Matters of State

Kay, W. David

doi:10.1007/978-1-349-23778-4_5

Cited by 2 publications

(4 citation statements)

References 7 publications

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“…Assignments for CspA in 90% DMSO/10% H2O were obtained from a 3D HNCACB spectrum 26 recorded at a temperature of 25 o C. The HNCACB experiment has proven extremely useful for assigning NMR spectra of denatured proteins, including the acid-and urea-denatured forms of CspA. 19 In the case of DMSO-denatured CspA, assignments could be established for 64 of the 67 backbone 1 H- 15 N correlations (96%) expected from the amino acid sequence of the protein (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In order to obtain residue-specific information on hydrogen exchange in CspA fibrils, 1 H and 15 N NMR resonances of denatured CspA in 90% DMSO were assigned using a 3D-HNCACB experiment. 26 The 3D-HNCACB experiment was recorded at 25 o C on a virgin 0.6 mM 13 C/ 15 N-labeled CspA sample dissolved in 90% d6-DMSO/10% H2O, 20 mM DCA, pH* 4.5. This buffer corresponds to the protonated analogue of the DMSO/D2O read-out buffer used to monitor hydrogen exchange in CspA fibrils.…”

Section: Methodsmentioning

confidence: 99%

“…The 1 H-15 N HSQC spectra employed pulse field gradients for coherence selection, and suppression of signals from the solvent. 26 Acquisition of each of the six 1 H-15 N HSQC spectra was completed in times randomly distributed between 26 and 31 min from the time of dissolving the exchange quenched samples in the 90% DMSO/10% D2O read-out buffer. In order to interfere with measurements of amide proton intensities, exchange in the 90% DMSO/10% D2O read-out buffer would have to be significant on time scales comparable to the <5 min differences in the recording of the HSQC spectra.…”

Section: Methodsmentioning

confidence: 99%

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An NMR-Based Quenched Hydrogen Exchange Investigation of Model Amyloid Fibrils Formed by Cold Shock Protein A

Alexandrescu

2000

Biocomputing 2001

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Acid-denatured cold shock protein A (CspA) self-assembles into polymers with properties typical of amyloid fibrils. In the present work, a quenched hydrogen exchange experiment was used to probe the interactions of CspA fibrils with solvent. Exchange was initiated by incubating suspensions of fibrils in D2O, and quenched by flash freezing. Following lyophilization, exchange-quenched samples were dissolved in 90% DMSO/10% D2O, giving DMSO-denatured monomers. Intrinsic exchange rates for denatured CspA in 90% DMSO/10% D2O (pH* 4.5) were sufficiently slow (~1 x 10-3 min-1) to enable quantification of NMR signal intensity decays due to H/D exchange in the fibrils. Hydrogen exchange rate constants for CspA fibrils were found to vary less than 3-fold from a mean value of 5 x 10-5 min-1. The uniformity of rate constants suggests that exchange is in the EX1 limit, and that the mechanism of exchange involves a cooperative dissociation of CspA monomers from fibrils, concomitant with unfolding. Previous studies indicated that the highest protection factors in native CspA are ~10 3 , and that protection factors for the acid-denatured monomer precursors of CspA fibrils are close to unity. Because exchange in is in the EX1 regime, it is only possible to place a lower limit of at least 10 5 on protection factors in CspA fibrils. The observation that all amide protons are protected from exchange indicates that the entire CspA polypeptide chain is structured in the fibrils.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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An NMR-Based Quenched Hydrogen Exchange Investigation of Model Amyloid Fibrils Formed by Cold Shock Protein A

Alexandrescu

2000

Biocomputing 2001

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“…The assignment strategy used is based on identifying an NH intraresidual C a , d\ C°, H a and HP signals and then comparing to another NH sequential C a , d 5 , C°, H a and HP [20][21][22]. When the intraresidual and sequential signals align, then the NHs are defined as sequential.…”

Section: Resultsmentioning

confidence: 99%

Leptin is a four‐helix bundle: secondary structure by NMR

et al. 1997

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Leptin is a signaling protein that in its mutant forms has been associated with obesity and Type II diabetes. The lack of sequence similarity has precluded analogies based on structural resemblance to known systems. Backbone NMR signals for mouse leptin ( 13 C/ 1B N -labeled) have been assigned and its secondary structure reveals it to be a four-helix bundle cytokine. Helix lengths and disulfide pattern are in agreement with leptin as a member of the short-helix cytokine family. A three-dimensional model was built verifying the mechanical consistency of the identified elements with a short-helix cytokine core.

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Matters of State

Cited by 2 publications

References 7 publications

An NMR-Based Quenched Hydrogen Exchange Investigation of Model Amyloid Fibrils Formed by Cold Shock Protein A

An NMR-Based Quenched Hydrogen Exchange Investigation of Model Amyloid Fibrils Formed by Cold Shock Protein A

Leptin is a four‐helix bundle: secondary structure by NMR

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