1H-15N Heteronuclear Single Quantum Coherence Research Articles

Interaction between peptide and solid lignin suggest mechanisms of abiotic covalent bond formation

The mechanisms behind the formation of soil organic matter (SOM) and associated nitrogen immobilization remain partially elusive, while ongoing research continues to shed light on carbon and nitrogen sequestration in the environment. Studies show that quinone-like structures within alkali-soluble humic extracts of SOM can bond covalently with nitrogen-containing molecules derived from proteinaceous organic matter. However, direct molecular evidence for this chemical bonding with lignin in the solid form and non-solvent-extracted SOM is lacking. Using gel-state 1H-15N heteronuclear single quantum coherence (HSQC) high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that a 15N-labeled peptide (glycine-glycine-glycine-arginine, GGGR), can covalently bind to solid, untreated, brown rotted wood as well as 1,2-naphthoquinone, a quinone-model molecule typically found in degraded lignin. The new peaks in both reactions represent a change in the environment surrounding the N-functional groups. Interaction of the 15N-labeled peptide with the model quinone produced NMR cross peaks that are similar to those of the solid lignin. This suggests that the quinone-like structures are the most likely functional groups to form covalent bonds with peptides in the degraded lignin. Both Michael addition and Schiff base formation is proposed when the peptide GGGR interacts with white oak lignin and 1,2-naphthoquinone. These processes are of considerable importance to N incorporation into SOM and offer insights into how proteinaceous molecules could potentially be preserved and sequestered through covalent bond formation.

The enzymatic oxygen sensor cysteamine dioxygenase binds its protein substrates through their N-termini

The non-heme iron-dependent dioxygenase 2-aminoethanethiol (aka cysteamine) dioxygenase (ADO) has recently been identified as an enzymatic oxygen sensor that coordinates cellular changes to hypoxia by regulating the stability of proteins bearing an N-terminal cysteine (Nt-cys) through the N-degron pathway. It catalyzes O2-dependent Nt-cys sulfinylation, which promotes proteasomal degradation of the target. Only a few ADO substrates have been verified, including regulators of G-protein signaling (RGS) 4 and 5, and the proinflammatory cytokine interleukin-32, all of which exhibit cell and/or tissue specific expression patterns. ADO, in contrast, is ubiquitously expressed, suggesting it can regulate the stability of additional Nt-cys proteins in an O2-dependent manner. However, the role of individual chemical groups, active site metal, amino acid composition, and globular structure on protein substrate association remains elusive. To help identify new targets and examine the underlying biochemistry of the system, we conducted a series of biophysical experiments to investigate the binding requirements of established ADO substrates RGS5 and interleukin-32. We demonstrate, using surface plasmon response and enzyme assays, that a free, unmodified Nt-thiol and Nt-amine are vital for substrate engagement through active site metal coordination, with residues next to Nt-cys moderately impacting association and catalytic efficiency. Additionally, we show, through 1H-15N heteronuclear single quantum coherence nuclear magnetic resonance titrations, that the globular portion of RGS5 has limited impact on ADO association, with interactions restricted to the N-terminus. This work establishes key features involved in ADO substrate binding, which will help identify new protein targets and, subsequently, elucidate its role in hypoxic adaptation.

Targeted and Logical Discovery of Piperazic Acid-Bearing Natural Products Based on Genomic and Spectroscopic Signatures.

