Locally Disordered Conformer of the Hamster Prion Protein:  A Crucial Intermediate to PrP<sup>Sc</sup>?

Kuwata, Kazuo; Li, Hua; Yamada, Hiroaki; Legname, Giuseppe; Prusiner, Stanley B.; Akasaka, Kazuyuki; James, Thomas Leroy

doi:10.1021/bi026129y

Cited by 158 publications

(175 citation statements)

References 30 publications

Supporting

Mentioning

149

Contrasting

Unclassified

Order By: Relevance

“…Furthermore, we note that the experimentally determined partial molar volume of ubiquitin decreases in the order of the decreasing conformational order, namely N 1 Ͼ N 2 Ͼ I Ͼ U. Along with the similar trend in other globular proteins (17)(18)(19)(20), the result clearly supports the volume theorem of protein, which can be stated as ''partial molar volume of a protein decreases in parallel with the decrease of its conformational order'' (8).…”

Section: Resultssupporting

confidence: 79%

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

Kitahara

Akasaka²

2003

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

103

122

View full text Add to dashboard Cite

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...

show abstract

Section: Resultssupporting

confidence: 79%

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

Kitahara

Akasaka²

2003

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

103

122

View full text Add to dashboard Cite

show abstract

“…Perhaps contradictory to the modeling based on 2D crystals, 46 these studies suggest that a largely intact helix B and C is unlikely. Indeed, the NMR studies suggest that helix A actually has slightly more stability than helix B or C. 32,53 Recent amide protection studies of prion fibrils (PrP Sc rather than PrP C ) have indicated that a relatively short stretch of the sequence constitutes the b-sheet rich core of PrP Sc between residues 169 and 214, 24 again supporting the assertion that helix B and C are not retained as helices in the PrP* precursor to the scrapie isoform.…”

Section: Prp C Folding Intermediates and Stabilitymentioning

confidence: 91%

“…Studies using hydrostatic pressure 53 have been used to study the unfolding of PrP C in an attempt to identify folding intermediates. These folding intermediates may represent conformations of the prion protein (PrP*) on a pathway to PrP Sc and amyloid formation.…”

Section: Prp C Folding Intermediates and Stabilitymentioning

confidence: 99%

Dynamics of a truncated prion protein, PrP(113–231), from ¹⁵N NMR relaxation: Order parameters calculated and slow conformational fluctuations localized to a distinct region

et al. 2009

View full text Add to dashboard Cite

Prion diseases are associated with the misfolding of the prion protein (PrP C ) from a largely a-helical isoform to a b-sheet rich oligomer (PrP Sc ). Flexibility of the polypeptide could contribute to the ability of PrP C to undergo the conformational rearrangement during PrP C -PrP Sc interactions, which then leads to the misfolded isoform. We have therefore examined the molecular motions of mouse PrP C , residues 113-231, in solution, using 15 N NMR relaxation measurements. A truncated fragment has been used to eliminate the effect of the 90-residue unstructured tail of PrP C so the dynamics of the structured domain can be studied in isolation. 15 N longitudinal (T 1 ) and transverse relaxation (T 2 ) times as well as the proton-nitrogen nuclear Overhauser effects have been used to calculate the spectral density at three frequencies, 0, x N, and 0.87x H . Spectral densities at each residue indicate various time-scale motions of the main-chain. Even within the structured domain of PrP C , a diverse range of motions are observed. We find that removal of the tail increases T 2 relaxation times significantly indicating that the tail is responsible for shortening of T 2 times in full-length PrP C . The truncated fragment of PrP has facilitated the determination of meaningful order parameters (S 2 ) from the relaxation data and shows for the first time that all three helices in PrP C have similar rigidity. Slow conformational fluctuations of mouse PrP C are localized to a distinct region that involves residues 171 and 172. Interestingly, residues 170-175 have been identified as a segment within PrP that will form a steric zipper, believed to be the fundamental amyloid unit. The flexibility within these residues could facilitate the PrP C -PrP Sc recognition process during fibril elongation.

show abstract

“…Recently, a metastable state of the PrP C was characterized by using a high-pressure NMR (14), where hydrostatic pressure was elevated up to 2,500 bar in an on-line high-pressure NMR cell. The thermodynamical stability profile shows that diverse residues in helices B and C are less stable, indicating the formation of the intermediate conformation (PrP*) (14).…”

mentioning

confidence: 99%

Hot spots in prion protein for pathogenic conversion

Kuwata

Nishida

Matsumoto

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

174

225

View full text Add to dashboard Cite

Prion proteins are key molecules in transmissible spongiform encephalopathies (TSEs), but the precise mechanism of the conversion from the cellular form (PrP C ) to the scrapie form (PrP Sc ) is still unknown. Here we discovered a chemical chaperone to stabilize the PrP C conformation and identified the hot spots to stop the pathogenic conversion. We conducted in silico screening to find compounds that fitted into a ''pocket'' created by residues undergoing the conformational rearrangements between the native and the sparsely populated high-energy states (PrP*) and that directly bind to those residues. Forty-four selected compounds were tested in a TSE-infected cell culture model, among which one, 2-pyrrolidin-1-yl-N-[4-[4-(2-pyrrolidin-1-yl-acetylamino)-benzyl]-phenyl]-acetamide, termed GN8, efficiently reduced PrP Sc . Subsequently, administration of GN8 was found to prolong the survival of TSE-infected mice. Heteronuclear NMR and computer simulation showed that the specific binding sites are the A-S2 loop (N159) and the region from helix B (V189, T192, and K194) to B-C loop (E196), indicating that the intercalation of these distant regions (hot spots) hampers the pathogenic conversion process. Dynamics-based drug discovery strategy, demonstrated here focusing on the hot spots of PrP C , will open the way to the development of novel anti-prion drugs.anti-prion compound ͉ binding sites ͉ chemical chaperone ͉ dynamicsbased drug discovery ͉ transmissible spongiform encephalopathy

show abstract

Locally Disordered Conformer of the Hamster Prion Protein: A Crucial Intermediate to PrP^Sc?

Cited by 158 publications

References 30 publications

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

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

Dynamics of a truncated prion protein, PrP(113–231), from ¹⁵N NMR relaxation: Order parameters calculated and slow conformational fluctuations localized to a distinct region

Hot spots in prion protein for pathogenic conversion

Contact Info

Product

Resources

About

Locally Disordered Conformer of the Hamster Prion Protein: A Crucial Intermediate to PrPSc?

Cited by 158 publications

References 30 publications

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

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

Dynamics of a truncated prion protein, PrP(113–231), from 15N NMR relaxation: Order parameters calculated and slow conformational fluctuations localized to a distinct region

Hot spots in prion protein for pathogenic conversion

Contact Info

Product

Resources

About

Locally Disordered Conformer of the Hamster Prion Protein: A Crucial Intermediate to PrP^Sc?

Dynamics of a truncated prion protein, PrP(113–231), from ¹⁵N NMR relaxation: Order parameters calculated and slow conformational fluctuations localized to a distinct region