Chain-selective isotopic labeling of the heterodimeric type iii secretion chaperone, scc4:Scc1, reveals the total structural rearrangement of the chlamydia trachomatis bi-functional protein, scc4

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Scc4 is an unusual bi-functional protein from Chlamydia trachomatis (CT) that functions as a type III secretion system (T3SS) chaperone and an RNA polymerase (RNAP)-binding protein. Both functions require interactions with protein partners during specific stages of the CT developmental cycle. As a T3SS chaperone, Scc4 binds Scc1 during the late stage of development to form a heterodimer complex, which chaperones the essential virulence effector, CopN. During the early-middle stage of development, Scc4 regulates T3SS gene expression by binding the σ66-containing RNAP holoenzyme. In order to study the structure and association mechanism of the Scc4:Scc1 T3SS chaperone complex using nuclear magnetic resonance (NMR) spectroscopy, we developed an approach to selectively label each chain of the Scc4:Scc1 complex with the15N-isotope. The approach allowed one protein to be visible in the NMR spectrum at a time, which greatly reduced resonance overlap and permitted comparison of the backbone structures of free and bound Scc4.1H,15N-heteronuclear single quantum coherence spectra of the15N-Scc4:Scc1 and Scc4:15N-Scc1 complexes showed a total structural rearrangement of Scc4 upon binding Scc1 and a dynamic region isolated to Scc1, respectively. Development of the chain-selective labeling approach revealed that the association of Scc4 and Scc1 requires partial denaturation of Scc1 to form the high affinity complex, while low affinity interactions occurred between the isolated proteins under non-denaturing conditions. These results provide new models for Scc4′s functional switching mechanism and Scc4:Scc1 association in CT

A Kazal-Type Serine Protease Inhibitor from the Defense Gland Secretion of the Subterranean Termite Coptotermes formosanus Shiraki

Coptotermes formosanus is an imported, subterranean termite species with the largest economic impact in the United States. The frontal glands of the soldier caste termites comprising one third of the body mass, contain a secretion expelled through a foramen in defense. The small molecule composition of the frontal gland secretion is well-characterized, but the proteins remain to be identified. Herein is reported the structure and function of one of several proteins found in the termite defense gland secretion. TFP4 is a 6.9 kDa, non-classical group 1 Kazal-type serine protease inhibitor with activity towards chymotrypsin and elastase, but not trypsin. The 3-dimensional solution structure of TFP4 was solved with nuclear magnetic resonance spectroscopy, and represents the first structure from the taxonomic family, Rhinotermitidae. Based on the structure of TFP4, the protease inhibitor active loop (Cys8 to Cys16) was identified

Three-dimensional structure of the weakly associated protein homodimer SeR13 using RDCs and paramagnetic surface mapping

The traditional NMR-based method for determining oligomeric protein structure usually involves distinguishing and assigning intra- and intersubunit NOEs. This task becomes challenging when determining symmetric homo-dimer structures because NOE cross-peaks from a given pair of protons occur at the same position whether intra- or intersubunit in origin. While there are isotope-filtering strategies for distinguishing intra from intermolecular NOE interactions in these cases, they are laborious and often prove ineffectual in cases of weak dimers, where observation of intermolecular NOEs is rare. Here, we present an efficient procedure for weak dimer structure determination based on residual dipolar couplings (RDCs), chemical shift changes upon dilution, and paramagnetic surface perturbations. This procedure is applied to the Northeast Structural Genomics Consortium protein target, SeR13, a negatively charged Staphylococcus epidermidis dimeric protein (Kd 3.4 ± 1.4 mM) composed of 86 amino acids. A structure determination for the monomeric form using traditional NMR methods is presented, followed by a dimer structure determination using docking under orientation constraints from RDCs data, and scoring under residue pair potentials and shape-based predictions of RDCs. Validation using paramagnetic surface perturbation and chemical shift perturbation data acquired on sample dilution is also presented. The general utility of the dimer structure determination procedure and the possible relevance of SeR13 dimer formation are discussed. Published by Wiley-Blackwell. © 2010 The Protein Society

