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. 2007 Nov 20;104(47):18473-7.
doi: 10.1073/pnas.0708296104. Epub 2007 Nov 15.

Measurement of bond vector orientations in invisible excited states of proteins

Pramodh Vallurupalli  1 , D Flemming Hansen, Elliott Stollar, Eva Meirovitch, Lewis E Kay
Affiliations

Measurement of bond vector orientations in invisible excited states of proteins

Pramodh Vallurupalli et al. Proc Natl Acad Sci U S A. .

Abstract

The focus of structural biology is on studies of the highly populated, ground states of biological molecules; states that are only sparsely and transiently populated are more difficult to probe because they are invisible to most structural methods. Yet, such states can play critical roles in biochemical processes such as ligand binding, enzyme catalysis, and protein folding. A description of these states in terms of structure and dynamics is, therefore, of great importance. Here, we present a method, based on relaxation dispersion NMR spectroscopy of weakly aligned molecules in a magnetic field, that can provide such a description by direct measurement of backbone amide bond vector orientations in transient, low populated states that are not observable directly. Such information, obtained through the measurement of residual dipolar couplings, has until now been restricted to proteins that produce observable spectra. The methodology is applied and validated in a study of the binding of a target peptide to an SH3 domain from the yeast protein Abp1p and subsequently used in an application to protein folding of a mutational variant of the Fyn SH3 domain where (1)H-(15)N dipolar couplings of the invisible unfolded state of the domain are obtained. The approach, which can be used to obtain orientational restraints at other sites in proteins as well, promises to significantly extend the available information necessary for providing a site-specific characterization of structural properties of transient, low populated states that have to this point remained recalcitrant to detailed analysis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Measurement of amide bond vector orientation in invisible excited protein states. (a) Energy level diagram for a two-state exchanging system, where the loop (green) can exist in two conformations. (b) Resulting 1H-decoupled 15N spectrum for a single amide probe of conformational exchange between two states whose populations are highly skewed. In weakly aligning media (c) and without 1H decoupling, each line is split by the sum of 1H-15N dipolar and scalar couplings (JNH ∼ −93 Hz). Spectra resulting from the 1H in the down and up spin-states are shown in d in red and blue, respectively. (e and f) Separate 15N CPMG relaxation dispersion experiments monitor exchange between ground and excited state conformations that are separated by Δν (black), Δν − 0.5ΔDNH (red), or Δν + 0.5ΔDNH (blue), from which ΔDNH can be extracted. There is a small contribution to the chemical shift that results from alignment (19) so that νA and νB are shifted slightly (≤5 Hz for the alignment parameters of the systems considered here at a field of 800 MHz) between b and d (not included for clarity). Thus, values of Δν include contributions from incomplete averaging of the anisotropic chemical shift, as described in the text. In f, intrinsic relaxation rates, R2,∞, have been subtracted from the dispersion profiles to emphasize their differences, ΔR2 = R2,eff − R2,∞. Note that the relative magnitude of TROSY and anti-TROSY dispersion profiles reverses with the sign of the product Δν × ΔDNH.
Fig. 2.
Fig. 2.
Pulse schemes of 15N constant-time TROSY and anti-TROSY CPMG relaxation dispersion experiments for measurement of ΔDNH in protein systems undergoing millisecond-time-scale exchange dynamics. All 1H and 15N 90° (180°) radiofrequency pulses are shown as narrow (wide) black bars and are applied at the highest possible power level, with the exception of the 15N refocusing pulses of the CPMG element, along with the 90° sandwiching pulses, which are applied at a slightly lower power level (≈6 kHz). Composite 180° pulses (34) are represented by “striped” rectangles. All pulse phases are assumed to be x, unless indicated otherwise. N can be any integer. Differences in the TROSY/anti-TROSY schemes are highlighted (in red and blue for TROSY and anti-TROSY, respectively; the 180° pulse at point c is omitted in the case of the TROSY experiment). Water-selective 90° 1H pulses (shaped pulses) are rectangular (≈1.6 ms). The phase cycling used is as follows (Varian): φ1 = {x, −x}; φ2 = 2{y}, 2{−y}; φ3 = 2{x}, 2{−x}; φ4 = 2{y}, 2{−y}, 2{−x}, 2{x}; φ5 = −y; φ6 = y; φ7 = −y; receiver = {y, −y, −y, y, x, −x, −x, x}. Sensitivity enhanced quadrature detection in the indirect dimension (–38) is obtained by recording a second data set with φ4 = 2{y}, 2{−y}, 2{x}, 2{−x}; φ5 = φ5 + π; φ6 = φ6 + π; φ7 = φ7 + π; and receiver = receiver + π for each t1 increment. In addition, phase φ4 is incremented along with the receiver by 180° for each complex t1 point (39). The delays used are τa = 2.25 ms, τb = 1/(4|JNH|) = 2.68 ms, and τeq = (2 − 3)/(kex) ∼ 5 ms. Gradient strengths G/cm (length in milliseconds) are as follows: g0 = −15(1), g1 = 5(1), g2 = 12(1), g3 = 8(0.3), g4 = 10(0.5), g5 = 0.5(t1), g6 = 6(0.3), g7 = 25(0.3). A spin-lock element is applied immediately after acquisition at the same power level and for the same duration (Trelax) as used for the experiment measuring |Δν| (SI Fig. 5) so that the heating effects are constant over all measurements.
Fig. 3.
Fig. 3.
Measuring 1H-15N dipolar couplings of the invisible peptide-bound state of the Abp1p SH3 domain and validation of the methodology. (a) Selected region of 1H-15N TROSY-HSQC spectra of Abp1p SH3 with 6.8% and 100% peptide bound, 800 MHz, 25°C. (b–d) Dispersion profiles of selected residues measured at 800 MHz, from which ΔDNH is obtained (as described in the text). Intrinsic relaxation rates have been subtracted from the dispersion curves. (e) Dipolar couplings of the invisible minor state, corresponding to the Ark1p peptide-bound form of Abp1p SH3, DNHB, agree well with dipolar couplings, DNH, measured directly from a fully bound sample. As couplings from separate samples are compared with small differences in the amounts of phage and hence slight differences in Aa values, the slope of the best-fit correlation is not 1 [(Aa, R) = ((−6.4 ± 0.3) × 10−4, 0.38 ± 0.07) and ((−7.35 ± 0.05) × 10−4, 0.36 ± 0.01) calculated from DNHB and DNH, respectively]. Alignment parameters Aa and R are as defined in ref. .
Fig. 4.
Fig. 4.
Measuring 1H-15N dipolar couplings of the excited, unfolded state of the G48M Fyn SH3 domain. (a) TROSY and anti-TROSY 15N dispersion profiles (800 MHz) are identical when measured in isotropic phase, with clear differences when protein is dissolved in aligning media (b). (c) Comparison of DNHB (red) and DNHA (green) values of the invisible unfolded state and the folded conformer measured on the same sample by means of relaxation dispersion and direct methods, respectively.
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