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Protein Science: a Publication of the Protein Society

Keshalini Sabaratnam, Max Renner, Guido Paesen, Karl Harlos, Venugopal Nair, Raymond J Owens, Jonathan M Grimes
cAMP response element binding protein 3 (CREB3) is an endoplasmic reticulum (ER) membrane-bound transcription factor, which belongs to the basic leucine zipper (bZIP) superfamily of eukaryotic transcription factors. CREB3 plays a role in the ER-stress induced unfolded protein response (UPR) and is a multifunctional cellular factor implicated in a number of biological processes including cell proliferation and migration, tumour suppression, and immune-related gene expression. To gain structural insights into the transcription factor, we determined the crystal structure of the conserved bZIP domain of chicken CREB3 (chCREB3) to a resolution of 3...
January 17, 2019: Protein Science: a Publication of the Protein Society
Enrique S Morales, Ivana L Parcerisa, Eduardo A Ceccarelli
The production of recombinant proteins in bacteria has increased significantly in recent years, becoming a common tool for both research and the industrial production of proteins. One of the requirements of this methodology is to obtain the desired protein without contaminants. However, this goal cannot always be readily achieved. Multiple strategies have been developed to improve the quality of the desired protein product. Nevertheless, contamination with molecular chaperones is one of the recalcitrant problems that still affect the quality of the obtained proteins...
January 17, 2019: Protein Science: a Publication of the Protein Society
Daisuke Takahashi, Kano Suzuki, Taiichi Sakamoto, Takeo Iwamoto, Takeshi Murata, Fumio Sakane
Diacylglycerol kinases (DGKs) are multi-domain lipid kinases that phosphorylate diacylglycerol into phosphatidic acid, modulating the levels of these key signaling lipids. Recently, increasing attention has been paid to DGKα isozyme as a potential target for cancer immunotherapy. We have previously shown that DGKα is positively regulated by Ca2+ binding to its N-terminal EF-hand domains (DGKα-EF). However, little progress has been made for the structural biology of mammalian DGKs and the molecular mechanism underlying the Ca2+ -triggered activation remains unclear...
January 17, 2019: Protein Science: a Publication of the Protein Society
Grzegorz Bereta, Theodoros Goulas, Mariusz Madej, Ewa Bielecka, Maria Solà, Jan Potempa, F Xavier Gomis-Rüth
Citrullination is an essential post-translational modification in which the guanidinium group of protein and peptide arginines is deiminated by peptidylarginine deiminases (PADs). When deregulated, excessive citrullination leads to inflammation as in severe periodontal disease (PD) and rheumatoid arthritis (RA). Porphyromonas gingivalis is the major periodontopathogenic causative agent of PD and also an etiological agent of RA. It secretes a PAD, termed Porphyromonas PAD (PPAD), which is a virulence factor that causes aberrant citrullination...
January 14, 2019: Protein Science: a Publication of the Protein Society
Allison W Wong, Anatoly Urisman, Alma L Burlingame, Kevan M Shokat
The proteomic mapping of enzyme-substrate interactions is challenged by their transient nature. A method to capture interacting protein kinases in complexes with a single substrate of interest would provide a new tool for mapping kinase signaling networks. Here we describe a nucleotide based substrate analog capable of reprogramming the wild-type phosphoryl-transfer reaction to produce a kinase-acrylamide based thioether crosslink to mutant substrates with a cysteine nucleophile substituted at the native phosphorylation site...
January 13, 2019: Protein Science: a Publication of the Protein Society
Yuka Kawahara-Nakagawa, Koji Nishikawa, Satoru Nakashima, Shota Inoue, Takehiro Ohta, Takashi Ogura, Yasuteru Shigeta, Katsuyuki Fukutani, Tatsuhiko Yagi, Yoshiki Higuchi
Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox-dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy...
January 4, 2019: Protein Science: a Publication of the Protein Society
Ki Baek Lee, Zachary Dunn, Xin Ge
Playing pivotal roles in tumor growth and metastasis, matrix metalloproteinase-14 (MMP-14) is an important cancer target. Potent inhibitory Fab 3A2 with therapy-desired high selectivity has been isolated from a synthetic antibody library carrying long CDR-H3s. However, like many standard mechanism protease inhibitors, Fab 3A2 can be cleaved by high concentrations of MMP-14 after extended incubation at acidic pH. Edman sequencing of generated 3A2 fragments indicated that cleavage occurred within its CDR-H3 between residues N100h (P1) and L100i (P1')...
December 28, 2018: Protein Science: a Publication of the Protein Society
Casper Bøjer Rasmussen, Gunna Christiansen, Brian Stougaard Vad, Carina Lynggaard, Jan J Enghild, Maria Andreasen, Daniel Otzen
Functional amyloid (FA) is widespread in bacteria and serves multiple purposes such as strengthening of biofilm and contact with eukaryotic hosts. Unlike pathological amyloid, FA has been subjected to evolutionary optimization which is likely to be reflected in the aggregation mechanism. FA from different bacteria, including E. coli (CsgA) and Pseudomonas (FapC), contains a number of imperfect repeats which may be key to efficient aggregation. Here we report on the aggregative behavior of FapC constructs which represent all single, double, and triple deletions of the protein's 3 imperfect repeats...
December 28, 2018: Protein Science: a Publication of the Protein Society
Jimin Wang
In the absence of protective reducing agents, Cys residues in purified proteins can be oxidized spontaneously by oxygen in the air, as frequently observed in protein crystal structures. However, formation of an O-bridge via dehydration mechanism between a peroxidized Cys side chain and a primary amine of Lys side chain in proteins has not yet been reported. When an electron density feature was observed for an extra group or an extra atom between side chains of Cys-245 and Lys-158 in the crystal structure of histidinol phosphate phosphatase, mass spectrometric analysis was carried out for its chemical identification...
December 28, 2018: Protein Science: a Publication of the Protein Society
Milo B Fasken, Anita H Corbett, Murray Stewart
The poly(A) RNA binding Zn finger ribonucleoprotein Nab2 functions to control the length of 3' poly(A) tails in Saccharomyces cerevisiae as well as contributing to the integration of the nuclear export of mature mRNA with preceding steps in the nuclear phase of the gene expression pathway. Nab2 is constructed from an N-terminal PWI-fold domain, followed by QQQP and RGG motifs and then seven CCCH Zn fingers. The nuclear pore-associated proteins Gfd1 and Mlp1 bind to opposite sides of the Nab2 N-terminal domain and function in the nuclear export of mRNA, whereas the Zn fingers, especially fingers 5-7, bind to A-rich regions of mature transcripts and function to regulate poly(A) tail length as well as mRNA compaction prior to nuclear export...
December 22, 2018: Protein Science: a Publication of the Protein Society
Xuejun C Zhang, Hang Li
Transmembrane electrostatic membrane potential is a major energy source of the cell. It determines the structures and functions of charge-carrying membrane proteins. Here, we discuss the relationship between membrane potential and the immersed membrane protein, in particular whether the conformation of the membrane protein is integrally connected to the membrane potential. These concepts provide a framework for understanding the electrostatic effects of membrane potential on the reversible or unidirectional processes of membrane proteins...
December 14, 2018: Protein Science: a Publication of the Protein Society
Jacob A Brockerman, Mark Okon, Stephen G Withers, Lawrence P McIntosh
T4 phage lysozyme (T4L) is an enzyme that cleaves bacterial cell wall peptidoglycan. Remarkably, the single substitution of the active site Thr26 to a His (T26H) converts T4L from an inverting to a retaining glycoside hydrolase with transglycosylase activity. It has been proposed that T26H-T4L follows a double displacement mechanism with His26 serving as a nucleophile to form a covalent glycosyl-enzyme intermediate (Kuroki et al. (1999) PNAS 96: 8949-8954). To gain further insights into this or alternative mechanisms, we used NMR spectroscopy to measure the acid dissociation constants (pKa values) and/or define the ionization states of the Asp, Glu, His, and Arg residues in the T4L mutant...
December 7, 2018: Protein Science: a Publication of the Protein Society
Xuping Jing, Yihui Yuan, Yan Wu, Dandan Wu, Peng Gong, Meiying Gao
Insecticidal crystal (Cry) proteins produced by Bacillus thuringiensis (Bt) are widely used as environmentally friendly insecticides. As the only known Cry protein with insecticidal activity against Locusta migratoria manilensis, a locust subspecies that causes extensive destruction of crops, the Cry7Ca1 protein from Bt strain BTH-13 identified in our previous study is of particular interest to locust prevention and control. However, the three-dimensional structure of Cry7Ca1 toxin (the active form of the Cry7Ca1 protein) and the mechanisms of the Cry7Cal insecticidal specificity remain largely elusive...
December 2, 2018: Protein Science: a Publication of the Protein Society
Sergio E Martinez, Joseph D Bauman, Kalyan Das, Eddy Arnold
Stavudine (d4T, 2',3'-didehydro-2',3'-dideoxythymidine) was one of the first chain-terminating nucleoside analogs used to treat HIV infection. We present the first structure of the active, triphosphate form of d4T (d4TTP) bound to a catalytic complex of HIV-1 RT/dsDNA template-primer. We also present a new strategy for disulfide (S-S) chemical cross-linking between N6 of a modified adenine at the second overhang base to I63C in the fingers subdomain of RT. The cross-link site is upstream of the duplex-binding region of RT, however, the structure is very similar to published RT structures with cross-linking to Q258C in the thumb, which suggests that cross-linking at either site does not appreciably perturb the RT/DNA structures...
