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Protein Science: a Publication of the Protein Society

Kelsey M Kean, P Andrew Karplus
Lactate monooxygenase (LMO) catalyzes the FMN-dependent "coupled" oxidation of lactate and O2 to acetate, carbon dioxide and water, involving pyruvate and hydrogen peroxide as enzyme-bound intermediates. Other α-hydroxy acid oxidase family members follow an "uncoupled pathway," wherein the α-keto acid product quickly dissociates before the reduced flavin reacts with oxygen. Here, we report the structures of Mycobacterium smegmatis wild-type LMO and a wild-type-like C203A variant at 2.1 Å and 1...
September 11, 2018: Protein Science: a Publication of the Protein Society
Curran Oi, John D Treado, Zachary A Levine, Christopher S Lim, Kirsten M Knecht, Yong Xiong, Corey S O'Hern, Lynne Regan
We present the structure of an engineered protein-protein interface between two beta barrel proteins, which is mediated by interactions between threonine (Thr) residues. This Thr zipper structure suggests that the protein interface is stabilized by close-packing of the Thr residues, with only one inter-monomer hydrogen bond (H-bond) between two of the Thr residues. This Thr-rich interface provides a unique opportunity to study the behavior of Thr in the context of many other Thr residues. In previous work, we have shown that the side chain (χ1 ) dihedral angles of interface and core Thr residues can be predicted with high accuracy using a hard sphere plus stereochemical constraint (HS) model...
September 10, 2018: Protein Science: a Publication of the Protein Society
Ryan L Hayes, Jonah Z Vilseck, Charles L Brooks
The estimation of changes in free energy upon mutation is central to the problem of protein design. Modern protein design methods have had remarkable success over a wide range of design targets, but are reaching their limits in ligand binding and enzyme design due to insufficient accuracy in mutational free energies. Alchemical free energy calculations have the potential to supplement modern design methods through more accurate molecular dynamics based prediction of free energy changes, but suffer from high computational cost...
September 2, 2018: Protein Science: a Publication of the Protein Society
Jeffrey S McFarlane, Richard Hagen, Annemarie S Chilton, Dianna Forbes, Audrey Lamb, Holly R Ellis
The π-helix located at the tetramer interface of two-component FMN-dependent reductases contributes to the structural divergence from canonical FMN-bound reductases within the NADPH:FMN reductase family. The π-helix in the SsuE FMN-dependent reductase of the alkanesulfonate monooxygenase system has been proposed to be generated by the insertion of a Tyr residue in the conserved α4-helix. Variants of Tyr118 were generated, and their X-ray crystal structures determined, to evaluate how these alterations affect the structural integrity of the π-helix...
August 31, 2018: Protein Science: a Publication of the Protein Society
Emily G Sweeney, Arden Perkins, Karen Kallio, S James Remington, Karen Guillemin
Bacteria use chemoreceptor proteins to sense and navigate their chemical environments. The most common class of chemoreceptors are transmembrane proteins that sense chemical cues through binding of a small molecule ligand to a periplasmic domain, which modulates the receptor's ability to stimulate reversal of the cell's flagella motors. The prevalent gastric pathogen Helicobacter pylori (H. pylori) uses such membrane-bound chemoreceptors, called transducer-like proteins (Tlp), to colonize and persist within the stomach...
August 31, 2018: Protein Science: a Publication of the Protein Society
Andrew J Miles, B A Wallace
Circular dichroism (CD) spectroscopy is a highly-used method for the examination and characterisation of proteins, including, amongst other features, their secondary and tertiary structures, thermal stability, comparisons of wildtype and mutant proteins, and monitoring the binding of small molecules, folding/unfolding pathways, and formation of macromolecular complexes. This paper describes CDtoolX, a new, user-friendly, free-to-download-and-use software program that enables processing, displaying, archiving, calibrating, comparisons, and analyses of circular dichroism and synchrotron radiation circular dichroism (SRCD) spectroscopic data...
August 30, 2018: Protein Science: a Publication of the Protein Society
Liezelle C Lopez, Nikita Mukhitov, Lindsey D Handley, Cristina S Hamme, Cristina R Hofman, Lindsay Euers, Jennifer R McKinney, Amani D Piers, Ellen Wadler, Laura M Hunsicker-Wang
The Sco protein from Thermus thermophilus has previously been shown to perform a disulfide bond reduction in the CuA protein from Thermus thermophilus, which is a soluble protein engineered from subunit II of cytochrome ba3 oxidase that lacks the transmembrane helix. The native cysteines on TtSco and TtCuA were mutated to serine residues to probe the reactivities of the individual cysteines. Conjugation of TNB to the remaining cysteine in TtCuA and subsequent release upon incubation with the complementary TtSco protein demonstrated formation of the mixed disulfide intermediate...
