Local stability identification and the role of a key aromatic amino acid residue in staphylococcal nuclease refolding

Su, Zhengding; Wu, Jianyu; Fang, Huey-Jen; Tsong, Tian Yow; Chen, Hueih-Min

doi:10.1111/j.1742-4658.2005.04814.x

Cited by 7 publications

(6 citation statements)

References 27 publications

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“…Detection of Conformational Changes with Trp Fluorescence and CD Spectroscopy. Acid-base titrations monitored with three different types of spectroscopic signals were used to detect conformational reorganization coupled to the ionization of internal groups: (1) intrinsic fluorescence of Trp-140, which is known to be an excellent reporter of global unfolding of SNase (21,28,29); (2) far-UV CD at 222 nm, which reports primarily on the R-helical contents of the protein, with some contribution from β-sheets; and (3) near-UV CD at 275 nm, which reports primarily on the microenvironments of the aromatic residues, which are abundant in SNase.…”

Section: Resultsmentioning

confidence: 99%

Conformational Consequences of Ionization of Lys, Asp, and Glu Buried at Position 66 in Staphylococcal Nuclease

2010

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The pKa values measured previously for the internal Lys-66, Asp-66 and Glu-66 in variants of a highly stable form of staphylococcal nuclease are shifted by as many as 5 pKa units relative to normal pKa values in water. These shifts cannot be reproduced with continuum electrostatics calculations with static structures unless the protein is treated with high dielectric constants near 10. These high apparent dielectric constants are inconsistent with the highly hydrophobic microenvironments of the ionizable moieties in crystal structures. To examine the origins of these high apparent dielectric constants we showed that the pKa of these internal residues are sensitive to the global stability of the protein; the shifts tend to be smaller in less stable forms of nuclease. This implies that the apparent dielectric constants reported by these internal ionizable groups are high because they reflect conformational reorganization coupled to their ionization. To detect this directly, acid/base titrations monitored with Trp fluorescence, near-UV and far-UV CD spectroscopy were performed on variants with Lys-66, Glu-66 or Asp-66 in background proteins with different stability. Conformational reorganization coupled to the ionization of the internal groups was spectroscopically detectable, especially in the less stable background proteins. The data show that to improve the accuracy of structure-based pKa calculations of internal groups the calculations will have to treat explicitly all structural reorganization coupled to ionization. The data also suggest a novel approach to mapping the folding free energy landscape of proteins by using internal ionizable groups to stabilize partially unfolded states.

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Section: Resultsmentioning

confidence: 99%

Conformational Consequences of Ionization of Lys, Asp, and Glu Buried at Position 66 in Staphylococcal Nuclease

2010

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“…NT Hi Nuc was most stable around pH 9 with a reported T onset of 53 °C. Comparatively, the T onset of staphylococcal thermonuclease is 45.8 °C at pH 7 (calculated from published data; [ 24 ]). At pH 7, excess pdTp increased NT Hi Nuc thermal stability, reported as a T onset , from 40 °C to 48 °C ( Table 3 ).…”

Section: Resultsmentioning

confidence: 99%

Characterization of a nontypeable Haemophilus influenzae thermonuclease

et al. 2018

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Nontypeable Haemophilus influenzae (NTHi) has been shown to form biofilms, comprised of extracellular DNA (eDNA), in the middle ear and bronchus during clinical infections. Studies in our laboratory have shown that NTHi possesses a homolog of Staphylococcus aureus thermonuclease (staphylococcal thermonuclease), NTHi nuclease (NTHi Nuc, HI_1296). This enzyme had similar size, heat stability, and divalent cation requirements to those of the staphylococcal homolog as determined by light scattering and circular dichroism spectroscopy. Small angle X-ray scattering (SAXS) analysis suggested an overall shape and substrate-binding site comparable to those of staphylococcal nuclease. However, NTHi Nuc was approximately 25-fold more active in fluorescence resonance energy transfer (FRET) activity assay than staphylococcal thermonuclease. Homology modeling implicates shorter NTHi Nuc loops near the active site for this enhanced activity.

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“…8,9 These weak forces supporting protein functions can be easily eliminated by changing the pH, temperature, or ionic strength. To overcome these problems, therefore, we present the requirements for fabricating efficient protein chips; the first requirement is to ensure that the protein functions are effectively performed with the 3D conformation (folded state) when the protein is either in solution 10,11 or on a chip. 12 For proteins immobilised on a chip, this is essential; otherwise, the protein chip could be rendered useless.…”

Section: Introductionmentioning

confidence: 99%

Efficiency optimisation of proteins on a chip

Huang

Hsu

et al. 2015

Lab Chip

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This study elucidates that the protein reorientation on a chip can be changed by an external electric field (EEF) and optimised for achieving strong effective binding between proteins. Protein A and its binding protein immunoglobulin G (IgG) were used as an example, in addition to an anticancer peptide (CB1a) and its antibody (anti-CB1a). The binding forces (BFs) were measured by atomic force microscopy (AFM) with EEFs applied at different angles (EEF°). The optimal angle (OA) of the EEF (OAEEF°) corresponding to the maximum binding force (BFmax) was obtained. The results showed that the BFmax values between IgG/Protein A and anti-CB1a/CB1a were 6424.2 ± 195.3 pN (OAEEF° = 45°) and 729.1 ± 33.2 pN (OAEEF° = 22.5°), respectively. Without an EEF, the BF values were only 730.0 ± 113.9 pN and 337.3 ± 35.0 pN, respectively. Based on these observations, we concluded that the efficient optimisation of protein-protein interaction on a chip is essential. This finding is applicable to the industrial fabrication of all protein chips.

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Local stability identification and the role of a key aromatic amino acid residue in staphylococcal nuclease refolding

Cited by 7 publications

References 27 publications

Conformational Consequences of Ionization of Lys, Asp, and Glu Buried at Position 66 in Staphylococcal Nuclease

Conformational Consequences of Ionization of Lys, Asp, and Glu Buried at Position 66 in Staphylococcal Nuclease

Characterization of a nontypeable Haemophilus influenzae thermonuclease

Efficiency optimisation of proteins on a chip

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