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

Yao, Jian; Dyson, H. Jane; Wright, Peter E.

doi:10.1016/s0014-5793(97)01474-9

Cited by 161 publications

(136 citation statements)

References 31 publications

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“…5(B)] looked much different than the control spectra and the signal resembled that of an unfolded protein with poorly dispersed peaks. 20 In sum, these experiments suggest that CA-GFP does not fold into a stable b-barrel conformation and is more similar to GFP 1-10 than to the mature GFP.…”

Section: B-barrel Of Ca-gfp Not Fully Formedmentioning

confidence: 71%

Structural basis of fluorescence quenching in caspase activatable‐GFP

Nicholls

Hardy

2013

Protein Science

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Apoptosis is critical for organismal homeostasis and a wide variety of diseases. Caspases are the ultimate executors of the apoptotic programmed cell death pathway. As caspases play such a central role in apoptosis, there is significant demand for technologies to monitor caspase function. We recently developed a caspase activatable-GFP (CA-GFP) reporter. CA-GFP is unique due to its ''dark'' state, where chromophore maturation of the GFP is inhibited by the presence of a C-terminal peptide. Here we show that chromophore maturation is prevented because CA-GFP does not fold into the robust b-barrel of GFP until the peptide has been cleaved by active caspase. Both CA-GFP and GFP 1-10 , a split form of GFP lacking the 11th strand, have similar secondary structure, different from mature GFP. A similar susceptibility to proteolytic digestion indicates that this shared structure is not the robust, fully formed GFP b-barrel. We have developed a model that suggests that as CA-GFP is translated in vivo it follows the same folding path as wild-type GFP; however, the presence of the appended peptide does not allow CA-GFP to form the barrel of the fully matured GFP. CA-GFP is therefore held in a ''pro-folding'' intermediate state until the peptide is released, allowing it to continue folding into the mature barrel geometry. This new understanding of the structural basis of the dark state of the CA-GFP reporter will enable manipulation of this mechanism in the development of reporter systems for any number of cellular processes involving proteases and potentially other enzymes.

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Section: B-barrel Of Ca-gfp Not Fully Formedmentioning

confidence: 71%

Structural basis of fluorescence quenching in caspase activatable‐GFP

Nicholls

Hardy

2013

Protein Science

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“…Consequently, to mini-mize the change in sample pH over the range of urea concentrations monitored, the titration was carried out in three separate titrations, with C ␣ and CЈ chemical shifts were determined at the following urea concentrations: 0. 61 N-labeled ␤-PrP in the same buffer described above were titrated in an analogous manner to the 13 CD Spectroscopy-Far-UV CD spectra were acquired on a JASCO J-715 spectropolarimeter using 0.1-mm path length cuvettes and typically 10 accumulations, with a bandwidth of 1 nm and integration 1 s Ϫ1 . CD spectra were acquired both before and after the ultracentrifugation, and at 50 M protein concentration in various concentrations of urea, as described below.…”

Section: Methodsmentioning

confidence: 99%

Conformational Properties of β-PrP

Hosszu

Trevitt

Jones

et al. 2009

Journal of Biological Chemistry

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Prion propagation involves a conformational transition of the cellular form of prion protein (PrP C ) to a disease-specific isomer (PrP Sc ), shifting from a predominantly ␣-helical conformation to one dominated by ␤-sheet structure. This conformational transition is of critical importance in understanding the molecular basis for prion disease. Here, we elucidate the conformational properties of a disulfide-reduced fragment of human PrP spanning residues 91-231 under acidic conditions, using a combination of heteronuclear NMR, analytical ultracentrifugation, and circular dichroism. We find that this form of the protein, which similarly to PrP Sc , is a potent inhibitor of the 26 S proteasome, assembles into soluble oligomers that have significant ␤-sheet content. The monomeric precursor to these oligomers exhibits many of the characteristics of a molten globule intermediate with some helical character in regions that form helices I and III in the PrP C conformation, whereas helix II exhibits little evidence for adopting a helical conformation, suggesting that this region is a likely source of interaction within the initial phases of the transformation to a ␤-rich conformation. This precursor state is almost as compact as the folded PrP C structure and, as it assembles, only residues 126 -227 are immobilized within the oligomeric structure, leaving the remainder in a mobile, random-coil state.

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“…1) that allows the use of a number of spectroscopic techniques to monitor its folding and unfolding both at equilibrium and in real time (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Optimally, to fully characterize the folding reaction of cyt c, NMR spectroscopy would be used to perform a more detailed analysis of denatured cyt c. The poor chemical shift dispersion shown in NMR spectra of denatured proteins, however, makes characterization of such states difficult (16). Isotope exchange (17, 18) and nuclear relaxation (19) studies have provided valuable information on structural fluctuations under partially denaturing conditions, but do not allow direct observation of structural contacts in unfolded states.…”

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confidence: 99%