A comparison of HSQC and HMQC pulse schemes for recording 1H−13C correlation maps protonated methyl groups in highly deuterated proteins is presented. It shown that can be as much a factor 3 more sensitive than their counterparts the sensitivity gains result from TROSY effect involves cancellation intra-methyl dipolar relaxation interactions. spectra are recorded on U-[15N,2H], Ileδ1-[13C,1H] samples (i) malate synthase G, 723 residue protein, at 37 5 °C, (ii) protease ClpP, comprising 14...

New NMR experiments are presented for the assignment of methyl 13C and 1H chemical shifts from Ile, Leu, Val residues in high molecular weight proteins. The first class pulse schemes transfers magnetization group to backbone amide spins detection, while second more sensitive uses an "out-and-back" transfer scheme which side-chain carbons or carbonyls correlated with spins. Both groups benefit a new isotopic labeling protonation Leu large deuterated approach makes use α-ketoisovalerate that...

A four-dimensional (4-D) NMR study of Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is described. Virtually complete backbone 1HN, 15N, 13C, and 13Cβ chemical shift assignments this largely α-helical protein are reported. The assignment strategy follows from our previously described approach based on TROSY triple resonance 4-D spectroscopy [Yang, D.; Kay, L. E. J. Am. Chem. Soc. 1999, 121, 2571−2575. Konrat, R; Yang, D; Biomol. 15, 309−313] with number...

A new CPMG-based multiple quantum relaxation dispersion experiment is presented for measuring millisecond dynamic processes at side-chain methyl positions in high molecular weight proteins. The benefits from a methyl-TROSY effect which cancellation of intramethyl dipole fields occurs, leading to 13C−1H correlation spectra sensitivity and resolution (Tugarinov, V.; Hwang, P. M.; Ollerenshaw, J. E.; Kay, L. E. Am. Chem. Soc. 2003, 125, 10420−10428). utility the methodology illustrated with an...

The size of proteins that can be studied by solution NMR spectroscopy has increased significantly because recent developments in methodology. Important experiments include those make use approaches increase the lifetimes signals or define orientation internuclear bond vectors with respect to a common molecular frame. advances techniques are strongly coupled isotope labeling methods sensitivity and reduce complexity spectra. We show these exploited structural studies high-molecular-weight,...

Abstract In TROSY experiments, relaxation interference effects are exploited to produce spectra with improved resolution and signal‐to‐noise. Such experiments cannot be explained using the standard product operator formalism, but must instead analyzed at level of individual density matrix elements. Herein we illustrate this point an example from our recent work on a 1 H– 13 C correlation experiment for methyl groups in large proteins. Methyl useful spectroscopic probes protein structure...

Significance Chaperones are integral components of the cellular machinery that assist protein folding and protect against misfolding aggregation. A bottleneck in understanding how chaperones work is relevant functional states too sparsely populated dynamic to be observed using conventional biophysical methods. NMR uniquely suited detect provide atomic resolution information on such “invisible” states. Here we quantitate kinetics chaperone GroEL binding a substrate exists metastable...

J-domain chaperones are involved in the efficient handover of misfolded/partially folded proteins to Hsp70 but also function independently protect against cell death. Due their high flexibility, mechanism by which they regulate cycle and how specific substrate recognition is performed remains unknown. Here we focus on DNAJB6b, has been implicated various human diseases represents a key player protection neurodegeneration protein aggregation. Using variant that exists mainly monomeric form,...

The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (htt NT ), a polyglutamine (Q n ) tract, and proline-rich sequence. Polyglutamine expansion results in aggregation-prone protein responsible for Huntington’s disease. Here, we study earliest events involved oligomerization minimalistic construct, htt Q 7 , which remains largely monomeric over sufficiently long period time to permit detailed quantitative NMR analysis kinetics structure sparsely...

An approach for recording four-dimensional (4D) methyl 1H−13C−13C−1H NOESY spectra with high resolution and sensitivity is presented applied to Malate Synthase G (723 residues, 82 kDa). Sensitivity have been optimized using a highly deuterated, methyl-protonated sample in concert methyl-TROSY, sparse data sampling the three indirect dimensions, 4D spectral reconstruction multidimensional decomposition (MDD). A acquisition protocol introduced that ensures sufficiently long times can be...

The two-body Slowly Relaxing Local Structure (SRLS) model was applied to 15N NMR spin relaxation in proteins and compared with the commonly used original extended model-free (MF) approaches. In MF, dynamic modes are assumed be decoupled, local ordering at N−H sites is represented by generalized order parameters, internal motions described effective correlation times. SRLS accounts for dynamical coupling between global diffusion of protein motion bond vector. associated potential tensors...

A pair of experiments is presented for measuring intra-methyl 1H−1H dipolar cross-correlated spin relaxation rates in highly deuterated, methyl protonated proteins with significantly improved sensitivity relative to previously developed that measure dynamics via 1H relaxation. In applications correlation times the macromolecular limit, these cross-correlation are related directly order parameters, characterizing amplitude motion methyl-containing side-chains. The experimental approach...

Relaxation violated coherence transfer NMR spectroscopy has emerged as a powerful experimental tool for the quantitative measurement of amplitudes motion methyl containing side-chains. Typically, experiments, performed on proteins that are highly deuterated and methyl-protonated, monitor build-up (1)H double-quantum magnetization. Because all three protons in group degenerate, such coherences can only result from differential relaxation transverse magnetization components, which turn reflect...

The global motions and exchange kinetics of a model protein, ubiquitin, bound to the surface negatively charged lipid-based nanoparticles (liposomes) are derived from combined analysis lifetime broadening arising binding differing size. relative contributions residence time rotational tumbling total effective correlation protein modulated by nanoparticle size, thereby permitting various motional parameters be determined. ubiquitin both large small unilamellar liposomes is ∼20 μs. Bound...

Abstract Pathogenic huntingtin exon‐1 protein (htt ex1 ), characterized by an expanded polyglutamine tract located between the N‐terminal amphiphilic region and a C‐terminal polyproline‐rich domain, forms fibrils that accumulate in neuronal inclusion bodies, is associated with fatal, autosomal dominant neurodegenerative condition known as Huntington's disease. Here complete kinetic model described for aggregation/fibril formation of htt construct 35‐residue repeat, Q 35 . Using exchange NMR...

An enhanced sensitivity zero-quantum correlation experiment is proposed for recording 1H-13C correlations of methyl groups in highly deuterated, protonated large proteins. The spectra benefit from TROSY-effects which both intra- and inter-methyl dipolar relaxation interactions are minimized. Applications to malate synthase G at 5 °C (81 kDa single polypeptide chain enzyme, time 118 ns) lysine decarboxylase 45 (810 decameric enzyme) presented showing significant improvements resolution...

The importance and utility of Alaβ methyl groups as NMR probes molecular structure dynamics in high-molecular-weight proteins is explored. Using 2H 13C relaxation measurements {U-2H; Alaβ-[13CHD2]}-labeled Malate Synthase G (MSG)—an 82-kDa monomeric enzyme that contains 73 groups—we show the vast majority selectively labeled methyls are highly ordered. A number applications used for solution studies large protein molecules can benefit from proximity to backbone their high degree ordering. In...