Projected [(1)H, (15)N]-HMQC-[ (1)H, (1)H]-NOESY for large molecular systems: application to a 121 kDa protein-DNA complex.
We present a projected [(1)H,(15)N]-HMQC-[(1)H,(1)H]-NOESY experiment for observation of NOE interactions between amide protons with degenerate (15)N chemical shifts in large molecular systems. The projection is achieved by simultaneous evolution of the multiple quantum coherence of the nitrogen spin and the attached proton spin. In this way NOE signals can be separated from direct-correlation peaks also in spectra with low resolution by fully exploiting both (1)H and (15)N frequency differences, such that sensitivity can be increased by using short maximum evolution times. The sensitivity of the experiment is not dependent on the projection angle for projections up to 45 degrees and no additional pulses or delays are required as compared to the conventional 2D [(1)H,(15)N]-HMQC-NOESY. The experiment provides two distinct 2D spectra corresponding to the positive and negative angle projections, respectively. With a linear combination of 1D cross-sections from the two projections the unavoidable sensitivity loss in projection spectra can be compensated for each particular NOE interaction. We demonstrate the application of the novel projection experiment for the observation of an NOE interaction between two sequential glycines with degenerate (15)N chemical shifts in a 121.3 kDa complex of the linker H1 histone protein with a 152 bp linear DNA.
Galius
Veniamin
V
Institute of Molecular Biology and Biophysics, ETH Zurich, 8093, Zurich, Switzerland.
Leontiou
Chrysoula
C
Richmond
Timothy
T
Wider
Gerhard
G
D000818:Animals; D002645:Chickens; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D006657:Histones / Q000737:chemistry* / Q000378:metabolism; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000737:chemistry*; D009587:Nitrogen Isotopes / Q000032:analysis / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011522:Protons; D013057:Spectrum Analysis
D004268:DNA-Binding Proteins; D006657:Histones; D046912:Multiprotein Complexes; D009587:Nitrogen Isotopes; D011522:Protons; D004247:DNA; D005998:Glycine