A targeted and logical discovery method was devised for natural products containing piperazic acid (Piz), which is biosynthesized from ornithine by l-ornithine N-hydroxylase (KtzI) and N-N bond formation enzyme (KtzT). Genomic signature-based screening of a bacterial DNA library (2020 strains) using polymerase chain reaction (PCR) primers targeting ktzT identified 62 strains (3.1%). The PCR amplicons of KtzT-encoding genes were phylogenetically analyzed to classify the 23 clades into two monophyletic groups, I and II. Cultivating hit strains in media supplemented with 15NH4Cl and applying 1H-15N heteronuclear multiple bond correlation (HMBC) along with 1H-15N heteronuclear single quantum coherence (HSQC) and 1H-15N HSQC-total correlation spectroscopy (HSQC-TOCSY) NMR experiments detected the spectroscopic signatures of Piz and modified Piz. Chemical investigation of the hit strains prioritized by genomic and spectroscopic signatures led to the identification of a new azinothricin congener, polyoxyperuin B seco acid (1), previously reported chloptosin (2) in group I, depsidomycin D (3) incorporating two dehydropiperazic acids (Dpz), and lenziamides A and B (4 and 5), structurally novel 31-membered cyclic decapeptides in group II. By consolidating the phylogenetic and chemical analyses, clade-structure relationships were elucidated for 19 of the 23 clades. Lenziamide A (4) inhibited STAT3 activation and induced G2/M cell cycle arrest, apoptotic cell death, and tumor growth suppression in human colorectal cancer cells. Moreover, lenziamide A (4) resensitized 5-fluorouracil (5-FU) activity in both in vitro cell cultures and the in vivo 5-FU-resistant tumor xenograft mouse model. This work demonstrates that the genomic and spectroscopic signature-based searches provide an efficient and general strategy for new bioactive natural products containing specific structural motifs.

Qualitative and Quantitative Characterization of Protein-Carbohydrate Interactions by NMR Spectroscopy.

Solution-state nuclear magnetic resonance (NMR) spectroscopy can be used to monitor protein-carbohydrate interactions. Two-dimensional 1H-15N heteronuclear single quantum coherence (HSQC)-based techniques described in this chapter can be used quickly and effectively to screen a set of possible carbohydrate-binding partners, to quantify the dissociation constant (Kd) of any identified interactions, and to the map the carbohydrate-binding site on the structure of a protein. Here, we describe the titration of a family 32 carbohydrate-binding module from Clostridium perfringens (CpCBM32) with the monosaccharide N-acetylgalactosamine (GalNAc), in which we calculate the apparent dissociation of the interaction and map the GalNAc binding site onto the structure of CpCBM32. This approach can be applied to other CBM- and protein-ligand systems.

Development of flavonoid probes and the binding mode of the target protein and quercetin derivatives

This study investigated the mechanism underlying anti-cancer cell migration activity of quercetin derivatives by investigating the binding mode of the target protein. Five flavonoid probes were newly synthesized, and pull down assay using synthesized flavonoid probes indicated matrix metalloproteinase-1 (MMP-1) as the target protein of quercetin derivatives. Quercetin and 3-O-methylquercetin (3MQ) inhibited MMP-1. SPR analysis demonstrated dose dependent interaction between quercetin derivatives and recombinant MMP-1 catalytic domain. And 1H-15N heteronuclear single quantum coherence (HSQC) NMR analysis using 15N-labeled MMP-1 catalytic domain indicated that 3MQ interacted around metal ions in the MMP-1. The development of flavonoid probes can broaden the possibility to discover the new target proteins and elucidate the core mechanisms of the multi bioactivity of flavonoids.

Protein-Ligand Binding Volume Determined from a Single 2D NMR Spectrum with Increasing Pressure.

Proteins undergo changes in their partial volumes in numerous biological processes such as enzymatic catalysis, unfolding–refolding, and ligand binding. The change in the protein volume upon ligand binding—a parameter termed the protein–ligand binding volume—can be extensively studied by high-pressure NMR spectroscopy. In this study, we developed a method to determine the protein–ligand binding volume from a single two-dimensional (2D) 1H–15N heteronuclear single quantum coherence (HSQC) spectrum at different pressures, if the exchange between ligand-free and ligand-bound states of a protein is slow in the NMR time-scale. This approach required a significantly lower amount of protein and NMR time to determine the protein–ligand binding volume of two carbonic anhydrase isozymes upon binding their ligands. The proposed method can be used in other protein–ligand systems and expand the knowledge about protein volume changes upon small-molecule binding.

Repurposing Acitretin as an Antipseudomonal Agent Targeting the Pseudomonas aeruginosa Iron-Regulated Heme Oxygenase.

Iron is an essential micronutrient for the survival and virulence of the bacterial pathogen Pseudomonas aeruginosa. To overcome iron withholding and successfully colonize a host, P. aeruginosa uses a variety of mechanisms to acquire iron, including the secretion of high-affinity iron chelators (siderophores) or the uptake and utilization of heme. P. aeruginosa heme oxygenase (HemO) plays pivotal roles in heme sensing, uptake, and utilization and has emerged as a therapeutic target for the development of antipseudomonal agents. Using a high-throughput fluorescence quenching assay combined with minimum inhibitory concentration measurements, we screened the Selleck Bioactive collection of 2100 compounds and identified acitretin, a Food and Drug Administration-approved oral retinoid, as a potent and selective inhibitor of HemO. Acitretin binds to HemO with a KD value of 0.10 ± 0.02 μM and inhibits the growth of P. aeruginosa PAO1 with an IC50 of 70 ± 18 μg/mL. In addition, acitretin showed good selectivity for HemO, which uniquely generates BVIXβ/δ, over human heme oxygenase (hHO1) and other BVIXα-producing homologues such as the heme oxygenases from Neisseria meningitidis (nmHO) and Acinetobacter baumannii (abHO). The binding of acitretin within the HemO active site was confirmed by 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance, and molecular modeling provided further insight into potential interactions of acitretin with residues specific for orienting heme in the β/δ selective HemO. Moreover, at 20 μM, acitretin inhibited the enzymatic activity of HemO in P. aeruginosa cells by >60% and effectively blocked the ability of P. aeruginosa to sense and acquire heme as demonstrated in the β-galactosidase transcriptional reporter assay.

Rapid Quantification of Protein-Ligand Binding via 19F NMR Lineshape Analysis

Fluorine incorporation is ideally suited to many NMR techniques, and incorporation of fluorine into proteins and fragment libraries for drug discovery has become increasingly common. Here, we use one-dimensional 19F NMR lineshape analysis to quantify the kinetics and equilibrium thermodynamics for the binding of a fluorine-labeled Src homology 3 (SH3) protein domain to four proline-rich peptides. SH3 domains are one of the largest and most well-characterized families of protein recognition domains and have a multitude of functions in eukaryotic cell signaling. First, we showe that fluorine incorporation into SH3 causes only minor structural changes to both the free and bound states using amide proton temperature coefficients. We then compare the results from lineshape analysis of one-dimensional 19F spectra to those from two-dimensional 1H-15N heteronuclear single quantum coherence spectra. Their agreement demonstrates that one-dimensional 19F lineshape analysis is a robust, low-cost, and fast alternative to traditional heteronuclear single quantum coherence-based experiments. The data show that binding is diffusion limited and indicate that the transition state is highly similar to the free state. We also measured binding as a function of temperature. At equilibrium, binding is enthalpically driven and arises from a highly positive activation enthalpy for association with small entropic contributions. Our results agree with those from studies using different techniques, providing additional evidence for the utility of 19F NMR lineshape analysis, and we anticipate that this analysis will be an effective tool for rapidly characterizing the energetics of protein interactions.

The influenza NS1 protein modulates RIG-I activation via a strain-specific direct interaction with the second CARD of RIG-I

The influenza NS1 protein modulates RIG-I activation via a strain-specific direct interaction with the second CARD of RIG-I

Ultra-thin free-floating carbon nanotube/gold nanoparticle hybrid film prepared with self-assembly protein of α-synuclein

Fabrication of ultra-thin 2D film of carbon nanotubes (CNTs) is achieved by employing a self-assembly protein of α-synuclein (αS). Electron energy loss spectroscopy-transmission electron microscopy and circular dichroism spectroscopy analyses indicate that αS binds to the hydrophobic surface of CNT through its N-terminal region by forming α-helix. 1H-15N heteronuclear single-quantum coherence correlation-nuclear magnetic resonance spectra show that the acidic C-terminus of αS is exposed outward from the CNT surface. Following a slow vacuum filtration of the CNTs complexed with αS through a polycarbonate membrane filter, chloroform is used to unleash the free-floating ultra-thin CNT film from the substrate by causing unique αS-αS interactions of the enhanced anti-parallel β-sheet and α-helix formation. To make a graphically patterned CNT film stable in the free-floating state, gold nanoparticles (AuNPs) encapsulated with αS are employed to generate the αS-mediated CNT/AuNP hybrid film. The CNT-AuNP hybrid pattern is also directly transferred onto a thermo-responsive hydrogel sheet and sandwiched with a passive polymer layer. The resulting triply structure exhibits a shape deformation upon the light exposure. The light-responsive controllable soft material, therefore, has been suggested to be utilized in various areas including not only soft robotics but also human-machine interface by providing stimuli-responsive electroconductive and biocompatible matrix.

Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A.

Numerous bacterial toxins and other virulence factors use low pH as a trigger to convert from water-soluble to membrane-inserted states. In the case of colicins, the pore-forming domain of colicin A (ColA-P) has been shown both to undergo a clear acidic unfolding transition and to require acidic lipids in the cytoplasmic membrane, whereas its close homologue colicin N shows neither behavior. Compared to that of ColN-P, the ColA-P primary structure reveals the replacement of several uncharged residues with aspartyl residues, which upon replacement with alanine induce an unfolded state at neutral pH. Here we investigate ColA-P’s structural requirement for these critical aspartyl residues that are largely situated at the N-termini of α helices. As previously shown in model peptides, the charged carboxylate side chain can act as a stabilizing helix N-Cap group by interacting with free amide hydrogen bond donors. Because this could explain ColA-P destabilization when the aspartyl residues are protonated or replaced with alanyl residues, we test the hypothesis by inserting asparagine, glutamine, and glutamate residues at these sites. We combine urea (fluorescence and circular dichroism) and thermal (circular dichroism and differential scanning calorimetry) denaturation experiments with 1H–15N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy of ColA-P at different pH values to provide a comprehensive description of the unfolding process and confirm the N-Cap hypothesis. Furthermore, we reveal that, in urea, the single domain ColA-P unfolds in two steps; low pH destabilizes the first step and stabilizes the second.

Biophysical and functional characterizations of recombinant RimI acetyltransferase from Mycobacterium tuberculosis.

Nα-acetylation is a universal protein modification related to a wide range of physiological processes in eukaryotes and prokaryotes. RimI, an Nα-acetyltransferase in Mycobacterium tuberculosis, is responsible for the acetylation of the α-amino group of the N-terminal residue in the ribosomal protein S18. Despite growing evidence that protein acetylation may be correlated with the pathogenesis of tuberculosis, no structural information is yet available for mechanistically understanding the MtRimI acetylation. To enable structural studies for MtRimI, we constructed a serial of recombinant MtRimI proteins and assessed their biochemical properties. We then chose an optimal construct MtRimIC21A4-153 and expressed and purified the truncated high-quality protein for further biophysical and functional characterizations. The 2D 1H-15N heteronuclear single quantum coherence spectrum of MtRimIC21A4-153 exhibits wider chemical shift dispersion and favorable peak isolation, indicating that MtRimIC21A4-153 is amendable for further structural determination. Moreover, bio-layer interferometry experiments showed that MtRimIC21A4-153 possessed similar micromolar affinity to full-length MtRimI for binding the hexapeptide substrate Ala-Arg-Tyr-Phe-Arg-Arg. Enzyme kinetic assays also exhibited that MtRimIC21A4-153 had almost identical enzymatic activity to MtRimI, indicating insignificant influence of the recombinant variations on enzymatic functions. Furthermore, binding sites of the peptide were predicted by molecular docking approach, suggesting that this substrate binds to MtRimI primarily through electrostatic and hydrogen bonding interactions. Our results lay a foundation for the further structural determination and dynamics detection of MtRimI.