November 30, 2018: Protein Science: a Publication of the Protein Society
Yuwei Tian, Jennifer L Lippens, Chawita Netirojjanakul, Iain Campuzano, Brandon T Ruotolo
Antibody-drug conjugates (ADCs) are antibody-based therapeutics that have proven to be highly effective cancer treatment platforms. They are comprised of monoclonal antibodies conjugated with highly potent drugs via chemical linkers. Compared to cysteine-targeted chemistries, conjugation at native lysine residues can lead to a higher degree of structural heterogeneity, and thus it is important to evaluate the impact of conjugation on antibody conformation. Here, we present a workflow involving native ion mobility (IM)-MS and gas-phase unfolding for the structural characterization of lysine-linked mAb-biotin conjugates...
November 30, 2018: Protein Science: a Publication of the Protein Society
Kriti Chopra, Shrankhla Bawaria, Radha Chauhan
Nuclear pore complex (NPC) is the largest multimeric protein assembly of the eukaryotic cell, which mediates the nucleocytoplasmic transport. The constituent proteins of this assembly (nucleoporins) are present in varying copy numbers to give a size from ~ 60MDa (yeast) to 112MDa (human) and share common ancestry with other membrane-associated complexes such as COPI/COPII and thus share the same structural folds. However, the nucleoporins across species exhibit very low percentage sequence similarity and this reflects in their distinct secondary structure and domain organization...
November 29, 2018: Protein Science: a Publication of the Protein Society
Dominique Mias-Lucquin, Angélique Chéron, Elisabeth Le Rumeur, Jean-François Hubert, Olivier Delalande
Coiled-coil domain is a structural motif found in proteins crucial for achievement of central biological processes, such as cellular cohesion or neuro-transmission. The coiled-coil fold consists of alpha-helices bundle that can be repeated to form larger filament. Hydrophobic residues, distributed following a regular seven-residues pattern, named heptad pattern, are commonly admitted to be essential for the formation and the stability of canonical coiled-coil repeats. Here we investigated the first three coiled-coil repeats (R1-3) of the central domain of dystrophin, a scaffolding protein in muscle cells whose deficiency leads to Duchenne and Becker Muscular Dystrophies...
November 23, 2018: Protein Science: a Publication of the Protein Society
R Kalaivani, T J Narwani, A G de Brevern, N Srinivasan
Many protein kinases are characterized by at least two structural forms corresponding to the highest level of activity (active) and low or no activity, (inactive). Further, protein dynamics is an important consideration in understanding the molecular and mechanistic basis of enzyme function. In this work, we use Protein kinase A (PKA) as the model system and perform microsecond range molecular dynamics (MD) simulations on 6 variants which differ from one another in terms of active and inactive form, with or without bound ligands, C-terminal tail and phosphorylation at activation loop...
November 23, 2018: Protein Science: a Publication of the Protein Society
Breann L Brown, Ellen F Vieux, Tejas Kalastavadi, SaRa Kim, James Z Chen, Tania A Baker
The protein quality control network (pQC) plays critical roles in maintaining protein and cellular homeostasis, especially during stress. Lon is a major pQC AAA+ protease, conserved from bacteria to human mitochondria. It is the principal enzyme that degrades most unfolded or damaged proteins. Degradation by Lon also controls cellular levels of several key regulatory proteins. Recently, our group determined that E. coli Lon, previously thought to be an obligate homo-hexamer, also forms a dodecamer. This larger assembly has decreased ATPase activity and displays substrate-specific alterations in degradation compared to the hexamer...
November 21, 2018: Protein Science: a Publication of the Protein Society
Yixin Cui, Gongqin Sun
Site-directed mutagenesis is a traditional approach for structure-function analysis of protein tyrosine kinases, and it requires the generation, expression, purification, and analysis of each mutant enzyme. In this study, we report a versatile high throughput bacterial screening system that can identify functional kinase mutants by immunological detection of tyrosine phosphorylation. Two key features of this screening system are noteworthy. First, instead of blotting bacterial colonies directly from Agar plates to nitrocellulose membrane, the colonies were cultured in 96-well plates, and then spotted in duplicate onto the membrane with appropriate controls...
November 21, 2018: Protein Science: a Publication of the Protein Society
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