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Full-Text Articles in Life Sciences
Role Of Protein Charge Density On Hepatitis B Virus Capsid Formation, Xinyu Sun, Dong Li, Zhaoshuai Wang, Panchao Yin, Rundong Hu, Rundong Hu, Hui Li, Qiao Liu, Yunyi Gao, Baiping Ren, Jie Zheng, Yinan Wei, Tianbo Liu
Role Of Protein Charge Density On Hepatitis B Virus Capsid Formation, Xinyu Sun, Dong Li, Zhaoshuai Wang, Panchao Yin, Rundong Hu, Rundong Hu, Hui Li, Qiao Liu, Yunyi Gao, Baiping Ren, Jie Zheng, Yinan Wei, Tianbo Liu
Chemistry Faculty Publications
The role of electrostatic interactions in the viral capsid assembly process was studied by comparing the assembly process of a truncated hepatitis B virus capsid protein Cp149 with its mutant protein D2N/D4N, which has the same conformational structure but four fewer charges per dimer. The capsid protein self-assembly was investigated under a wide range of protein surface charge densities by changing the protein concentration, buffer pH, and solution ionic strength. Lowering the protein charge density favored the capsid formation. However, lowering charge beyond a certain point resulted in capsid aggregation and precipitation. Interestingly, both the wild-type and D2N/D4N mutant displayed …
Informing Efforts To Develop Nitroreductase For Amine Production, Anne-Frances Miller, Jonathan T. Park, Kyle L. Ferguson, Warintra Pitsawong, Andreas S. Bommarius
Informing Efforts To Develop Nitroreductase For Amine Production, Anne-Frances Miller, Jonathan T. Park, Kyle L. Ferguson, Warintra Pitsawong, Andreas S. Bommarius
Chemistry Faculty Publications
Nitroreductases (NRs) hold promise for converting nitroaromatics to aromatic amines. Nitroaromatic reduction rate increases with Hammett substituent constant for NRs from two different subgroups, confirming substrate identity as a key determinant of reactivity. Amine yields were low, but compounds yielding amines tend to have a large π system and electron withdrawing substituents. Therefore, we also assessed the prospects of varying the enzyme. Several different subgroups of NRs include members able to produce aromatic amines. Comparison of four NR subgroups shows that they provide contrasting substrate binding cavities with distinct constraints on substrate position relative to the flavin. The unique architecture …
Quaternary Interactions And Supercoiling Modulate The Cooperative Dna Binding Of Agt, Manana Melikishvili, Michael G. Fried
Quaternary Interactions And Supercoiling Modulate The Cooperative Dna Binding Of Agt, Manana Melikishvili, Michael G. Fried
Center for Structural Biology Faculty Publications
Human O6-alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. The search for these lesions, through a vast excess of competing, unmodified genomic DNA, is a mechanistic challenge that may limit the repair rate in vivo. Here, we examine influences of DNA secondary structure and twist on protein–protein interactions in cooperative AGT complexes formed on lesion-free DNAs that model the unmodified parts of the genome. We used a new approach to resolve nearest neighbor (nn) and long-range (lr) components from the ensemble-average cooperativity, ωave. We found …
Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode For Catalytically Competent Nitroreductase, Warintra Pitsawong, Chad A. Haynes, Ronald L. Koder, David W. Rodgers, Anne-Frances Miller
Mechanism-Informed Refinement Reveals Altered Substrate-Binding Mode For Catalytically Competent Nitroreductase, Warintra Pitsawong, Chad A. Haynes, Ronald L. Koder, David W. Rodgers, Anne-Frances Miller
Chemistry Faculty Publications
Nitroreductase from Enterobacter cloacae (NR) reduces diverse nitroaromatics including herbicides, explosives and prodrugs, and holds promise for bioremediation, prodrug activation and enzyme-assisted synthesis. We solved crystal structures of NR complexes with bound substrate or analog for each of its two half-reactions. We complemented these with kinetic isotope effect (KIE) measurements elucidating H-transfer steps essential to each half-reaction. KIEs indicate hydride transfer from NADH to the flavin consistent with our structure of NR with the NADH analog nicotinic acid adenine dinucleotide (NAAD). The KIE on reduction of p-nitrobenzoic acid (p-NBA) also indicates hydride transfer, and requires revision of …
