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The monoclonal antibody (mAb) protein class has become a primary therapeutic platform for the production of new life saving drug products. MAbs are comprised of two domains: the antigen-binding fragment (Fab) and crystallizable fragment (Fc). Despite the success in the clinic, NMR assignments of the complete Fab domain have been elusive, in part due to problems in production of properly folded, triply-labeled 2 H, 13 C, 15 N Fab domain. Here, we report the successful recombinant expression of a triply-labeled Fab domain, derived from the standard IgG1κ known as NISTmAb, in yeast. Using the 2 H, 13 C, 15 N Fab domain, we assigned 94% of the 1 H, 13 C, and 15 N backbone atoms., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Metalloproteins perform a diverse array of redox-related reactions facilitated by the increased chemical functionality afforded by their metallocofactors. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that are responsible for the breakdown of recalcitrant polysaccharides via oxidative cleavage at the glycosidic bond. The activated copper-oxygen intermediates and their mechanism of formation remains to be established. Neutron protein crystallography which permits direct visualization of protonation states was used to investigate the initial steps of oxygen activation directly following active site copper reduction in Neurospora crassa LPMO9D. Herein, we cryo-trap an activated dioxygen intermediate in a mixture of superoxo and hydroperoxo states, and we identify the conserved second coordination shell residue His157 as the proton donor. Density functional theory calculations indicate that both superoxo and hydroperoxo active site states are stable. The hydroperoxo formed is potentially an early LPMO catalytic reaction intermediate or the first step in the mechanism of hydrogen peroxide formation in the absence of substrate. We observe that the N-terminal amino group of the copper coordinating His1 remains doubly protonated directly following molecular oxygen reduction by copper. Aided by molecular dynamics and mining minima free energy calculations we establish that the conserved second-shell His161 in Mt PMO3* displays conformational flexibility in solution and that this flexibility is also observed, though to a lesser extent, in His157 of Nc LPMO9D. The imidazolate form of His157 observed in our structure following oxygen intermediate protonation can be attributed to abolished His157 flexibility due steric hindrance in the crystal as well as the solvent-occluded active site environment due to crystal packing. A neutron crystal structure of Nc LPMO9D at low pH further supports occlusion of the active site since His157 remains singly protonated even at acidic conditions., Competing Interests: S. P. W. declares an equity interest in VeraChem LLC, which develops the VM2 free energy software package. The remaining authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)

Revealing the positions of all the atoms in large macromolecules is powerful but only possible with neutron macromolecular crystallography (NMC). Neutrons provide a sensitive and gentle probe for the direct detection of protonation states at near-physiological temperatures and clean of artifacts caused by x rays or electrons. Currently, NMC use is restricted by the requirement for large crystal volumes even at state-of-the-art instruments such as the macromolecular neutron diffractometer at the Spallation Neutron Source. EWALD's design will break the crystal volume barrier and, thus, open the door for new types of experiments, the study of grand challenge systems, and the more routine use of NMC in biology. EWALD is a single crystal diffractometer capable of collecting data from macromolecular crystals on orders of magnitude smaller than what is currently feasible. The construction of EWALD at the Second Target Station will cause a revolution in NMC by enabling key discoveries in the biological, biomedical, and bioenergy sciences.

Lytic polysaccharide monooxygenases (LPMOs) are copper-center enzymes that are involved in the oxidative cleavage of the glycosidic bond in crystalline cellulose and other polysaccharides. The LPMO reaction is initiated by the addition of a reductant and oxygen to ultimately form an unknown activated copper-oxygen species that is responsible for polysaccharide-substrate H-atom abstraction. Given the sensitivity of metalloproteins to radiation damage, neutron protein crystallography provides a nondestructive technique for structural characterization while also informing on the positions of H atoms. Neutron cryo-crystallography permits the trapping of catalytic intermediates, thereby providing insight into the protonation states and chemical nature of otherwise short-lived species in the reaction mechanism. To characterize the reaction-mechanism intermediates of LPMO9D from Neurospora crassa, a cryo-neutron diffraction data set was collected from an ascorbate-reduced crystal. A second neutron diffraction data set was collected at room temperature from an LPMO9D crystal exposed to low-pH conditions to probe the protonation states of ionizable groups involved in catalysis under acidic conditions.

