¹H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

Abstract

1H detection can significantly improve solid‐state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for 1H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water‐inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high‐resolution 1H‐detected solid‐state NMR (ssNMR) studies of water‐inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K+ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K+ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

“…2 D, right), possibly due to local motion. Previously, we detected such increased mobility of selectivity filter residues, in particular for V76, in ssNMR studies using synthetic (45) as well as native bacterial membranes (46,47), in line with results of previous molecular-dynamics studies (48).…”

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

“…[9][10][11][12][13][14] Fast MAS and deuteration extend the coherence lifetimes T 2 ' of 1 H, 15 Na nd 13 Cn uclei, resulting in two beneficial effects:1 )the associated narrow line widths increase resolution and intensity,and 2) the coherence transfer between nuclei becomes more efficient, which furtheri ncreases sensitivity in correlation experiments.D ifferent 3D experimentsh ave been proposed to correlate the amide site ( 1 H-15 N) to the 13 Ca, 13 Cb and 13 CO nuclei of the same amino acid residue (intra-residue H i -N i -C i )a nd preceding residue (H i -N i -C iÀ1 ). [15][16][17] Sequential assignment of amides is achievedb ym atching the 13 C i and 13 C iÀ1 frequencies of different amide sites. Furthermore,i th as been proposed to correlatet he 1 H- 15 Nf requency pair to the 15 Nf requency of the neighboring residue.…”

“…Overlap of the H-N frequencies, that is, peak overlap in the two-dimensional" root" spectrum, increases the ambiguity of connecting ag iven H-N pair to the corresponding intra-and inter-residue 13 Cf requencies, and, therefore, severely complicates the assignment process, particularly for large (> 20 kDa) proteins.W ea ddress this problem here by introducing two complementary approaches for correlating the amide sites with either the 13 Ca and 13 CO carbon atoms flanking the given amide site, resultingi napairo f4 D( H i -N i -Ca i -CO i ,H i -N iCa iÀ1 -CO iÀ1 )s pectra, or to the amide protons of either of the neighboring residues,r esulting in another set of correlation spectra (H i -N i -H iÀ1 ,H i -N i -H i+ +1 ).…”

“…For CÀ Ct ransfers,d ifferent schemes have been proposed, using either scalar-coupling-based INEPT steps [16,17] or dipolar-based transfers. [20,21] Under conditions of fast MAS (> 50 kHz) and deuteration, the 13 Cc oherence lifetimes are long (typically of the order of > 10 ms for 13 Ca and > 25 ms for 13 CO [22] ). Under such conditions, INEPT-based transfers generally outcompete those that are dipolar-based.…”

“…Under such conditions, INEPT-based transfers generally outcompete those that are dipolar-based. Of central importance to the presents tudy is the realizationt hat the INEPT-based transfer scheme allows the simultaneous labeling of the 13 Ca or 13 CO resonance frequency during the transfer delay,aproperty that dipolar-based transfer schemesd on ot have. Therefore, the addition of 13 Cf requency dimensions is without further loss of sensitivity (except for the p 2l oss inherentt oq uadrature detection in any indirect dimension) because frequency editing and transfer are achieved during the same delay,w hich represents as ignificant advantage over dipolar-transfer based schemes.…”