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Thermal-induced dissociation and unfolding of homodimeric DsbC revealed by temperature-jump time-resolved infrared spectra.

ABSTRACT: The thermal stability of DsbC, a homodimeric protein disulfide isomerase in prokaryotic periplasm, has been studied by using temperature-dependent Fourier transformation infrared and time-resolved infrared spectroscopy coupled with temperature-jump initiation. The infrared absorbance thermal titration curves for thermal-induced unfolding of DsbC in D(2)O exhibit a three-state transition with the first transition midpoint temperature at 37.1 +/- 1.1 degrees C corresponding to dissociation, and the second at >74.5 degrees C corresponding to global unfolding and aggregation. The dissociation midpoint temperature of DsbC in phosphate buffer shifts to 49.2 +/- 0.7 degrees C. Temperature-jump time-resolved infrared spectra in D(2)O shows that DsbC dissociates into the corresponding germinate monomeric encounter pair with a time constant of 40 +/- 10 ns independent of the protein concentration and 77% of the newly formed monomeric encounter pair undergoes further coil to helix/loop transition with a time constant of 160 +/- 10 ns. The encounter pair is expected to proceed with further dissociation into monomers. The dissociation of DsbC is confirmed by size-exclusion chromatography and subunit hybridization. The in vivo oxidase activity of DsbC attributed to the monomer has also been observed by using cadmium sensitivity and the oxidative state of beta-lactamase.

SUBMITTER: Li H

PROVIDER: S-EPMC2776246 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Thermal-induced dissociation and unfolding of homodimeric DsbC revealed by temperature-jump time-resolved infrared spectra.

Li Heng H Ke Huimin H Ren Guoping G Qiu Xianggang X Weng Yu-Xiang YX Wang Chih-Chen CC

Biophysical journal 20091101 10

The thermal stability of DsbC, a homodimeric protein disulfide isomerase in prokaryotic periplasm, has been studied by using temperature-dependent Fourier transformation infrared and time-resolved infrared spectroscopy coupled with temperature-jump initiation. The infrared absorbance thermal titration curves for thermal-induced unfolding of DsbC in D(2)O exhibit a three-state transition with the first transition midpoint temperature at 37.1 +/- 1.1 degrees C corresponding to dissociation, and th ...[more]

PMID: 19917236

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Project description:Infrared photodissociation analyses are supported by theoretical calculations that allow a trustworthy interpretation of experimental spectra of gaseous ions. B3LYP calculations are the most prominent method used to model IR spectra, as detailed in our bibliographic survey. However, this and other commonly used methods are known to provide inaccurate energy values and geometries, especially when it comes to long-range interactions, such as intramolecular H-bonds, which show increased anharmonicity. Therefore, we evaluated some of the most commonly used density functional theory methods (B3LYP, CAM-B3LYP, and M06-2X) and basis sets (6-31+G(d,p), 6-311++G(d,p), 6-311++G(3df,2pd), aug-cc-pVDZ, and aug-cc-pVTZ), including anharmonicity and dispersion corrections. The results were compared to MP2 calculations and to experimental high-frequency (2000-4000 cm-1) IR multiphotonic dissociation (IRMPD) spectra of two protonated model molecules containing intramolecular hydrogen bonds: biotin and tryptophan. M06-2X/6-31+G(d,p) was shown to be the most cost-effective level of theory, whereas CAM-B3LYP was the most efficient method to describe the van der Waals interactions. The use of the dispersion correction D3, proposed by Grimme, improved the description of O-H vibrations involved in H-bonding but worsened the description of N-H stretches. Anharmonic calculations were shown to be extremely expensive when compared to other approaches. The efficiencies of well-established scaling factors (SFs) in opposition to sample-dependent SFs were also discussed and the use of fitted SFs were shown to be the most cost-effective approach to predict IRMPD spectra. M06-2X/6-31+G(d,p) and CAM-B3LYP/aug-cc-pVDZ were also tested against the fingerprint region. Our results suggest that these methods can also be used for analysis in this lower frequency range and should be regarded as the methods of choice for cost-effective IRMPD simulations rather than the ubiquitous B3LYP method, especially when further molecular properties are needed.

Dataset Information

Thermal-induced dissociation and unfolding of homodimeric DsbC revealed by temperature-jump time-resolved infrared spectra.

Publications

Thermal-induced dissociation and unfolding of homodimeric DsbC revealed by temperature-jump time-resolved infrared spectra.

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