A protocol for increasing soluble protein expression by fusing the chaperone GroEL apical domain with a gene of interest is described herein. GroEL apical domain, the minichaperone that functions independently of GroES and ATP in protein folding, is cloned downstream of the lambda CII ribosome binding site in the parent pRE vector. The pRE vector has tightly controlled transcription suitable for expressing toxic proteins. The GroEL minichaperone is fused to a glycine-serine rich linker followed by the enterokinase protease recognition sequence. A number of genes that are recalcitrant to protein production in the parent pRE vector 5were cloned into the pRE:GroEL fusion vector and successfully expressed as fusion proteins in Escherichia coli., Competing Interests: Disclaimer Certain commercial equipment, instruments or materials are identified in this paper in order to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by NIST, nor is it intended to imply that the materials or equipment identified are necessarily the best available for the purpose., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Replicative DNA helicases are essential cellular enzymes that unwind duplex DNA in front of the replication fork during chromosomal DNA replication. Replicative helicases were discovered, beginning in the 1970s, in bacteria, bacteriophages, viruses, and eukarya, and, in the mid-1990s, in archaea. This year marks the 20th anniversary of the first report on the archaeal replicative helicase, the minichromosome maintenance (MCM) protein. This minireview summarizes 2 decades of work on the archaeal MCM., (This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.)

Monoclonal antibodies (mAbs) represent an important platform for the development of biotherapeutic products. Most mAbs are produced in mammalian cells, but several mAbs are made in Escherichia coli , including therapeutic fragments. The NISTmAb is a well-characterized reference material made widely available to facilitate the development of both originator biologics and biosimilars. Here, when expressing NISTmAb from codon-optimized constructs in E. coli (eNISTmAb), a truncated variant of its heavy chain was observed. N-terminal protein sequencing and mutagenesis analyses indicated that the truncation resulted from an internal translation initiation from a GTG codon (encoding Val) within eNISTmAb. Using computational and biochemical approaches, we demonstrate that this translation initiates from a weak Shine-Dalgarno sequence and is facilitated by a putative ribosomal protein S1-binding site. We also observed similar internal initiation in the mAb adalimumab (the amino acid sequence of the drug Humira) when expressed in E. coli Of note, these internal initiation regions were likely an unintended result of the codon optimization for E. coli expression, and the amino acid pattern from which it is derived was identified as a Pro-Ser- X-X-X -Val motif. We discuss the implications of our findings for E. coli protein expression and codon optimization and outline possible strategies for reducing the likelihood of internal translation initiation and truncated product formation.

The scattering of neutrons can be used to provide information on the structure and dynamics of biological systems on multiple length and time scales. Pursuant to a National Science Foundation-funded workshop in February 2018, recent developments in this field are reviewed here, as well as future prospects that can be expected given recent advances in sources, instrumentation and computational power and methods. Crystallography, solution scattering, dynamics, membranes, labeling and imaging are examined. For the extraction of maximum information, the incorporation of judicious specific deuterium labeling, the integration of several types of experiment, and interpretation using high-performance computer simulation models are often found to be particularly powerful.

Neutron diffraction is exquisitely sensitive to the positions of H atoms in protein crystal structures. IMAGINE is a high-intensity, quasi-Laue neutron crystallography beamline developed at the High Flux Isotope Reactor (HFIR) at Oak Ridge National Laboratory. This state-of-the-art facility for neutron diffraction has enabled detailed structural analysis of macromolecules. IMAGINE is especially suited to resolve individual H atoms in protein structures, enabling neutron protein structures to be determined at or near atomic resolutions from crystals with volumes of less than 1 mm 3 and unit-cell edges of less than 150 Å. Beamline features include elliptical focusing mirrors that deliver neutrons into a 2.0 × 3.2 mm focal spot at the sample position, and variable short- and long-wavelength cutoff optics that provide automated exchange between multiple wavelength configurations. This review gives an overview of the IMAGINE beamline at the HFIR, presents examples of the scientific questions being addressed at this beamline, and highlights important findings in enzyme chemistry that have been made using the neutron diffraction capabilities offered by IMAGINE.

