3D NOE-NOE spectroscopy of proteins. Observation of sequential 3D NOE cross peaks in arc repressor

Abstract

2D NMR spectroscopy has been shown to be of great value to the study of biomolecular structures in solution ( 1, 2). Recently 3D NMR was introduced as an extension of the standard 2D techniques (3-5). It is expected that 3D NMR will extend the range of biomolecules that can be studied successfully by high-resolution NMR (cf. Refs. ( 6, 7) for reviews). 3D NMR combines two separate 2D experiments where the detection period of the first 2D experiment acts as the evolution period of the second. In this way, three independent time domains are obtained, which after a 3D Fourier transform leads to a complete 3D spectrum. The three time domains are separated by two mixing periods in which the spins can exchange magnetization leading to the 3D cross peaks. Initially, data matrices and measuring time were restricted by the use of selective pulses (5, 8-1 I ) , but recently it has been demonstrated that nonselective 3D NMR spectra can be obtained within a reasonable time (6, 7, 1224). The obvious goal of 3D NMR is to diminish overlap in crowded 2D spectra. A logical choice is the combination of two different magnetization transfer processes, such as one coherent and one incoherent transfer step. 3D NMR experiments that combine two identical mixing processes, however, have been advocated with the purpose of identifying unambiguously two-step transfer cross peaks, i.e., a COSY-COSY experiment for relayed Jconnectivities (5) and a 3D NOE-NOE experiment to identify the contribution of spin diffusion to NOES ( 23). Here we want to report on the application of 3D NOE-NOE spectroscopy for analyzing networks of NOE cross peaks, in particular those related to the problem of sequential resonance assignment. Results are presented for the Arc repressor of bacteriophage P22, which was studied earlier by 2D NMR ( 1.5, 16). The cross peaks in a 3D spectrum are objects in a 3D space. However, the discussion of the present 3D spectrum will be restricted to cross sections through this spectrum. Especially cross sections perpendicular to the o3 axis ( w3 planes) are suitable for the analysis, since the digital resolution along the w3 axis (the acquisition domain) is generally higher than that along the or and w2 axes. Therefore, these planes contain fewer artifacts due to cross talk between planes, and furthermore, the distribution of noise in o3 planes is more uniform than that in the other cross sections ( 14). The discussion will be focused on w3 cross sections at the amide resonances of the spectrum, since the NH signals are generally the basis for the sequential ‘H NMR assignment of proteins ( I ) and since the chemical-shift dispersion of these protons is large