Deuterium Spin Probes of Backbone Order in Proteins: ²H NMR Relaxation Study of Deuterated Carbon α Sites

Abstract

Abstract2 H spin relaxation NMR experiments to study the dynamics of deuterated backbone α-positions, D α , are developed. To date, solution-state 2 H relaxation measurements in proteins have been confined to side-chain deuterons -primarily 13 CH 2 D or 13 CHD 2 methyl groups. It is shown that quantification of 2 H relaxation rates at D α backbone positions and the derivation of associated order parameters of C α -D α bond vector motions in small [U-15 N, 13 C, 2 H]-labeled proteins is feasible with reasonable accuracy. The utility of the developed methodology is demonstrated on a pair of proteins -ubiquitin (8.5 kDa) at 10°C, 27°C, and 40°C, and a variant of GB1 (6.5 kDa) at 22°C. In both proteins, the D α -derived parameters of the global rotational diffusion tensor are in good agreement with those obtained from 15 N relaxation rates. Semi-quantitative solution state NMR measurements yield an average value of the quadrupolar coupling constant, QCC, for D α sites in proteins equal to 174 kHz. Using the uniform value of QCC for all D α sites, we show that C α -D α bond vectors are motionally distinct from the backbone amide N-H bond vectors, with 2 H-derived squared order parameters of C α -D α bond vector motions, S 2 CαDα , on average slightly higher than their N-H amides counterparts, S 2 NH . For ubiquitin, the 2 H-derived backbone mobility compares well with that found in a 1-μs molecular dynamics simulation.

Estimating quadrupole couplings of amide deuterons in proteins from direct measurements of 2 H spin relaxation rates

“…1 are approximately 2.5-fold less sensitive compared to the HN(COCA)-based 2 H relaxation measurements at D a sites [10]. This is a direct consequence of (i) smaller 1 J ND coupling constants ($14 Hz) compared to 1 J CaDa ($22 Hz) leading to longer magnetization transfer periods to and from amide 2 H, and (ii) faster 2 H spin flipping rates in the 15 N-D spin systems during these magnetization transfer steps (two 2s d periods in the scheme of Fig.…”

“…where S is the generalized order parameter describing the fluctuations of N-D bond vectors, [10], D N R Q (D + ) rates are 1.84-fold higher on average than their D a counterparts (Fig. 3b).…”

“…The availability of increasingly sensitive NMR instrumentation allows the quantification of 2 H relaxation rates at protein backbone sites. Very recently, we developed NMR methodology that extends the utility of deuterium as a probe of motions to a positions (D a ) in the backbones of [U-15 N, 13 C, 2 H]-labeled proteins [10]. Using the D a 2 H-derived measures of backbone order, it has been shown that in agreement with 1-ls molecular dynamics simulations, the backbone C a -D a bond vectors are motionally distinct from the amide N-H bond vectors, with average order parameters of C a -D a fluctuations slightly higher than their N-H counterparts [10].…”

Estimating side-chain order in methyl-protonated, perdeuterated proteins via multiple-quantum relaxation violated coherence transfer NMR spectroscopy

“…The correlation time of ubiquitin (D 2 O; 10°C) determined previously in H 2 O at 10°C from 15 N relaxation data (Sheppard et al 2009) has been scaled by the ratio of D 2 O/H 2 O viscosities at 10°C (Cho et al 1999…”

Four‐bond deuterium isotope effects on the chemical shifts of amide nitrogens in proteins

“…[36] Here, NMR measurements of the effects of 1 H-to-D substitution at several positions in a protein on the chemical shifts of backbone amide nitrogens located four bonds away are described. Three different types of four-bond 15 [37] and (ii) the [U-15 N; 13 C;60%-2 H]-labeled ubiquitin. [27] The~60% deuteration level in the latter sample has been achieved by the use of the 60%D 2 O/40%H 2 O mixture of solvents in the M9 minimal medium during protein production.…”