Toward solving the folding pathway of barnase: the complete backbone 13C, 15N, and 1H NMR assignments of its pH-denatured state.

Abstract

The structures of the major folding intermediate, the transition state for folding, and the folded state of barnase have been previously characterized. We now add a further step toward a complete picture ofthe folding of barnase by reporting the backbone 15N, 13C, and 'H NMR assignments for barnase unfolded at pH 1.8 resonances and has very recently been applied to the assignment and structural characterization of the urea-and guanidinium chloride-denatured states of the FK506-binding protein (8).Barnase has a cooperative and reversible unfolding transition between pH 2.8 and pH 2.0 at 300C (M. Oliveberg, S.V., and A.R.F., unpublished results). Here we report the sequence-specific NMR assignment of barnase in the pHdenatured form, which constitutes the first major step in the characterization of this unfolded state by NMR. Magnetization transfer methods at the transition midpoint (pH = 2.4) provided sequence-specific assignments for 30%o of the protein, and these assignments were confirmed and augmented, and the full assignment was completed by using threedimensional (3D) backbone triple-resonance experiments. MATERIALS AND METHODSBarnase was purified from cultures of Escherichia coli (BL21) containing the plasmid pMT410 (9) as described previously (10,11). Separate samples were produced of uniformly 15N-labeled and uniformly 15N, '3C-labeled protein. NMR samples at pH 2.9 and pH 1.8 were prepared by dissolving lyophilized protein in 0.5 ml of 90% H20/10% 2H20 (vol/vol) with appropriate concentrations of HCl. A sample at pH 2.4 additionally contained 50 mM glycine-d5 buffer. The final concentrations for samples of uniformly 15N-labeled barnase were 2.4 mM and for 15N, 13C-labeled barnase, 1.4 mM.All NMR spectra were recorded at 300C on a Bruker AMX500 spectrometer fitted with a fourth channel. Spectra were processed by using the Felix software package (Biosym Technologies, San Diego). The '5N and 'H amide resonances for native barnase at pH 2.9 were assigned by reference to the full barnase assign-

Chemical shift dispersion and secondary structure prediction in unfolded and partly folded proteins

“…Fig. 4 shows a comparison of the chemical shift dispersion of different nuclei, averaged over three unfolded proteins, apomyoglobin [19], barnase [5] and staphylococcal nuclease [6,9]. It is immediately obvious that the chemical shift dispersions for the backbone amide proton and backbone 15 N are large.…”

“…Chemical shift dispersion of various nucleus types averaged over all residue types for folded and unfolded proteins. The assigned chemical shifts are averaged for unfolded proteins apomyoglobin [19], barnase [5] and staphylococcal nuclease [6,9]. The assigned chemical shifts are averaged for five folded proteins, staphylococcal nuclease [10], human carbonic anhydrase [11], mutant dihydrofolate reducíase [12], recoverin [13] and ribonuclease A [14] The chemical shift dispersion is calculated as described in the text.…”

“…When the 13 C a , 13 C$, 1 H a and : HP are badly dispersed, it becomes necessary to find alternative nuclei with better overall chemical shift dispersion to be used for sequential assignment purposes. The dispersion of the 15 N and : HN resonances has been successfully used for this purpose (see for example, [1,[4][5][6]) but proved inadequate, in our hands, for unfolded apomyoglobin. A different strategy was therefore required, which utilized the high intrinsic dispersion of the 13 CO resonance.…”

“…These are present in a very similar solution environment in the unfolded protein, unlike a folded protein, where many nuclei are sequestered from solvent and are part of elements of secondary structure. Finally, a comprehensive calculation of chemical shift dispersion has been made, including data from two other unfolded proteins, barnase [5] and staphylococcal nuclease [6,9], and from five folded proteins, staphylococcal nuclease [10], human carbonic anhydrase [11], mutant dihydrofolate 0014-5793/97/S17.00 © 1997 Federation of European Biochemical Societies. All rights reserved.…”

Characterization of residual structure in the thermally denatured state of barnase by simulation and experiment: Description of the folding pathway

“…It is only recently that structural information for the denatured state has become available at atomic resolution because of developments in high field NMR spectroscopy (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). These studies have focused on areas of persistent structure, with the aim of obtaining insight into the relationship between sequence, structure, and in particular, the mechanism of folding.…”