A Comparison Of Nucleosome Organization In Drosophila Cell Lines, Rebecca L. Martin, John Maiorano, Greg J. Beitel, John F. Marko, Graham Mcvicker, Yvonne N. Fondufe-Mittendorf
A Comparison Of Nucleosome Organization In Drosophila Cell Lines, Rebecca L. Martin, John Maiorano, Greg J. Beitel, John F. Marko, Graham Mcvicker, Yvonne N. Fondufe-Mittendorf
Molecular and Cellular Biochemistry Faculty Publications
Changes in the distribution of nucleosomes along the genome influence chromatin structure and impact gene expression by modulating the accessibility of DNA to transcriptional machinery. However, the role of genome-wide nucleosome positioning in gene expression and in maintaining differentiated cell states remains poorly understood. Drosophila melanogastercell lines represent distinct tissue types and exhibit cell-type specific gene expression profiles. They thus could provide a useful tool for investigating cell-type specific nucleosome organization of an organism’s genome. To evaluate this possibility, we compared genome-wide nucleosome positioning and occupancy in five different Drosophila tissue-specific cell lines, and in reconstituted chromatin, and then …
An Arginine Finger Regulates The Sequential Action Of Asymmetrical Hexameric Atpase In The Double-Stranded Dna Translocation Motor, Zhengyi Zhao, Gian Marco De-Donatis, Chad T. Schwartz, Huaming Fang, Jingyuan Li, Peixuan Guo
An Arginine Finger Regulates The Sequential Action Of Asymmetrical Hexameric Atpase In The Double-Stranded Dna Translocation Motor, Zhengyi Zhao, Gian Marco De-Donatis, Chad T. Schwartz, Huaming Fang, Jingyuan Li, Peixuan Guo
Pharmaceutical Sciences Faculty Publications
Biological motors are ubiquitous in living systems. Currently, how the motor components coordinate the unidirectional motion is elusive in most cases. Here, we report that the sequential action of the ATPase ring in the DNA packaging motor of bacteriophage ϕ29 is regulated by an arginine finger that extends from one ATPase subunit to the adjacent unit to promote noncovalent dimer formation. Mutation of the arginine finger resulted in the interruption of ATPase oligomerization, ATP binding/hydrolysis, and DNA translocation. Dimer formation reappeared when arginine mutants were mixed with other ATPase subunits that can offer the arginine to promote their interaction. Ultracentrifugation …
An Extended Polyanion Activation Surface In Insulin Degrading Enzyme, Eun Suk Song, Mehmet Ozbil, Tingting Zhang, Michael Sheetz, David Lee, Danny Tran, Sheng Li, Rajeev Prabhakar, Louis B. Hersh, David W. Rodgers
An Extended Polyanion Activation Surface In Insulin Degrading Enzyme, Eun Suk Song, Mehmet Ozbil, Tingting Zhang, Michael Sheetz, David Lee, Danny Tran, Sheng Li, Rajeev Prabhakar, Louis B. Hersh, David W. Rodgers
Molecular and Cellular Biochemistry Faculty Publications
Insulin degrading enzyme (IDE) is believed to be the major enzyme that metabolizes insulin and has been implicated in the degradation of a number of other bioactive peptides, including amyloid beta peptide (Aβ), glucagon, amylin, and atrial natriuretic peptide. IDE is activated toward some substrates by both peptides and polyanions/anions, possibly representing an important control mechanism and a potential therapeutic target. A binding site for the polyanion ATP has previously been defined crystallographically, but mutagenesis studies suggest that other polyanion binding modes likely exist on the same extended surface that forms one wall of the substrate-binding chamber. Here we use …
Intra-Domain Cross-Talk Regulates Serine-Arginine Protein Kinase 1-Dependent Phosphorylation And Splicing Function Of Transformer 2Β1, Michael A. Jamros, Brandon E. Aubol, Malik M. Keshwani, Zhaiyi Zhang, Stefan Stamm, Joseph A. Adams
Intra-Domain Cross-Talk Regulates Serine-Arginine Protein Kinase 1-Dependent Phosphorylation And Splicing Function Of Transformer 2Β1, Michael A. Jamros, Brandon E. Aubol, Malik M. Keshwani, Zhaiyi Zhang, Stefan Stamm, Joseph A. Adams