O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis

Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human OGT recognizes the sugar donor and acceptor peptide and uses a new catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base as well as an essential lysine. This mechanism seems to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate and explains the unexpected specificity of a recently reported metabolic OGT inhibitor. © 2012 Nature America, Inc. All rights reserved

Multiple -coil nuclear magnetic resonance probes for high -throughput and difference spectroscopy

Advances in nuclear magnetic resonance (NMR) spectroscopy are often driven by the development of novel hardware and software. In this thesis, three new probes for high-throughput and difference spectroscopy are presented. The Multiplex NMR Probe is a four-coil NMR probe that can analyze four samples simultaneously and in rapid succession. An automated sample handling and analysis routine for high-throughput NMR was developed to simultaneously inject four samples into the Multiplex Probe and achieved an analysis rate of almost two samples per minute for 1D 1H NMR. Two probes were developed for NMR difference spectroscopy. The Difference Probes have the ability to acquire a single, difference spectrum from two samples simultaneously. The dual-coil probes have a unique radiofrequency circuit that generates a phase difference between the two samples\u27 NMR signals by switching the relative orientation of the radiofrequency transmitter and receiver during the NMR experiment. Crossed-diodes can be used as passive switches or single diodes can be used with an active bias circuit to create the necessary switching function during the NMR experiment. One Difference Probe was constructed with solenoidal-geometry micro detection coils and flow capabilities. Two samples containing a common analyte, were used to demonstrate signal cancellation in a difference spectrum collected with a single pulse experiment. The cancellation was over 96% effective. The approach described has applications in the areas of solvent subtraction, spectral simplification, and liquid chromatography (LC)-NMR. The second Difference Probe was constructed with saddle-shaped detection coils and uses a specially modified 3 mm NMR tube to hold the two samples. The degree of cancellation of common signals was determined to be approximately 90%, and the application of the probe to relaxation-edited difference spectroscopy for identifying protein-ligand interactions was demonstrated

Acid dissociation constants of uridine-5\u27-diphosphate compounds determined by \u3csup\u3e31\u3c/sup\u3ephosphorus nuclear magnetic resonance spectroscopy and internal pH referencing

The acid dissociation constant (pKa) of small, biological molecules is an important physical property used for investigating enzyme mechanisms and inhibitor design. For phosphorus-containing molecules, the 31P nuclear magnetic resonance (NMR) chemical shift is sensitive to the local chemical environment, particularly to changes in the electronic state of the molecule. Taking advantage of this property, we present a 31P NMR approach that uses inorganic phosphate buffer as an internal pH reference to determine the pKa values of the imide and second diphosphate of uridine-5\u27-diphosphate compounds, including the first reported values for UDP-GlcNAc and UDP-S-GlcNAc. New methods for using inorganic phosphate buffer as an internal pH reference, involving mathematical correction factors and careful control of the chemical shift reference sample, are illustrated. A comparison of the newly determined imide and diphosphate pKa values of UDP, UDP-GlcNAc, and UDP-S-GlcNAc with other nucleotide phosphate and thio-analogs reveals the significance of the monosaccharide and sulfur position on the pKa values. © 2012 Elsevier B.V

Microscopy basics and the study of actin-actin-binding protein interactions

Actin is a multifunctional eukaryotic protein with a globular monomer form that polymerizes into a thin, linear microfilament in cells. Through interactions with various actin-binding proteins (ABPs), actin plays an active role in many cellular processes, such as cell motility and structure. Microscopy techniques are powerful tools for determining the role and mechanism of actin-ABP interactions in these processes. In this article, we describe the basic concepts of fluorescent speckle microscopy, total internal reflection fluorescence microscopy, atomic force microscopy, and cryoelectron microscopy and review recent studies that utilize these techniques to visualize the binding of actin with ABPs. © 2013 Elsevier Inc. All rights reserved

Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights

© 2015 Elsevier Inc. Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins\u27 molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed