Paramagnetic relaxation enhancement (PRE) measurements on 1H nuclei have the potential to play an important role in NMR structure determination of macromolecules by providing unique long-range (10−35 Å) distance information. Recent methodological advances for covalently attaching paramagnetic groups at specific sites both proteins and nucleic acids permitted application PRE various biological macromolecules. However, because artificially introduced are exposed solvent linked macromolecule...

Surface proteins of Gram-positive bacteria play important roles during the pathogenesis human infections and require sortase for anchoring to cell-wall envelope. Sortase cleaves surface at LPXTG motif catalyzes formation an amide bond between carboxyl group threonine (T) amino crossbridges. The NMR structure reveals a unique β-barrel structure, in which active-site sulfhydryl cysteine-184 is poised ionization by histidine-120, presumably enabling resultant thiolate attack peptide. Calcium...

Nonspecific protein-DNA interactions are inherently dynamic and involve both diffusion of the protein along DNA hopping from one molecule or segment to another. Understanding how gene regulatory proteins interact nonspecifically with in terms structure dynamics is challenging because experimental observables an ensemble average many rapidly exchanging states. By using a variety NMR spectroscopic techniques, including relaxation analysis, paramagnetic enhancement, residual dipolar couplings,...

Significance Many transcription factors and DNA repair/modifying enzymes must first locate the target sites through stochastic scanning of in vast presence nonspecific sites. In this work, we demonstrate that search by these proteins can be accelerated via engineering based on structural dynamic knowledge DNA-scanning process. Our biophysical data for Egr-1 zinc-finger protein its nuclease derivatives reveal kinetic thermodynamic roles conformational equilibrium between two modes process:...

In eukaryotes, many DNA/RNA-binding proteins possess intrinsically disordered regions (IDRs) with large negative charge, some of which involve a consecutive sequence aspartate (D) or glutamate (E) residues. We refer to them as D/E repeats. The functional role repeats is not well understood, though are known cause autoinhibition through intramolecular electrostatic interaction domains. this work, we investigated the impacts on target DNA search kinetics for high-mobility group box 1 (HMGB1)...

The backbone 1H, 13C, and 15N resonances of the c-Ha-Ras protein [a truncated version consisting residues 1−171, Ras(1−171)] bound with GMPPNP (a slowly hydrolyzable analogue GTP) were assigned compared those GDP-bound Ras(1−171). amide amino acid 10−13, 21, 31−39, 57−64, 71 Ras(1−171)·GMPPNP, but not Ras(1−171)·GDP, extremely broadened, whereas other Ras(1−171)·GMPPNP exhibited nearly as sharp Ras(1−171)·GDP. exhibiting extreme broadening, except for 21 71, are localized in three functional...

In this paper, we present a series of heteronuclear NMR experiments for the direct observation and characterization lysine NH3 groups in proteins. context HoxD9 homeodomain bound specifically to DNA were able directly observe three cross-peaks, arising from groups, with 15N chemical shifts around ∼33 ppm at pH 5.8 35 °C. Measurement water-exchange rates various types transverse relaxation these reveals that rapid water exchange dominates antiphase coherence respect 1H through scalar second...

Egr-1 is an inducible transcription factor that recognizes 9-bp target DNA sites via three zinc finger domains and activates genes in response to cellular stimuli such as synaptic signals vascular stresses. Using spectroscopic computational approaches, we have studied structural, dynamic, kinetic aspects of the DNA-scanning process which nonspecifically bound perpetually changes its location on DNA. Our NMR data indicate undergoes highly dynamic domain motions when scanning In particular, 1...

The inducible transcription factor Egr-1, which recognizes a 9-bp target DNA sequence via three zinc-finger domains, rapidly activates particular genes upon cellular stimuli such as neuronal signals and vascular stresses. Here, using the stopped-flow fluorescence method, we measured search kinetics of Egr-1 protein at various ionic strengths between 40 400 mM KCl found most efficient 150 KCl. We further investigated intersegment transfer, dissociation, sliding this on distinct concentrations...