Molecular and Cellular Biochemistry Faculty Publications
Transformer 2β1 (Tra2β1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2β1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2β1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated …
Thermodynamics Of Binding By Calmodulin Correlates With Target Peptide Α-Helical Propensity, Tori B. Dunlap, Jessime M. Kirk, Emily A. Pena, Meghan S. Yoder, Trevor P. Creamer
Thermodynamics Of Binding By Calmodulin Correlates With Target Peptide Α-Helical Propensity, Tori B. Dunlap, Jessime M. Kirk, Emily A. Pena, Meghan S. Yoder, Trevor P. Creamer
Center for Structural Biology Faculty Publications
In this work, we have examined contributions to the thermodynamics of calmodulin (CaM) binding from the intrinsic propensity for target peptides to adopt an α-helical conformation. CaM target sequences are thought to commonly reside in disordered regions within proteins. Using the ability of TFE to induce α-helical structure as a proxy, the six peptides studied range from having almost no propensity to adopt α-helical structure through to a very high propensity. This despite all six peptides having similar CaM-binding affinities. Our data indicate there is some correlation between the deduced propensities and the thermodynamics of CaM binding. This finding implies …
Assembly Of The Type Ii Secretion System Such As Found In Vibrio Cholerae Depends On The Novel Pilotin Asps, Rhys A. Dunstan, Eva Heinz, Lakshmi C. Wijeyewickrema, Robert N. Pike, Anthony W. Purcell, Timothy J. Evans, Judyta Praszkier, Roy M. Robins-Browne, Richard A. Strugnell, Konstantin V. Korotkov, Trevor Lithgow
Assembly Of The Type Ii Secretion System Such As Found In Vibrio Cholerae Depends On The Novel Pilotin Asps, Rhys A. Dunstan, Eva Heinz, Lakshmi C. Wijeyewickrema, Robert N. Pike, Anthony W. Purcell, Timothy J. Evans, Judyta Praszkier, Roy M. Robins-Browne, Richard A. Strugnell, Konstantin V. Korotkov, Trevor Lithgow
Molecular and Cellular Biochemistry Faculty Publications
The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of …
C6 Pyridinium Ceramide Influences Alternative Pre-Mrna Splicing By Inhibiting Protein Phosphatase-1, Chiranthani Sumanasekera, Olga Kelemen, Monique Beullens, Brandon E. Aubol, Joseph A. Adams, Manjula Sunkara, Andrew J. Morris, Mathieu Bollen, Athena Andreadis, Stefan Stamm
C6 Pyridinium Ceramide Influences Alternative Pre-Mrna Splicing By Inhibiting Protein Phosphatase-1, Chiranthani Sumanasekera, Olga Kelemen, Monique Beullens, Brandon E. Aubol, Joseph A. Adams, Manjula Sunkara, Andrew J. Morris, Mathieu Bollen, Athena Andreadis, Stefan Stamm
Molecular and Cellular Biochemistry Faculty Publications
Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic …
Direct Cloning Of Double-Stranded Rnas From Rnase Protection Analysis Reveals Processing Patterns Of C/D Box Snornas And Provides Evidence For Widespread Antisense Transcript Expression, Manli Shen, Eduardo Eyras, Jie Wu, Amit Khanna, Serene Josiah, Mathieu Rederstorff, Michael Q. Zhang, Stefan Stamm
Direct Cloning Of Double-Stranded Rnas From Rnase Protection Analysis Reveals Processing Patterns Of C/D Box Snornas And Provides Evidence For Widespread Antisense Transcript Expression, Manli Shen, Eduardo Eyras, Jie Wu, Amit Khanna, Serene Josiah, Mathieu Rederstorff, Michael Q. Zhang, Stefan Stamm
Molecular and Cellular Biochemistry Faculty Publications
We describe a new method that allows cloning of double-stranded RNAs (dsRNAs) that are generated in RNase protection experiments. We demonstrate that the mouse C/D box snoRNA MBII-85 (SNORD116) is processed into at least five shorter RNAs using processing sites near known functional elements of C/D box snoRNAs. Surprisingly, the majority of cloned RNAs from RNase protection experiments were derived from endogenous cellular RNA, indicating widespread antisense expression. The cloned dsRNAs could be mapped to genome areas that show RNA expression on both DNA strands and partially overlapped with experimentally determined argonaute-binding sites. The data suggest a conserved processing pattern …