR spectroscopy will be presented. Sample preparation is challenging because membrane proteins are generally low expressing in heterologous systems, hydrophobic, and difficult to re‐fold into their active conformations – any variation from the native structure results in inactive and/or mis‐folded protein. Typically, the purification or extraction of these proteins requires the use of detergents, which can be detrimental to their structure and function. Therefore, an important goal is the development and implementation of detergent‐free environments for NMR studies of membrane proteins.The main membrane environments used for NMR studies include micelles, bicelles, liposomes, and nano‐ and macro‐discs. Studying the same protein in different environments can reveal significant differences in the structure, conformation, and orientation of the protein. For example, the HIV‐1 protein Vpu, studied using static oriented sample solid state NMR, in different lipid bicelle environments resulted in different tilt angles of the protein in the model membrane as well as the absence (or presence) of a kink in the transmembrane helix, depending on the bilayer‐forming phospholipids. Another example is the structure obtained for the hepatitis C protein p7. In DPC micelles, the protein is a hexamer whereas in DHPC micelles the protein is a monomer, having the typical two‐transmembrane spanning protein topology.A macrodisc is a detergent‐free model membrane system composed of phospholipid bilayers surrounded by a peptide, protein, or styrene‐maleic acid (SMA) polymer belt, with a diameter of > 30nm. They serve to immobilize and magnetically align embedded membrane proteins. Examples of proteins having between one and seven trans‐membrane helices will be described, including G‐protein coupled receptors (GPCRs).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TPS domain folding causes activation of a newly defined hemolytic functional domain
Authors: Wade Gina Mary , Weaver Todd Michael , Grilley Daniel Patrick 
Published: June 2021

Proteus mirabilis is a pathogenic opportunistic Gram‐negative bacterium that produces and secretes a toxic protein called hemolysin A (HpmA). P. mirabilis utilizes a two‐partner secretion (TPS) pathway to simultaneously transport and activate HpmA across its outer membrane and release it into the blood of a host where it destroys red blood cells (hemolysis). Like all other TPS proteins, HpmA contains an N‐terminal TPS domain (≈300 residues) essential for secretion and a separate C‐terminal functional domain (>1000 residues) responsible for virulent functions. To understand the functional relationship between these domains and define the boundaries of the virulent hemolytic domain in HpmA, we made N‐terminal, internal, and C‐terminal deletions. In the absence of secretion, the unfolded full‐length HpmA requires a folded TPS domain to act as a template protein to achieve its hemolytically active form. Using a Template Assisted Hemolytic Activity (TAHA) assay, we can quantitate the effect of the deletions on HpmA function. Unfolded proteins lacking a TPS domain at the N‐terminus show no activity in TAHA assays. This suggests that TPS domain folding is required for activation of hemolytic activity. To determine if there is a direct linkage between TPS domain folding and hemolytic activation, we created internal deletions immediately C‐terminal to the TPS domain. These demonstrate how TPS domain folding is coupled to activation of hemolytic function. We also show that a significant segment (>300 residues) can be removed from the C‐terminus and not affect the hemolytic function. These results have allowed us to define the C‐terminal boundary of the hemolytic functional domain. Results of this research have allowed us to locate regions of HpmA necessary for its hemolytic function and characterize how TPS domain folding is required to induce hemolytic activation. These studies have expanded our working model for HpmA secretion, activation, and function.Support or Funding InformationThis research was generously supported by a University of Wisconsin ‐ La Crosse Undergraduate Research and Creativity Grant (GMW).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.