Amino Acid Selective 13C Labeling and 13C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance.

Amino acid selective isotope labeling is an important nuclear magnetic resonance technique, especially for larger proteins, providing strong bases for the unambiguous resonance assignments and information concerning the structure, dynamics, and intermolecular interactions. Amino acid selective 15N labeling suffers from isotope dilution caused by metabolic interconversion of the amino acids, resulting in isotope scrambling within the target protein. Carbonyl 13C atoms experience less isotope scrambling than the main-chain 15N atoms do. However, little is known about the side-chain 13C atoms. Here, the 13C scrambling profiles of the Cα and side-chain carbons were investigated for 15N scrambling-prone amino acids, such as Leu, Ile, Tyr, Phe, Thr, Val, and Ala. The level of isotope scrambling was substantially lower in 13Cα and 13C side-chain labeling than in 15N labeling. We utilized this reduced scrambling-prone character of 13C as a simple and efficient method for amino acid selective 13C labeling using an Escherichia coli cold-shock expression system and high-cell density fermentation. Using this method, the 13C labeling efficiency was >80% for Leu and Ile, ∼60% for Tyr and Phe, ∼50% for Thr, ∼40% for Val, and 30-40% for Ala. 1H-15N heteronuclear single-quantum coherence signals of the 15N scrambling-prone amino acid were also easily filtered using 15N-{13Cα} spin-echo difference experiments. Our method could be applied to the assignment of the 55 kDa protein.

Identification of major matrix metalloproteinase-20 proteolytic processing products of murine amelogenin and tyrosine-rich amelogenin peptide using a nuclear magnetic resonance spectroscopy based method

ObjectiveThe aim of this study was to identify major matrix metalloproteinase-20 (MMP20) proteolytic processing products of amelogenin over time and determine if the tyrosine-rich amelogenin peptide (TRAP) was a substrate of MMP20. DesignRecombinant15N-labeled murine amelogenin and 13C,15N-labeled TRAP were incubated with MMP20 under conditions where amelogenin self-assembles into nanospheres. Digestion products were fractionated by reverse-phase high-performance liquid chromatography at various time points. Product identification took advantage of the intrinsic disorder property of amelogenin that results in little change to its fingerprint 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance spectrum in 2% acetic acid upon removing parts of the protein, allowing cleavage site identification by observing which amide cross peaks disappear. ResultsThe primary product in five out of the six major reverse-phase high-performance liquid chromatography bands generated after a 24 h incubation of murine amelogenin with MMP20 were: S55-L163, P2-L147, P2-E162, P2-A167, and P2-R176. After 72 h these products were replaced with five major reverse-phase high-performance liquid chromatography bands containing: L46-A170, P2-S152, P2-F151, P2-W45, and short N-terminal peptides. TRAP was completely digested by MMP20 into multiple small peptides with the initial primary site of cleavage between S16 and Y17. ConclusionsIdentification of the major MMP20 proteolytic products of amelogenin confirm a dynamic process, with sites towards the C-terminus more rapidly attacked than sites near the N-terminus. This observation is consistent with nanosphere models where the C-terminus is exposed and the N-terminus more protected. One previously reported end-product of the MMP20 proteolytic processing of amelogenin, TRAP, is shown to be an in vitro substrate for MMP20.

Mechanism of Competitive Inhibition and Destabilization of Acidothermus cellulolyticus Endoglucanase 1 by Ionic Liquids.