ConspectusMolecular association of proteins with nucleic acids is required for many biological processes essential to life. Electrostatic interactions via ion pairs (salt bridges) acid phosphates and protein side chains are crucial bind DNA or RNA. Counterions around the macromolecules also key constituents thermodynamics protein–nucleic association. Until recently, there had been only a limited amount experiment-based information about how ions ionic moieties behave in macromolecular...

Significance Electrostatic potentials are important information for our understanding of biomolecular functions as well protein engineering and drug design. Typically, electrostatic around biomolecules computed from three-dimensional structures. However, the theory behind this computation is approximate with some limitations. Validation through experiments therefore essential yet remains inadequate. This paper presents a unique spectroscopic method to experimentally determine near molecular...

NMR spectroscopy is an important tool for the measurement of electrostatic properties biomolecules. To this point, paramagnetic relaxation enhancements (PREs) 1H nuclei arising from nitroxide cosolutes in biomolecular solutions have been used to measure effective near-surface potentials (ϕENS) proteins and nucleic acids. Here, we present a gadolinium (Gd)-based method, exploiting Gd chelates with different net charges, measuring ϕENS values demonstrate its utility through applications number...

A novel approach is presented for studying the kinetics of specific protein−DNA interactions by NMR exchange spectroscopy. The experimental design involves direct observation translocation a homeodomain between cognate sites on two oligonucleotide duplexes, differing only single base pair at edge DNA recognition sequence. base-pair change perturbs 1H−15N correlation spectrum number residues, while leaving affinity unchanged. process has apparent rate constants in 5−20 s-1 range which are...

Despite their importance in macromolecular interactions and functions, the dynamics of lysine side-chain amino groups proteins are not well understood. In this study, we have developed methodology for investigations NH3(+) by NMR spectroscopy computation. By using 1H−15N heteronuclear correlation experiments optimized 15NH3(+) moieties, analyzed dynamic behavior individual human ubiquitin at 2 °C pH 5. We modified theoretical framework previously CH3 used it to analyze 15N relaxation data...

Ion pairing is one of the most fundamental chemical interactions and essential for molecular recognition by biological macromolecules. From an experimental standpoint, very little known to date about ion-pair dynamics in macromolecular systems. Absorption, infrared, Raman spectroscopic methods were previously used characterize dynamic properties ion pairs, but these can be applied only small compounds. Here, using NMR (15)N relaxation hydrogen-bond scalar (15)N-(31)P J-couplings ((h3)J(NP)),...

Basic side chains play major roles in recognition of nucleic acids by proteins. However, dynamic properties these positively charged are not well understood. In this work, we studied changes conformational dynamics basic upon protein–DNA association for the zinc-finger protein Egr-1. By nuclear magnetic resonance (NMR) spectroscopy, characterized all side-chain cationic groups free and complex with target DNA. Our NMR order parameters indicate that arginine guanidino interacting DNA bases...

Nonspecific protein−DNA interactions play an important role in a variety of contexts related to DNA packaging, nucleoprotein complex formation, and gene regulation. Biophysical characterization nonspecific at the atomic level poses significant challenges owing dynamic nature such complexes. Although NMR spectroscopy represents powerful tool for analysis systems, conventional techniques have provided little information on interactions. We show that intermolecular 1H paramagnetic relaxation...

We report direct evidence for deprotonation of a lysine side chain buried in the hydrophobic core protein, demonstrating heteronuclear 1H−15N NMR data on Lys-66 amine (Nζ) group Δ-PHS/V66K variant staphylococcal nuclease. Previous crystallographic study has shown that Nζ is completely core. On basis double and triple resonance experiments, we found 1Hζ 15Nζ chemical shifts at pH 8.0 6 °C are 0.81 23.3 ppm, respectively, which too abnormal to correspond protonated (NH3+) state. Further...