Close identity of a pressure-stabilized intermediate with a kinetic intermediate in protein folding

Abstract

U biquitin, consisting of only 76 amino acid residues (8.6 kDa) with no disulfide bonds, is a signaling protein for an ATP-dependent protein degradation but also for other cellular processes (1). The basic folded structure of ubiquitin has been determined by X-ray crystallography (2) and NMR analysis (3-5). The ubiquitin fold, consisting of a four-stranded, antiparallel ␤-sheet and two short helices, is widely found in many proteins (6). Our previous NMR investigation, carried out on uniformly 15 N-labeled ubiquitin in the pressure range between 1 and 3,500 bar (1 bar ϭ 100 kPa) at pH 4.5 at 20°C, indicated that folded ubiquitin consists of two major conformers mutually interconverting with lifetimes of much less than ms (7): first, the well known conformer N 1 has a rigid C-terminal backbone up to residue 72, and second, the conformer N 2 has an increased freedom of motion for C-terminal residues 70-76 (7).In the present work, we extend our high pressure 15 N͞ 1 H heteronuclear single-quantum coherence (HSQC) NMR experiments under equilibrium conditions to 0°C at pH 4.5 in the pressure range between 30 and 3,700 bar. Lowering the temperature substantially increased the pressure effect on ubiquitin and allowed conformational search of ubiquitin from the native to the totally unfolded conformer. We detected a series of higher energy conformers of ubiquitin, whose partial molar volumes decrease in parallel with the loss of its conformational order, in accordance with the postulated volume theorem of a globular protein (8). In particular, a unique, locally unfolded conformer was detected, whose structure is nearly identical with that of the kinetically trapped folding intermediate found earlier in a guadinium chloride concentration-jump experiment (9). Generality of close structural identity of a pressure-trapped intermediate with a kinetic folding intermediate is discussed. Materials and MethodsNMR Sample. Uniformly 15 N-labeled ubiquitin (VLI, Malvern, PA) was dissolved in 30 mM acetate buffer (pH 4.5 at 20°C) containing 5% 2 H 2 O, resulting in a protein concentration of 2.0 mM.High-Pressure NMR Measurements. High-pressure NMR experiments were performed on a DMX-750 spectrometer (Bruker) by using the on-line cell high-pressure NMR technique (10) equipped with a pressure-resistive quartz cell (11). 15 N͞ 1 H HSQC spectra were measured with a standard HSQC sequence (12) combined with a WATERGATE technique with a 3-9-19 pulsed field gradient (13) at a 1 H-frequency of 750.13 MHz and a 15 N-frequency of 76.01 MHz. The 15 N dimension was acquired with 256 increments, and for the 1 H dimension, 2,048 complex points were collected with an offset at the residual water signal. Data were processed with the XWIN-NMR package (Bruker) and NMRPIPE running on a Silicon Graphics O2 workstation. Spectra were zero-filled to give a final matrix of 4,096 ϫ 512 real data points and apodized with a 90°shifted sine-bell window function in both dimensions.Thermodynamic Analysis. The Gibbs free-energy difference (⌬G) between any two con...

Cold denaturation of ubiquitin at high pressure

“…The structure is likely to be similar to that of the locally disordered conformer detected at high pressure (3.7 kbar and 0°C). 4 However, they did not report full unfolding of ubiquitin by this method.…”

“…E-mail: akasaka@waka.kindai.ac.jp recognition. 4,5 Thus, the study of a conformational fluctuation of ubiquitin in a wider perspective is useful for delineating the molecular mechanism of its function.…”

Atomic-level description of ubiquitin folding

“…Ubiquitin has no prosthetic groups or disulfide bonds, is highly soluble, and is thermostable (T M > 360 K) (27)(28), making it an ideal target for experimental folding studies. The N and C termini form consecutive strands in the β sheet and are in close contact, a common feature in two-state folders (29); indeed, early investigations established that ubiquitin folds on the millisecond timescale with an apparent two-state behavior (30)(31)(32)(33), but later studies suggested that additional on-or off-pathway intermediates may be populated depending on the experimental conditions (34)(35)(36)(37)(38)(39)(40)(41)(42). A protein engineering Φ-value analysis of ubiquitin folding is consistent with a transition state ensemble (TSE) characterized by a well-defined folding nucleus localized in the N-terminal region of the protein and encompassing the helix and first two β strands (43,44).…”

High-pressure NMR reveals close similarity between cold and alcohol protein denaturation in ubiquitin

“…Thus, water can be kept liquid down to 251 K at a pressure of 2.07 kbar (3). Pressure-assisted cold denaturation has been followed for ubiquitin (15)(16)(17), using high-pressure quartz NMR tubes. The analysis of chemical shift data, resonance intensities, and 15 N relaxation showed that the pressure-assisted cold unfolding of ubiquitin is not a simple two-state process, but that several intermediates exist at 3 kbar and pH 4.6.…”