Background: Cellobiose dehydrogenases have gained interest due to their potential applications in sectors from biofuel production to biomedical devices. The CDHIIA variant is comprised of a cytochrome domain (CYT), a dehydrogenase domain (DH), and a carbohydrate-binding module (CBM) that are connected by two flexible linkers. Upon cellobiose oxidation at the DH, intramolecular electron transfer (IaET) occurs from the DH to the CYT. In vivo, CDHIIA CYT subsequently performs intermolecular electron transfer (IeET) to a lytic polysaccharide monooxygenase (LPMO). The relevant solution-state CDH domain conformations for IaET and IeET have not been fully characterized., Methods: Small-angle X-ray and neutron scattering measurements of oxidized CDHIIA from Myriococcum thermophilum and Neurospora crassa were performed to investigate the structural landscape explored in solution by MtCDHIIA and NcCDHIIA in response to cations, pH, and the presence of an electron acceptor, LPMO9D from N. crassa., Results: The scattering data complemented by modeling show that, under oxidizing conditions, MtCDHIIA undergoes global conformational rearrangement in the presence of Ca 2+ . Oxidized NcCDHIIA exhibits conformational changes upon pH variation and, in the presence of NcLPMO9D, primarily adopts a compact conformation., Conclusions: These results demonstrate different conformational responses of oxidized MtCDHIIA and NcCDHIIA to changes in environment. The results also reveal a shift in the oxidized NcCDHIIA conformational landscape toward interdomain compaction upon co-incubation with NcLPMO9D., General Significance: The present study is the first report on the structural landscapes explored in solution by oxidized cellobiose dehydrogenases under various cation concentrations, pH conditions and in the presence of an electron-accepting LPMO., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sensitivity to hydrogen/deuterium and lack of observable radiation damage makes cold neutrons an ideal probe the structural studies of proteins with highly photosensitive groups such as the copper center of lytic polysaccharide monooxygenases (LPMOs) and flavin adenine dinucleotide (FAD) and heme redox cofactors of cellobiose dehydrogenases (CDHs). Here, neutron crystallography and small-angle neutron scattering are used to investigate Neurospora crassa LPMO9D (NcLPMO9D) and CDHIIA (NcCDHIIA), respectively. The presence of LPMO greatly enhances the efficiency of commercial glycoside hydrolase cocktails in the depolymerization of cellulose. LPMOs can receive electrons from CDHs to activate molecular dioxygen for the oxidation of cellulose resulting in chain cleavage and disruption of local crystallinity. Using neutron protein crystallography, the hydrogen/deuterium atoms of NcLPMO9D could be located throughout the structure. At the copper active site, the protonation states of the side chains of His1, His84, His157 and Tyr168, and the orientation of water molecules could be determined. Small-angle neutron scattering measurements provided low resolution models of NcCDHIIA with both the dehydrogenase and cytochrome domains in oxidized states that exhibited elongated conformations. This work demonstrates the suitability of neutron diffraction and scattering for characterizing enzymes critical to oxidative cellulose deconstruction., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bonds via an oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 from Neurospora crassa (NcPMO-2) was heterologously expressed in Pichia pastoris to facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressing NcPMO-2 from a glycoengineered strain of P. pastoris and by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and the production of a large NcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.

Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. We have determined high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed "pre-bound" molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygen activation. These results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme-substrate complex., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Neutron protein crystallography is a powerful tool for investigating protein chemistry because it directly locates hydrogen atom positions in a protein structure. The visibility of hydrogen and deuterium atoms arises from the strong interaction of neutrons with the nuclei of these isotopes. Positions can be unambiguously assigned from diffraction at resolutions typical of protein crystals. Neutrons have the additional benefit to structural biology of not inducing radiation damage in protein crystals. The same crystal could be measured multiple times for parametric studies. Here, we review the basic principles of neutron protein crystallography. The information that can be gained from a neutron structure is presented in balance with practical considerations. Methods to produce isotopically-substituted proteins and to grow large crystals are provided in the context of neutron structures reported in the literature. Available instruments for data collection and software for data processing and structure refinement are described along with technique-specific strategies including joint X-ray/neutron structure refinement. Examples are given to illustrate, ultimately, the unique scientific value of neutron protein crystal structures., (Copyright © 2015 Elsevier Inc. All rights reserved.)