August 30, 2018: Protein Science: a Publication of the Protein Society
Jakub Ptacek, Jana Nedvedova, Michal Navratil, Barbora Havlinova, Jan Konvalinka, Cyril Barinka
Calcium ions are required for proper function of a wide spectrum of proteins within cells. X-ray crystallography of human glutamate carboxypeptidase II (GCPII) revealed the presence of a Ca2+ -binding site, but its importance for the structure and function of this metallopeptidase has not been elucidated to date. Here, we prepared a panel of mutants targeting residues that form the Ca2+ coordination sphere of GCPII and analyzed their structural and enzymatic properties using an array of complementary biophysical and biochemical approaches...
August 30, 2018: Protein Science: a Publication of the Protein Society
Abhinandan V Murthy, Ramita Sulu, M Kristian Koski, Hongmin Tu, Jothi Anantharajan, Shiv K Sah-Teli, Johanna Myllyharju, Rik K Wierenga
The peptide-substrate-binding (PSB) domain of collagen prolyl 4-hydroxylase (C-P4H, an α2 β2 tetramer) binds proline-rich procollagen peptides. This helical domain (the middle domain of the α subunit) has an important role concerning the substrate binding properties of C-P4H, although it is not known how the PSB domain influences the hydroxylation properties of the catalytic domain (the C-terminal domain of the α subunit). The crystal structures of the PSB domain of the human C-P4H isoform II (PSB-II) complexed with and without various short proline-rich peptides are described...
August 30, 2018: Protein Science: a Publication of the Protein Society
Judy P Hines, Matthew R Dent, Daniel J Stevens, Judith N Burstyn
The transcriptional activator CooA belongs to the CRP/FNR (cAMP receptor protein/fumarate and nitrate reductase) superfamily of transcriptional regulators and uses heme to sense carbon monoxide (CO). Effector-driven allosteric activation is well understood in CRP, a CooA homologue. A structural allosteric activation model for CooA exists which parallels that of CRP; however, the role of protein dynamics, which is crucial in CRP, is not well understood in CooA. We employed site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) spectroscopy to probe CooA motions on the μs-ms timescale...
August 30, 2018: Protein Science: a Publication of the Protein Society
Jon D Faughn, William L Dean, Christine E Schaner Tooley
Protein, DNA, and RNA methyltransferases have an ever-expanding list of novel substrates and catalytic activities. Even within families and between homologs, it is becoming clear the intricacies of methyltransferase specificity and regulation are far more diverse than originally thought. In addition to specific substrates and distinct methylation levels, methyltransferase activity can be altered by complex formation with close homologs. We work with the N-terminal methyltransferase homologs NRMT1 and NRMT2...
August 27, 2018: Protein Science: a Publication of the Protein Society
Euiyeon Lee, Kyoungmi Min, Youngtae Chang, Youngeun Kwon
An efficient and wash-free method to conjugate a fluorescent tag to a target membrane protein is developed, using engineered Npu DnaE split-inteins. This approach allowed fast labeling while avoiding the strenuous synthesis of a long polypeptide. Two different modes of labeling, namely specific binding and covalent conjugation, are observed. The covalent labeling was monitored within 5 min, without background staining. This article is protected by copyright. All rights reserved.
August 27, 2018: Protein Science: a Publication of the Protein Society
Kazuhide Miyamoto, Arisa Nakatani, Mayumi Sunagawa, Kazuki Saito
Ubiquitin-conjugating (E2) enzymes in protein ubiquitination are associated with various diseases. An artificial RING finger (ARF) is a useful tool, and E2 activities are conveniently estimated based on ARF reactivities. To extend the use of ARF in cells, we constructed a TAT-ARF using a cell-penetrating TAT peptide. An in vitro ubiquitination assay without substrates showed auto-ubiquitination of TAT-ARF via its TAT region. TAT-ARF was translocated into MCF7 breast cancer cells, and then TAT-ARF ubiquitinated itself via its ARF...
