Magic Angle Spinning Solid-State NMR Spectroscopy for Structural Studies of Protein Interfaces. Resonance Assignments of Differentially Enriched Escherichia coli Thioredoxin Reassembled by Fragment Complementation

Abstract

De novo site-specific backbone and side-chain resonance assignments are presented for U-15N(1-73)/U-13C,15N(74-108) reassembly of Escherichia coli thioredoxin by fragment complementation, determined using solid-state magic angle spinning NMR spectroscopy at 17.6 T. Backbone dihedral angles and secondary structure predicted from the statistical analysis of 13C and 15N chemical shifts are in general agreement with solution values for the intact full-length thioredoxin, confirming that the secondary structure is retained in the reassembled complex prepared as a poly(ethylene glycol) precipitate. The differential labeling of complementary thioredoxin fragments introduced in this work is expected to be beneficial for high-resolution structural studies of protein interfaces formed by protein assemblies by solid-state NMR spectroscopy.

Sensitivity enhancement in 13C solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing 1H T1 relaxation

“…It is probable that the present approach can be adopted in CPMAS or static experiments of other insoluble proteins. The successful reduction of 1 H T 1 relaxation times in the present experiments will also open new possibilities of sensitivity enhancements in more advanced experiments such as multi-dimensional 13 C SSNMR of uniformly 13 C-labeled proteins [18][19][20][21][22][23][24][25] and various distance measurements [3;11;13;47-50] for selectively 13 C-labeled protein samples in our future studies. (a-d) 13 (a-d) 13 C CPMAS spectra of protein micro crystals for (a, b) lysozyme and (c, d) ubiquitin prepared in D 2 O obtained (a, c) with and (b, d) without 10 mM Cu-EDTA at 13 C NMR frequency of 100.6 MHz.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] Particularly, use of protein micro-/nano-crystals [17] has significantly improved resolution in high-resolution SSNMR of dilute spins such as 13 C and 15 N, permitting signal assignment and structural determination of various uniformly 13 C and/or 15 N-labeled proteins by SSNMR. [18][19][20][21][22][23][24][25] However, restricted sensitivity in 13 C and 15 N SSNMR has been still one of the major limiting factors in SSNMR analysis of proteins. In an experimental time required for 13 C SSNMR of proteins, more than 95 % is typically consumed for recycle delays to retrieve spin polarization by 1 H T 1 relaxation, to protect a probe from arcing due to RF irradiation, or to avoid sample degradation due to heating.…”

Magic angle spinning NMR spectroscopy of thioredoxin reassemblies

“…16,21,30 -32 In our recent studies, we have demonstrated that 108-residue Escherichia coli thioredoxin is an excellent model protein for high-resolution structural studies by SSNMR spectroscopy. 31,33,34 Thioredoxin, a disulfide reductase and a member of a large superfamily of multifunctional protein modules, has been known since 1964, when it was isolated by Laurent and coworkers. 35 Thioredoxins are ubiquitous in many organisms from Archea to humans, and owing to their key role in the redox regulation of protein function and signaling via thiol redox center, their biochemistry and structure have been the subject of multiple investigations.…”

“…31,33 We presented complete or nearly complete resonance assignments of the intact thioredoxin and of the 74-108( 13 C, 15 N) fragment in the reassembled protein, using a combination of two-dimensional homoand heteronuclear correlation experiments at 17.6 T; we had conducted analysis of the secondary structure based on Torsion Angle Likelihood Obtained from Shift and sequence similarity (TALOS)-derived chemical shifts, and for the intact thioredoxin, examined the tertiary constraints available from the dipolar-assisted rotational resonance (DARR) spectra acquired at different mixing times. 31,33 In this report, we present resonance assignments, and secondary and tertiary structure analysis of the differentially enriched 1-73( 13 …”

Characterization of the Conjugation Pattern in Large Polysaccharide–Protein Conjugates by NMR Spectroscopy

“…For the solid‐state NMR investigation, ANSII (either free or its MenC conjugate) was freeze‐dried, packed into the magic‐angle spinning (MAS) rotors, then rehydrated to restore the resolution 30, 34, 35, 36. The spectra collected on the pelleted ANSII‐MenC conjugate (Figure 2) are largely superimposable to those of the native and PEGylated forms of the enzyme (see Figure S2 in the Supporting Information) making possible the assessment of the preservation of the protein fold after conjugation with the MenC polysaccharide 37, 38, 39, 40. The resonances of the protein residues were easily reassigned starting from the assignment of the native form of ANSII, by using a carbon‐detection approach (Figure S3) 41.…”