29 research outputs found
The threat of instability: neurodegeneration predicted by protein destabilization and aggregation propensity.
No full textMisfolding and Aggregation of SOD1
No full text<p>Wang et al.'s correlation of mutant SOD1 properties with disease duration [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060193#pbio-0060193-b006" target="_blank">6</a>] implicates two steps in the formation of aggregates of SOD1 in ALS: (1) native homodimeric metalloprotein, shown in ribbon representation (left) partially or completely unfolds to form a range of possible dimeric or monomeric aggregation-prone species, shown as a general grey irregular shape (middle), followed by (2) progressive assembly of the aggregation-prone species to form initially small soluble and later fibrillar aggregates (right panels). The structure of native SOD1 was generated using MolMol [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060193#pbio-0060193-b029" target="_blank">29</a>] and Protein Data Bank accession code 1n18. Sites of ALS-associated mutations studied by Wang et al. are shown in red; bound copper and zinc ions are shown as blue and black spheres, respectively. The right panels are transmission electron microscopy images of apo SOD1 aggregates formed in vitro that strongly resemble granular and granule-coated fibrillar SOD1 aggregates observed in ALS [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0060193#pbio-0060193-b019" target="_blank">19</a>].</p
Two-dimensional <sup>1</sup>H and <sup>15</sup>N NMR titration studies of hisactophilin
No full text We have used two-dimensional 1H-15N heteronuclear single quantum correlation spectroscopy to measure the pH dependence of backbone amide group chemical shifts in the actin binding protein hisactophilin over the pH range 5.7-11.1. Most of the resonances can be analyzed using a simple equation involving a single apparent ionization constant, pKapp. The majority of resonances in the protein titrate with pKapp values of 5.6-7.4. The results can be rationalized in terms of titration of many histidine residues in hisactophilin. The titration data provide direct experimental support for the proposed models of the atomic basis of actin and membrane binding by hisactophilin.Key words: hisactophilin, histidine, ionization constants, nuclear magnetic resonance, NMR. </jats:p
Spectrophotometric method for simultaneous measurement of zinc and copper in metalloproteins using 4-(2-pyridylazo)resorcinol
No full textThe final publication is available at Elsevier via https://doi.org/10.1016/j.ab.2019.03.007. © 2019.
This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/Bound metals are observed in a great many natural proteins, where they perform diverse roles in determining protein folding, stability and function. Due to the broad impact of bound metals on biophysical and biochemical properties of proteins, it is valuable to have accurate and facile methods for determining the metal content of proteins. Here we describe an optimized methodology using 4-(2-pyridylazo)resorcinol (PAR) to simultaneously quantify two metal ions in solution. The assay is demonstrated for quantification of Cu2+ and Zn2+ ions in human Cu, Zn superoxide dismutases (SOD1s); however, the method is general and can be applied to various combinations of metal ions. Advantages of the assay are that it is rapid and inexpensive, requires little sample and preparation, and has simple data analysis. We show that spectral decomposition software can accurately resolve the absorption bands of Cu2+ and Zn2+ with high accuracy and precision. Using the PAR assay, we determined that metal binding is altered in disease-associated mutants of SOD1, with comparable results to those determined by ICP-AES. In addition, we highlight key issues for using spectrophotometric chelators such as PAR for metal analysis of proteins.This work was supported by the Canadian Institutes of Health Research (CIHR) and National Sciences and Engineering Research Council of Canada (NSERC)
pH and Urea Dependence of Amide Hydrogen−Deuterium Exchange Rates in the β-Trefoil Protein Hisactophilin<sup>†</sup>
No full textAmide hydrogen/deuterium exchange rates were measured as a function of pH and urea for
37 slowly exchanging amides in the β-trefoil protein hisactophilin. The rank order of exchange rates is
generally maintained under different solution conditions, and trends in the pH and urea dependence of
exchange rates are correlated with the rank order of exchange rates. The observed trends are consistent
with the expected behavior for exchange of different amides via global and/or local unfolding. Analysis
of the pH dependence of exchange in terms of rate constants for structural opening and closing reveals
a wide range of rates in different parts of the hisactophilin structure. The slowest exchanging amides
have the slowest opening and closing rates. Many of the slowest exchanging amides are located in trefoil
2, but there are also some slow exchanging amides in trefoils 1 and 3. Slow exchangers tend to be near
the interface between the β-barrel and the β-hairpin triplet portions of this single-domain structure. The
pattern of exchange behaviour in hisactophilin is similar to that observed previously in interleukin-1β,
indicating that exchange properties may be conserved among β-trefoil proteins. Comparisons of opening
and closing rates in hisactophilin with rates obtained for other proteins reveal clear trends for opening
rates; however, trends in closing rates are less apparent, perhaps due to inaccuracies in the values used
for intrinsic exchange rates in the data fitting. On the basis of the pH and urea dependence of exchange
rates and optical measurements of stability and folding, EX2 is the main exchange mechanism in
hisactophilin, but there is also evidence for varying levels of EX1 exchange at low and high pH and high
urea concentrations. Equilibrium intermediates in which subglobal portions of structure are cooperatively
disrupted are not apparent from analysis of the urea dependence of exchange rates. There is, however, a
strong correlation between the Gibbs free energy of opening and the denaturant dependence of opening
for all amides, which suggests exchange from a continuum of states with different levels of structure.
Intermediates are not very prominent either in equilibrium exchange experiments or in quenched-flow
kinetic studies; hence, hisactophilin may not form partially folded states as readily as IL-1β and other
β-trefoil proteins
Energetics and mechanisms of folding and flipping the myristoyl switch in the β-trefoil protein, hisactophilin
No full text
Myristoylation, the covalent linkage of a saturated, C
14
fatty acyl chain to the N-terminal glycine in a protein, plays a vital role in reversible membrane binding and signaling by the modified proteins. Currently, little is known about the effects of myristoylation on protein folding and stability, or about the energetics and molecular mechanisms of switching involving states with sequestered versus accessible myristoyl group. Our analysis of these effects in hisactophilin, a histidine-rich protein that binds cell membranes and actin in a pH-dependent manner, shows that myristoylation significantly increases hisactophilin stability, while also markedly increasing global protein folding and unfolding rates. The switching between sequestered and accessible states is pH dependent, with an apparent pK
switch
of 6.95, and an apparent free energy change of 2.0 kcal·mol
-1
. The myristoyl switch is linked to the reversible uptake of ∼1.5 protons, likely by histidine residues. This pH dependence of switching appears to be the physical basis of the sensitive, pH-dependent regulation of membrane binding observed in vivo. We conclude that an increase in protein stability upon modification and burial of the attached group is likely to occur in numerous proteins modified with fatty acyl or other hydrophobic groups, and that the biophysical effects of such modification are likely to play an important role in their functional switches. In addition, the increased global dynamics caused by myristoylation of hisactophilin reveals a general mechanism whereby hydrophobic moieties can make nonnative interactions or relieve strain in transition states, thereby increasing the rates of interconversion between different states.
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Wild‐type and mutant SOD1 localizes to RNA‐rich structures in cells and mice but does not bind RNA
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