August 26, 2018: Protein Science: a Publication of the Protein Society
Garry W Buchko, Surya V S R K Pulavarti, Victor Ovchinnikov, Elizabeth A Shaw, Stephen A Rettie, Peter J Myler, Martin Karplus, Thomas Szyperski, David Baker, Christopher D Bahl
Disulfide-rich peptides represent an important protein family with broad pharmacological potential. Recent advances in computational methods have made it possible to design new peptides which adopt a stable conformation de novo. Here, we describe a system to produce disulfide-rich de novo peptides using Escherichia coli as the expression host. The advantage of this system is that it enables production of uniformly 13 C- and 15 N-labeled peptides for solution nuclear magnetic resonance (NMR) studies. This expression system was used to isotopically label two previously reported de novo designed peptides, and to determine their solution structures using NMR...
August 26, 2018: Protein Science: a Publication of the Protein Society
Robert W Hepler, Debbie D Nahas, Bob Lucas, Robin Kaufhold, Jessica A Flynn, Jennifer D Galli, Ryan Swoyer, James M Wagner, Amy Espeseth, Joseph G Joyce, James C Cook, Eberhard Durr
Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. C. trachomatis serovars D-K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C...
August 25, 2018: Protein Science: a Publication of the Protein Society
Prakash Kulkarni, Tsega Solomon, Yanan He, Yihong Chen, Philip N Bryan, John Orban
The classical view of the structure-function paradigm advanced by Anfinsen in the 1960s is that a protein's function is inextricably linked to its three-dimensional structure and is encrypted in its amino acid sequence. However, it is now known that a significant fraction of the proteome consists of intrinsically disordered proteins (IDPs). These proteins populate a polymorphic ensemble of conformations rather than a unique structure but are still capable of performing biological functions. At the boundary between well-ordered and inherently disordered states are proteins that are on the brink of stability, either weakly stable ordered systems or disordered but on the verge of being stable...
August 24, 2018: Protein Science: a Publication of the Protein Society
Kristen M Ramsey, Dominic Narang, Elizabeth A Komives
The ankyrin repeat (AR) structure is a common protein-protein interaction motif and ankyrin repeat proteins comprise a vast family across a large array of different taxa. Natural AR proteins adopt a conserved fold comprised of several repeats with the N- and C-terminal repeats generally being of more divergent sequences. Obtaining experimental crystal structures for natural ARD can be difficult and often requires complexation with a binding partner. Homology modeling is an attractive method for creating a model of AR proteins due to the highly conserved fold, however modeling the divergent N- and C-terminal 'capping' repeats remains a challenge...
August 22, 2018: Protein Science: a Publication of the Protein Society
Harry Scott, Wei Huang, James G Bann, Derek J Taylor
The beta pore-forming proteins (β-PFPs) are a large class of polypeptides that are produced by all Kingdoms of life to contribute to their species' own survival. Pore assembly is a sophisticated multi-step process that includes receptor/membrane recognition and oligomerization events, that is ensued by large-scale structural rearrangements that facilitate maturation of a prepore into a functional, membrane spanning pore. A full understanding of pore formation, assembly, and maturation has traditionally been hindered by a lack of structural data; particularly for assemblies representing differing conformations of functional pores...
August 20, 2018: Protein Science: a Publication of the Protein Society
Gaurav Ghag, Nemil Bhatt, Daniel V Cantu, Marcos J Guerrero-Munoz, Anna Ellsworth, Urmi Sengupta, Rakez Kayed
Several studies have proposed that fibrillary aggregates of tau and other amyloidogenic proteins are neurotoxic and result in numerous neurodegenerative diseases. However, these studies usually involve sonication or extrusion through needles prior to the experimentation. As a consequence, these methods may fragment large aggregates producing a mixture of aggregated species rather than intact fibrils. Therefore, the results of these experiments may be reflective of other amyloidogenic species, such as oligomers and/or protofibrils/short fibrils...
August 20, 2018: Protein Science: a Publication of the Protein Society
Dimitri Niks, Russ Hille
An overview is provided of the molybdenum- and tungsten-containing enzymes that catalyze the interconversion of formate and CO2 , focusing on common structural and mechanistic themes, as well as a consideration of the manner in which the mature Mo- or W-containing cofactor is inserted into apoprotein. This article is protected by copyright. All rights reserved.
August 17, 2018: Protein Science: a Publication of the Protein Society
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