Two-dimensional proton NMR study of human ubiquitin: a main chain directed assignment and structure analysis

Abstract

The 1H resonances of human ubiquitin were studied by two-dimensional nuclear magnetic resonance techniques. A recently introduced assignment algorithm termed the main chain directed (MCD) assignment [Englander, S. W., & Wand, A. J. (1987) Biochemistry 26, 5953-5958] was applied. This approach relies on an ordered series of searches for prescribed patterns of connectivities in two-dimensional J-correlated and nuclear Overhauser effect spectra and centers on the dipolar interactions involving main-chain amide NH, alpha-CH, and beta-CH. Unlike the sequential assignment procedure, the MCD approach does not rest upon definition of side-chain J-coupled networks and is generally not sequential with the primary sequence of the protein. The various MCD patterns and the general algorithm are reiterated and applied to the analysis of human ubiquitin. With this algorithm, the vast majority of amino acid residue amide NH-C alpha H-C beta H J-coupled subspin systems could be associated with and aligned within units of secondary structure without any knowledge of the identity of the side chains. This greatly simplified recognition of side-chain spin systems by restricting their identity. Essentially complete resonance assignments are presented. The MCD method is compared with the sequential assignment method in some detail. The MCD method is highly amenable to automation. Human ubiquitin is found, at pH 5.8 and 30 degrees C, to be composed of an extensive beta-sheet structure involving five strands. Three of these strands form an antiparallel set sharing a common strand and have a parallel orientation to two antiparallel strands. Two helical segments were also observed. The largest, spanning 13 residues, shows dipolar interactions consistent with an alpha-helix while the smaller 4-residue helical segment appears, on the basis of observed nuclear Overhauser effects, to be a 3(10) helix. Five classical tight turns could be demonstrated.

High-resolution NMR of encapsulated proteins dissolved in low-viscosity fluids

“…Pulsed field gradient diffusion studies of the hydrodynamic behavior of these preparations in liquid pentane, for example, indicate that each reverse micelle has the radius expected for the encapsulation of a single minimally hydrated protein per reverse micelle. Overhauser effect spectrum of ubiquitin in AOT͞pentane shows the same pattern of nuclear Overhauser effects among main chain hydrogens as observed when the protein is dissolved directly in water (32). These observations confirm earlier optical and fluorescence studies indicating that proteins can be encapsulated in a reverse micelle environment without significant distortion of their native structure.…”

Specificity of the Ubiquitin Isopeptidase in the PA700 Regulatory Complex of 26 S Proteasomes

“…In DQF-COSY experiments to compare Ub and di-Ub, we were unable to detect any significant chemical shift changes for these amide hydrogen resonances. Similarly, the chemical shifts for side chain hydrogen atoms of Leu 8 , Ile 44 , and Val 70 in di-Ub were nearly identical to those observed previously for the Ub monomer by others (42,43) and confirmed here (Fig. 4, compare A and C; and Table I 44 , and Val 70 in di-Ub would not be affected by HyTEMPO relative to those in mono-Ub.…”

Selective Correlation of Amide Groups to Glycine Alpha Protons and of Arginine Guanidine Groups to Delta Protons in Proteins by Multiple Quantum Spectroscopy

“…The peaks appear (advantageously) as singlets in this spectrum since homonuclear geminal coupling is not effective. The resulting chemical shifts are in general agreement with previously published data (11,12).…”