The ability of ionic liquids (ILs) to solubilize cellulose has sparked interest in their use for enzymatic biomass processing. However, this potential is yet to be realized, primarily because ILs inactivate requisite cellulases by mechanisms that are yet to be identified. We used a combination of enzymology, circular dichroism (CD), nuclear magnetic resonance (NMR), and molecular dynamics (MD) methods to investigate the molecular basis for the inactivation of the endocellulase 1 (E1) from Acidothermus cellulolyticus by the imidazolium IL 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]). Enzymatic studies revealed that [BMIM][Cl] inactivates E1 in a biphasic manner that involves rapid, reversible inhibition, followed by slow, irreversible deactivation. Backbone NMR signals of the 40.5 kDa E1 were assigned by triple resonance NMR methods, enabling monitoring of residue-specific perturbations. 1H-15N NMR titration experiments revealed that [BMIM][Cl] binds reversibly to the E1 active site, indicating that reversible deactivation is due to competitive inhibition of substrate binding. Prolonged incubation with [BMIM][Cl] led to substantial global changes in the 1H-15N heteronuclear single quantum coherence NMR and CD spectra of E1 indicative of protein denaturation. Notably, weak interactions between [BMIM][Cl] and residues at the termini of several helices were also observed, which, together with MD simulations, suggest that E1 denaturation is promoted by [BMIM][Cl]-induced destabilization of helix capping structures. In addition to identifying determinants of E1 inactivation, our findings establish a molecular framework for engineering cellulases with improved IL compatibility.

Deciphering Conformational Changes Associated with the Maturation of Thrombin Anion Binding Exosite I.

Thrombin participates in procoagulation, anticoagulation, and platelet activation. This enzyme contains anion binding exosites, ABE I and ABE II, which attract regulatory biomolecules. As prothrombin is activated to thrombin, pro-ABE I is converted into mature ABE I. Unexpectedly, certain ligands can bind to pro-ABE I specifically. Moreover, knowledge of changes in conformation and affinity that occur at the individual residue level as pro-ABE I is converted to ABE I is lacking. Such changes are transient and were not captured by crystallography. Therefore, we employed nuclear magnetic resonance (NMR) titrations to monitor development of ABE I using peptides based on protease-activated receptor 3 (PAR3). Proton line broadening NMR revealed that PAR3 (44-56) and more weakly binding PAR3G (44-56) could already interact with pro-ABE I on prothrombin. 1H-15N heteronuclear single-quantum coherence NMR titrations were then used to probe binding of individual 15N-labeled PAR3G residues (F47, E48, L52, and D54). PAR3G E48 and D54 could interact electrostatically with prothrombin and tightened upon thrombin maturation. The higher affinity for PAR3G D54 suggests the region surrounding thrombin R77a is better oriented to bind D54 than the interaction between PAR3G E48 and thrombin R75. Aromatic PAR3G F47 and aliphatic L52 both reported on significant changes in the chemical environment upon conversion of prothrombin to thrombin. The ABE I region surrounding the 30s loop was more affected than the hydrophobic pocket (F34, L65, and I82). Our NMR titrations demonstrate that PAR3 residues document structural rearrangements occurring during exosite maturation that are missed by reported X-ray crystal structures.

Synthesis and properties of polyester imides based on 2,2-bis(4-hydroxyphenyl)propanedibenzoate-3,3ʹ,4,4ʹ-tetracarboxylic acid dianhydride

Evidence for the Higgs boson, for which the Nobel Prize in physics was awarded in 2013, was discovered by the European Organization for Nuclear Research (CERN) in 2012, using the Large Hadron Collider (LHC). PIXEO BP-S exhibited superior resistance to both radiation and cryogenics and this polymer played a crucial role in the operation of the LHC, thus contributing to the Higgs boson discovery. Herein, we report the synthesis and characterization of various polyester imides for a cryogenic and radiation-resistant polyimide adhesive tape. These polyester imides (3) were synthesized from 2,2-bis(4-hydroxyphenyl)propanedibenzoate-3,3ʹ,4,4ʹ-tetracarboxylic acid anhydride (ESDA) and various diamines (1a–1l). All polymers showed high solubility in various solvents, especially 3a, which was synthesized from ESDA and 4,4ʹ-bis(3-aminophenoxy)diphenyl sulfone and dissolved even in ether solvents such as dioxane and dioxolane. Dynamic mechanical analysis and X-ray diffraction studies indicated that the excluded volume of the polyester imides had a length of 150 A, and that this volume affected the thermoplastic properties and solubilities of the polymers. The degree of imidization of 3a dissolved in dimethyl sulfoxide was examined by 1H-Nuclear magnetic resonance(NMR) imaging utilizing a 15N-enriched amine model compound in conjunction with 1H–15N heteronuclear single quantum coherence 2-dimensional NMR and was found to be 99.5%. PIXEO BP-S tape used for Large Hadron Collider and its chemical structure.

