NMR Spectroscopic Detection of Protein Protons and Longitudinal Relaxation Rates between 0.01 and 50 MHz

Abstract

Nuclear magnetic relaxation data of water nuclei at variable fields provide valuable information on the dynamics of watersolute interactions. [1][2][3] However, information can be collected only within certain field ranges in which the nuclear relaxation rates are field-dependent owing to the dispersion of the spectral density, J(w,t). The dispersion depends on the type of motion and on the observed nucleus. The most informative 1 H NMR spectroscopic frequency range for rotational motions is centered around 10 MHz for small proteins (M W % 10 4 Da) and smaller frequencies for larger proteins and protein aggregates. Of course, resolution is low at these fields and in practice only one signal (or an unresolved signal envelope) can be detected. Furthermore, only the abundant water protons can be conveniently studied under such low sensitivity.Relaxation of the isotopes 17 O and 2 H in enriched water can also be studied; [1] in these cases the centers of the informative field ranges increase by a factor % 7 with respect to 1 H as a result of the magnetogyric ratios of 17 O and 2 H. Because water interacts with proteins, such relaxation studies provide direct information on the nature of water-protein dynamics, but give only indirect information on the protein itself.[4-6] It would be highly desirable to have complementary information directly from the protons of the protein, but this has been impractical so far owing to the low sensitivity of the available instrumentation. Protein 1 H relaxation data have been reported only on protein solutions of concentrations ! 35 % by mass, [7,8] far from physiological conditions. Improvements in field-cycling technology [9][10][11][12] have led to the production of instrumentation [13] with a % 10-fold increase in the signal-to-noise ratio, [14] with which direct protein 1 H detection can be attempted for protein concen- Figure 1. Protein 1 H relaxation dispersions for lysozyme solutions (2.8 mm in D 2 O) at a) pH* 3.5, and b) pH* 9.0 (pH=pH-meter reading in D20 solution). The time decay of the collective protein 1 H magnetization at 0.1 MHz and its single-exponential fit are shown in the inset of part a). Theoretical relaxation rates were obtained through singleexponential fits of time decays of collective protein 1 H magnetization calculated from the known protein structure of lysozyme [17] and a complete relaxation matrix (CORMA) analysis.[18] Only exchangeable NH protons from secondary structure elements ( % 50 % of total) were included, whereas all other exchangeable proton positions were assumed to be deuterated. Inclusion of either all or none of the NH protons affected the calculated rates by AE 2 %. The theoretical lowfield hE 2 i values obtained in this way were used to fit Equation (1) to the data (solid lines). The dotted lines represent data fits with two J-(w,t) terms in Equation (1) ( Table 1). Panel c) shows the protein 1 H relaxation dispersion for a solution of a-synuclein in D 2 O (1.4 mm, pH* 7.1). The solid and dotted lines represent fits with one or two J-(...

Long-Range Correlated Dynamics in Intrinsically Disordered Proteins

“…29,31,32 Surprisingly, measurements performed on the IDP α-synuclein also showed a small dispersion at ωτ R = 1 with a correlation time comparable to the reorientation time calculated for a folded protein with the same molecular weight. 29 The dispersion should not be observed if α-synuclein was only subject to segmental motions with correlation times much shorter than the reorientation time of the protein, 16,33 since the dispersions corresponding to such times occur at much larger fields. The observed dispersion therefore suggested that a residual correlated motion is present in α-synuclein.…”

“…The presence of a dispersion at low fields in the relaxation rate profile of protein protons (performed in D 2 O solutions in such a way as to only observe the nonexchangeable protein protons) can in fact directly provide the largest correlation time values that modulate the dipolar interactions between protein protons. 29 FFC measurements are now feasible, due to the availability of high-sensitivity fast fieldcycling relaxometers, with improved sensitivity with respect to that used in Bertini et al 29 The new instrument can directly detect signals of protein protons in millimolar solutions in D 2 O over a very wide field range (from a few kilohertz to tens of megahertz of proton Larmor frequency), being thus able to provide the collective relaxation dispersion profile of protein protons in solution. 30 This collective relaxation dispersion profile can then be analyzed in terms of an overall "collective" order parameter and an overall rotational correlation time.…”

“…At the same time, a small Lipari−Szabo S C 2 parameter 34 of 0.05−0.1 was observed for the collective protein protons, in line with the disordered nature of α-synuclein: the collective relaxation rate of protein protons at low fields, which are hundreds or thousands of s −1 in regularly folded proteins, drastically reduces to few tens of s −1 in the case of α-synuclein. 29,31 The observation of a long reorientational time in α-synuclein suggests that relaxation measurements of protein protons at low fields provide access to residual correlated motions in IDPs. However, the question remained if long correlation time is unique to α-synuclein or if it is a common feature of disordered proteins.…”

Cross‐relaxation bottleneck in water–lysozyme proton magnetization exchange

“…The P-protons in D-HEWL (or S-protons in N-HEWL, Figure 1) have T 1 values on the low temperature side of the T 1 minimum as evidenced by the plot of lnT 1 versus 1000/T being a straight line with positive slope (Figure 3) and 1/T 1 of HEWL protons being dispersive. 34 In addition, CH 3 can be considered independent of , so that it is reasonable to expect 38 T 1 to be approximately proportional to . Thus, if we assume that the labile protein protons, absent in D-HEWL, have similar T 1 values as those remaining on the HEWL molecule, the protein proton relaxation time in N-HEWL can be approximated as (1/1.2)T 1 (D-HEWL).…”

High resolution NMR study of T1 magnetic relaxation dispersion. III. Influence of spin 1/2 hetero-nuclei on spin relaxation and polarization transfer among strongly coupled protons

“…[1][2][3][4][5][6][7][8][9][10][11] In fieldcycling NMR experiments, the nuclear magnetic relaxation dispersion (NMRD) curve is measured, which is the magnetic field dependence of the longitudinal relaxation rate, R 1 , i.e., the inverse of the longitudinal relaxation time, T 1 : R 1 = 1/T 1 . In general, the rate of the relaxation transition between the μ-th and ν-th levels, R μν is given in second-order perturbation theory by 12 R μν = ∞ 0 V μν (t)V νμ (t + τ )e iω νμ τ dτ…”