Comprehensive determination of 3JHNHα for unfolded proteins using 13C′-resolved spin-echo difference spectroscopy

Abstract

An experiment is presented to determine 3 J HNHa coupling constants, with significant advantages for applications to unfolded proteins. The determination of coupling constants for the peptide chain using 1D 1 H, or 2D and 3D 1 H-15 N correlation spectroscopy is often hampered by extensive resonance overlap when dealing with flexible, disordered proteins. In the experiment detailed here, the overlap problem is largely circumvented by recording 1 H-13 C 0 correlation spectra, which demonstrate superior resolution for unfolded proteins. J-coupling constants are extracted from the peak intensities in a pair of 2D spin-echo difference experiments, affording rapid acquisition of the coupling data. In an application to the cytoplasmic domain of human neuroligin-3 (hNlg3cyt) data were obtained for 78 residues, compared to 54 coupling constants obtained from a 3D HNHA experiment. The coupling constants suggest that hNlg3cyt is intrinsically disordered, with little propensity for structure.

Structural Ensembles of Intrinsically Disordered Proteins Depend Strongly on Force Field: A Comparison to Experiment

“…Figure 5 shows 3 J HNHα scalar couplings, which report on the φ backbone dihedral angle, and are the mostinformative (and most-commonly used) for backbone conformation. 91 As can be seen, two force fields agree well with the measured couplings, CHARMM 22* with charmm-modified TIP3P and TIP4P-D water models. The 3 J HNHα scalar couplings for both CHARMM 36 ensembles are outside of experimental error.…”

An Approach to NMR Assignment of Intrinsically Disordered Proteins

“…h, j, l Considering that [ 1 H, 15 N]‐resolved triple‐resonance experiments may not always lead to satisfactory results in the study of disordered systems, either due to poor dispersion of cross‐peaks in the 15 N– 1 H correlation spectra or due to fast exchange of the solvent‐exposed labile protons with water protons, a variety of alternative strategies involving 1 H α and 13 C direct detection have been reported for the study of IDPs 5. From these studies it is apparent that dispersion of inter‐residue cross‐correlation peaks observed in spectra such as 15 N– 13 C′ and 13 C′– 1 H N is typically better than the intra‐residue cross peaks′ dispersion in conventional [ 1 H, 15 N]‐correlation spectra of disordered proteins 4i. k Hence, triple‐resonance experiments based on such heteronuclear chemical shift correlations are expected to bear considerable impact on to the study of IDPs and IDPRs.…”

Measurement and Applications of Long‐Range Heteronuclear Scalar Couplings: Recent Experimental and Theoretical Developments

“…The experiment begins with a 2D INADEQUATE module with a fixed delay of D CC = 0.25/( 1 J CC ) to create homonuclear 13 C double quantum coherences during t 1 evolution. [34] BIRD-J-resolved HMBC Long-range homonuclear and heteronuclear J-couplings; J-resolved Ad: Scaling of couplings; swelling of cross peaks is too small to affect line shape Lim: Sensitivity [26] BIRD-Jhigh-resolution-HMBC Long-range homonuclear and heteronuclear J-couplings; J-resolved Ad: Scaling of couplings; swelling of cross peaks is too small to affect line shape Lim: Sensitivity [26] IPAP-HSQMBC Measurement of small proton-carbon ( n J CH ) and proton-nitrogen ( n J NH ) coupling constants; spin-state-selective cross peaks Ad: Works well even for the measurement of small coupling values in both protonated and non-protonated carbon atoms Lim: Excessive cross talk and undesired differential J HH modulation give complex multiplets [33] HR-PANA-CEA INADEQUATE, HSQC and 3D HMBC have been combined into a single experiment Ad: Gives complete assignment or structure of small organic molecules Lim: Need for multiple receivers [35] Selective Jresolved HMBC Employs selective refocusing pulse and constant time; J-resolved Ad: Accurate measurement of long-range J-couplings in complex molecules ; sensitivity Lim: Use of selective pulse, that is, its duration should be short to reduce the decrease in the signal intensity due to short T 2 [36] 3D HSQC-TCOSY Based on the optimized random sampling of the evolution time space followed by multidimensional Fourier transformation Ad: Shorter experimental time; resolution of overlapped spectra observed in 2D experiments Lim: No information of coupling involving quaternary carbon [28] C-HetSERF Spin-selective correlation of 13 C-bound proton magnetization; provides a/b cross peaks displaced by n J CH Ad: Spin-selective a/b cross peaks allow the measurement of small coupling; relative signs; homonuclear couplings Lim: Sensitivity problem due to the application of small-angle mixing pulse [23,37,38] 13 C-resolved spin-echo J-coupling constants are extracted from the peak intensities in a pair of 2D spin-echo difference experiments…”

“…The coupling can be measured precisely from the shifting of two in-phase multiplet components, which are separated in the indirect dimension and therefore have no interfering influence, such as spectral overlap, on each other. Before proceeding with detailed discussions of some of the chosen techniques, a brief summary of the important experimental techniques, [26,28,[33][34][35][36][37][38][39] their applications, advantages and limitations is given in Table 1.…”

“…3D HSQC-TCOSY Based on the optimized random sampling of the evolution time space followed by multidimensional Fourier transformation Ad: Shorter experimental time; resolution of overlapped spectra observed in 2D experiments Lim: No information of coupling involving quaternary carbon [28] C-HetSERF Spin-selective correlation of 13 C-bound proton magnetization; provides a/b cross peaks displaced by n J CH Ad: Spin-selective a/b cross peaks allow the measurement of small coupling; relative signs; homonuclear couplings Lim: Sensitivity problem due to the application of small-angle mixing pulse [23,37,38] 13 C-resolved spin-echo J-coupling constants are extracted from the peak intensities in a pair of 2D spin-echo difference experiments Ad: Rapid acquisition of the coupling data; superior resolution for unfolded protein Lim: Not a robust sequence for medium-sized or large folded proteins [40] CPMG HSQMBC Method implements adiabatic inversion and refocusing pulses on the heteronucleus; allows determination of long-range heteronuclear couplings of very small magnitudes Ad: Provides couplings for both quaternary and protonated carbon atoms Lim: The narrow bandwidth of the low-power pulses in the CPMG cycle can give non-uniform excitation [39] Selective 1D CPMG HSQMBC Same as for CPMG HSQMBC except the G-BIRD module Ad: Shorter experimental time Lim: Same as for CPMG HSQMBC but more experiments have to be carried out to derive all the coupling parameters. Necessity of well-resolved multiplet for the selective pulse to be applied [39] The F 1 /F 2 plane pertains to the INADEQUATE spectrum with all the expected 13 C-13 C correlations.…”