Narrow carbonyl resonances in proton-diluted proteins facilitate NMR assignments in the solid-state

Abstract

HNCO/HNCACO type correlation experiments are an alternative for assignment of backbone resonances in extensively deuterated proteins in the solid-state, given the fact that line widths on the order of 14-17 Hz are achieved in the carbonyl dimension without the need of high power decoupling. The achieved resolution demonstrates that MAS solid-state NMR on extensively deuterated proteins is able to compete with solution-state NMR spectroscopy if proteins are investigated with correlation times s c that exceed 25 ns.

Solid‐State NMR H–N–(C)–H and H–N–C–C 3D/4D Correlation Experiments for Resonance Assignment of Large Proteins

“…The coherence lifetimes observed in these various samples of oligomeric complexes are similart op reviously reported values on smaller and most often crystalline protein samples. [11,22,27,28] The calculated line widths L,b ased on these coherence lifetimes Figure 2). For 15 Na nd 13 CO the predicted line widths (6-10 Hz) are about two-to threefold lower than experimentally observed ones, suggesting the presence of additional line broadening due to sample heterogeneity and/orm agnetic field inhomogeneity.A lthough thesec alculated and experimentally observed line widths are similar to well-behaving crystalline protein samples, the sheer number of cross-peaks in these samples of large proteins (ca.…”

Proton-detected MAS NMR experiments based on dipolar transfers for backbone assignment of highly deuterated proteins

“…The first essential step in structural NMR studies of proteins is the sequential assignment of the resonances. Even though deuterated proteins have only been studied for a relatively short time by solid-state NMR [19,20], already a plethora of different methods for 1 H, 13 C and 15 N assignment exists [15,18,[21][22][23][24][25][26]. These approaches can be classified into two categories according to the protonation level and the accompanied typical experimental parameters: (1) fully protonated proteins [21,26,27] or perdeuterated proteins with complete reprotonation on exchangeable sites [15,18], which require ultrafast MAS frequencies (40-60 kHz) and very high external magnetic fields; (2) perdeuterated proteins with partial reprotonation on exchangeable sites (ca.…”

“…These approaches can be classified into two categories according to the protonation level and the accompanied typical experimental parameters: (1) fully protonated proteins [21,26,27] or perdeuterated proteins with complete reprotonation on exchangeable sites [15,18], which require ultrafast MAS frequencies (40-60 kHz) and very high external magnetic fields; (2) perdeuterated proteins with partial reprotonation on exchangeable sites (ca. 20-30%) [22][23][24][25], which can be studied even at low external magnetic fields of 9.4 T and moderately high MAS rates between 20 and 32 kHz, achievable with 3.2 mm and 2.5 mm probe heads. The rotor size is an essential parameter in protein studies, since they are often limited by the sensitivity and require therefore the highest possible sample amount [28].…”

Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure

“…These are based on MAS solidstate NMR experiments, [5][6][7][8][9][10][11] and cryo-electron microscopic image reconstructions. 12 Even though there is some controversy concerning the conformational space that Ab fibrils can adopt, all published models, including the 2-fold 7,10 and 3-fold 9 symmetric Ab 1-40 fibril structure suggested by Tycko, and Bertini and co-workers, agree on the basic building block which involves a b-sheet (b1, residues 12-24), a turn, and a second b-sheet (b2, residues [28][29][30][31][32][33][34][35][36][37][38][39][40]. Biophysical studies indicate a certain degree of conformational plasticity for the N-terminal b-sheet.…”

“…25 At the same time, scalar coupling based experiments yield reliable resonance assignments in the solid-state. [26][27][28][29] Fibril samples prepared from deuterated Ab peptides allow us to collect long range distance restraints which will help to refine the quarternary structure of the fibril. 30,31 In the long run, this labeling scheme will allow an artifact-free quantification of dynamics in the solid-state without spin diffusion as a potential source of error.…”