Unfolding of CPR3 Gets Initiated at the Active Site and Proceeds via Two Intermediates

Cyclophilin catalyzes the ubiquitous process “peptidyl-prolyl cis-trans isomerization,” which plays a key role in protein folding, regulation, and function. Here, we present a detailed characterization of the unfolding of yeast mitochondrial cyclophilin (CPR3) induced by urea. It is seen that CPR3 unfolding is reversible and proceeds via two intermediates, I1 and I2. The I1 state has native-like secondary structure and shows strong anilino-8-naphthalenesulphonate binding due to increased exposure of the solvent-accessible cluster of non-polar groups. Thus, it has some features of a molten globule. The I2 state is more unfolded, but it retains some residual secondary structure, and shows weak anilino-8-naphthalenesulphonate binding. Chemical shift perturbation analysis by 1H-15N heteronuclear single quantum coherence spectra reveals disruption of the tertiary contacts among the regions close to the active site in the first step of unfolding, i.e., the N-I1 transition. Both of the intermediates, I1 and I2, showed a propensity to self-associate under stirring conditions, but their kinetic profiles are different; the native protein did not show any such tendency under the same conditions. All these observations could have significant implications for the function of the protein.

Characterization of Protein-Carbohydrate Interactions by NMR Spectroscopy.

Solution-state nuclear magnetic resonance (NMR) spectroscopy can be used to monitor protein-carbohydrate interactions. Two-dimensional 1H-15N heteronuclear single quantum coherence (HSQC)-based techniques described in this chapter can be used quickly and effectively to screen a set of possible carbohydrate binding partners, to quantify the dissociation constant (K d) of any identified interactions, and to map the carbohydrate binding site on the structure of the protein. Here, we describe the titration of a family 32 carbohydrate binding module from Clostridium perfringens (CpCBM32) with the monosaccharide N-acetylgalactosamine (GalNAc), in which we calculate the apparent dissociation of the interaction, and map the GalNAc binding site onto the structure of CpCBM32.

Entire-Dataset Analysis of NMR Fast-Exchange Titration Spectra: A Mg2+ Titration Analysis for HIV-1 Ribonuclease H Domain.

This article communicates our study to elucidate the molecular determinants of weak Mg2+ interaction with the ribonuclease H (RNH) domain of HIV-1 reverse transcriptase in solution. As the interaction is weak (a ligand-dissociation constant >1 mM), nonspecific Mg2+ interaction with the protein or interaction of the protein with other solutes that are present in the buffer solution can confound the observed Mg2+-titration data. To investigate these indirect effects, we monitored changes in the chemical shifts of backbone amides of RNH by recording NMR 1H-15N heteronuclear single-quantum coherence spectra upon titration of Mg2+ into an RNH solution. We performed the titration under three different conditions: (1) in the absence of NaCl, (2) in the presence of 50 mM NaCl, and (3) at a constant 160 mM Cl- concentration. Careful analysis of these three sets of titration data, along with molecular dynamics simulation data of RNH with Na+ and Cl- ions, demonstrates two characteristic phenomena distinct from the specific Mg2+ interaction with the active site: (1) weak interaction of Mg2+, as a salt, with the substrate-handle region of the protein and (2) overall apparent lower Mg2+ affinity in the absence of NaCl compared to that in the presence of 50 mM NaCl. A possible explanation may be that the titrated MgCl2 is consumed as a salt and interacts with RNH in the absence of NaCl. In addition, our data suggest that Na+ increases the kinetic rate of the specific Mg2+ interaction at the active site of RNH. Taken together, our study provides biophysical insight into the mechanism of weak metal interaction on a protein.