Fractional 13C enrichment of isolated carbons using [1-13C]- or [2-13C]-glucose facilitates the accurate measurement of dynamics at backbone Cα and side-chain methyl positions in proteins

Lundström, Patrik; Teilum, Kaare; Carstensen, Tommy; Bezsonova, Irina; Wiesner, Silke; Hansen, D. Flemming; Religa, Tomasz L.; Akke, Mikael; Kay, Lewis E.

doi:10.1007/s10858-007-9158-6

Cited by 161 publications

(227 citation statements)

References 40 publications

Supporting

Mentioning

221

Contrasting

Unclassified

Order By: Relevance

“…We expressed gdLRR-2-A-cap and, as a reference, the unfolded gdLRR-3-A-cap (see below) with [1][2][3][4][5][6][7][8][9][10][11][12][13] C]-glucose as the sole carbon source. Expression results in specific labeling of isolated sites, including methyl groups (29). In agreement with the FTIR results, the 2D 1 H- 13 C NMR spectrum of gdLRR-3-A-cap is characteristic of a random coil, with no more than 5% folded conformation (Fig.…”

Section: −1supporting

confidence: 82%

Computational design of a leucine-rich repeat protein with a predefined geometry

Rämisch¹,

Weininger

Martinsson³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Structure-based protein design offers a possibility of optimizing the overall shape of engineered binding scaffolds to match their targets better. We developed a computational approach for the structure-based design of repeat proteins that allows for adjustment of geometrical features like length, curvature, and helical twist. By combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeats (LRRs) from the ribonuclease inhibitor (RI) family that assemble into structures with a predefined geometry. The repeat proteins were built from self-compatible LRRs that are designed to interact to form highly curved and planar assemblies. We validated the geometrical design approach by engineering a ring structure constructed from 10 self-compatible repeats. Protein design can also be used to increase our structural understanding of repeat proteins. We use our design constructs to demonstrate that buried Cys play a central role for stability and folding cooperativity in RI-type LRR proteins. The computational procedure presented here may be used to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired.binding scaffold | Rosetta | buried cysteines | computational protein design | geometrical design

show abstract

Section: −1supporting

confidence: 82%

Computational design of a leucine-rich repeat protein with a predefined geometry

Rämisch¹,

Weininger

Martinsson³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Indeed, a sparse array of 13 C spins can increase the efficiency of long-range TEDOR experiments nearly threefold (35,42). We used [2-13 C]glucose to achieve 13 C labeling at the Cα-position and eliminate labeling at the adjacent C′ and Cβ-sites (45). A mixed sample made with this labeling pattern will have about one-eighth of the intermolecular 13 C-15 N pairs of a uniformly labeled sample due to the random incorporation of monomers into the polymer (∼25%) as well as the inefficiency of 13 Cα-labeling by [2-13 C]glucose (∼45%) (45).…”

Section: Resultsmentioning

confidence: 99%

“…We used [2-13 C]glucose to achieve 13 C labeling at the Cα-position and eliminate labeling at the adjacent C′ and Cβ-sites (45). A mixed sample made with this labeling pattern will have about one-eighth of the intermolecular 13 C-15 N pairs of a uniformly labeled sample due to the random incorporation of monomers into the polymer (∼25%) as well as the inefficiency of 13 Cα-labeling by [2-13 C]glucose (∼45%) (45). Combined with the order-ofmagnitude drop in experimental efficiency of measurement of long-range versus short-range interactions, this experiment sets a high bar for sensitivity.…”

Section: Resultsmentioning

confidence: 99%

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Frederick

Michaelis

Caporini

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The yeast prion protein Sup35NM is a self-propagating amyloid. Despite intense study, there is no consensus on the organization of monomers within Sup35NM fibrils. Some studies point to a β-helical arrangement, whereas others suggest a parallel inregister organization. Intermolecular contacts are often determined by experiments that probe long-range heteronuclear contacts for fibrils templated from a 1:1 mixture of 13 C-and 15 N-labeled monomers. However, for Sup35NM, like many large proteins, chemical shift degeneracy limits the usefulness of this approach. Segmental and specific isotopic labeling reduce degeneracy, but experiments to measure long-range interactions are often too insensitive. To limit degeneracy and increase experimental sensitivity, we combined specific and segmental isotopic labeling schemes with dynamic nuclear polarization (DNP) NMR. Using this combination, we examined an amyloid form of Sup35NM that does not have a parallel in-register structure. The combination of a small number of specific labels with DNP NMR enables determination of architectural information about polymeric protein systems. (2), is an amyloid form of the translation termination factor Sup35 (3). The amyloid fold is polymeric fiber of protein monomers that is rich in β-sheets that run perpendicular to the fiber axis. Amyloid formation sequesters Sup35 from the ribosome, changing the rate of stop-codon read-through and creating a variety of new yeast phenotypes (4,5). Different regions of the Sup35 protein are responsible for prion templating, prion inheritance, and translation termination. The N-terminal domain "N" is Q/N-rich and required for the formation and templating of amyloid. The highly charged K/E-rich middle domain "M" promotes solubility in the nonprion form and is required for chaperone-mediated prion inheritance (6, 7). The C-terminal domain is necessary and sufficient for translation termination. The N and M domains together, NM, contain all of the necessary features to act as an amyloid-based prion.A wide variety of approaches have been used to investigate Sup35 prion structures, but a consensus structural picture has yet to emerge. Amyloids are notoriously difficult to study, mainly because they are insoluble yet noncrystalline and are therefore not easily amenable to traditional structural biology methods. In all models, at least some part of the N domain is involved in the amyloid fold (8-11), and most of the M domain is thought to be solvent-accessible and intrinsically disordered (11-13). However, there are two conflicting models of how monomers of NM are arranged in amyloid fibers. In one model, called the β-helical model, monomers make intermolecular contacts in discrete regions, with the region in-between making intramolecular contacts. This model is supported by the patterns of interaction and cross-linking determined from a series of site-directed mutants (8). The work has the caveat that mutations may perturb the fibril structure. However, fibers made from the mutated versions of the protein ca...

show abstract

“…13 C]-glucose as the sole carbon source (Lundström et al 2007a). A maximum of 50 % enrichment of the C α positions can be achieved using [2-…”

Section: Alpha-carbon Relaxation Dispersionsmentioning

confidence: 99%

Relaxation Dispersion NMR Spectroscopy

Sauerwein

Hansen

2015

Protein NMR

Self Cite

View full text Add to dashboard Cite

Fractional 13C enrichment of isolated carbons using [1-13C]- or [2-13C]-glucose facilitates the accurate measurement of dynamics at backbone Cα and side-chain methyl positions in proteins

Cited by 161 publications

References 40 publications

Computational design of a leucine-rich repeat protein with a predefined geometry

Computational design of a leucine-rich repeat protein with a predefined geometry

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

Relaxation Dispersion NMR Spectroscopy

Contact Info

Product

Resources

About