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Title: The sole tryptophan of amicyanin enhances its thermal stability but does not influence the electronic properties of the type 1 copper site

Authors:
; ; ; ; ; ;  [1];  [2];  [2]
  1. (Central Florida)
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1132101
Resource Type:
Journal Article
Resource Relation:
Journal Name: Arch. Biochem. Biophys.; Journal Volume: 550-551; Journal Issue: 05, 2014
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Dow, Brian A., Sukumar, Narayanasami, Matos, Jason O., Choi, Moonsung, Schulte, Alfons, Tatulian, Suren A., Davidson, Victor L., Cornell), and SNU). The sole tryptophan of amicyanin enhances its thermal stability but does not influence the electronic properties of the type 1 copper site. United States: N. p., 2014. Web. doi:10.1016/j.abb.2014.03.010.
Dow, Brian A., Sukumar, Narayanasami, Matos, Jason O., Choi, Moonsung, Schulte, Alfons, Tatulian, Suren A., Davidson, Victor L., Cornell), & SNU). The sole tryptophan of amicyanin enhances its thermal stability but does not influence the electronic properties of the type 1 copper site. United States. doi:10.1016/j.abb.2014.03.010.
Dow, Brian A., Sukumar, Narayanasami, Matos, Jason O., Choi, Moonsung, Schulte, Alfons, Tatulian, Suren A., Davidson, Victor L., Cornell), and SNU). Thu . "The sole tryptophan of amicyanin enhances its thermal stability but does not influence the electronic properties of the type 1 copper site". United States. doi:10.1016/j.abb.2014.03.010.
@article{osti_1132101,
title = {The sole tryptophan of amicyanin enhances its thermal stability but does not influence the electronic properties of the type 1 copper site},
author = {Dow, Brian A. and Sukumar, Narayanasami and Matos, Jason O. and Choi, Moonsung and Schulte, Alfons and Tatulian, Suren A. and Davidson, Victor L. and Cornell) and SNU)},
abstractNote = {},
doi = {10.1016/j.abb.2014.03.010},
journal = {Arch. Biochem. Biophys.},
number = 05, 2014,
volume = 550-551,
place = {United States},
year = {Thu Jul 10 00:00:00 EDT 2014},
month = {Thu Jul 10 00:00:00 EDT 2014}
}
  • The effects of replacing the axial methionine ligand of the type 1 copper site with leucine on the structure and function of amicyanin have been characterized. The crystal structures of the oxidized and reduced forms of the protein reveal that the copper site is now tricoordinate with no axial ligand, and that the copper coordination distances for the two ligands provided by histidines are significantly increased. Despite these structural changes, the absorption and EPR spectra of M98L amicyanin are only slightly altered and still consistent with that of a typical type 1 site. The oxidation-reduction midpoint potential (E{sub m}) valuemore » becomes 127 mV more positive as a consequence of the M98L mutation, most likely because of the increased hydrophobicity of the copper site. The most dramatic effect of the mutation was on the electron transfer (ET) reaction from reduced M98L amicyanin to cytochrome c{sub 551i} within the protein ET complex. The rate decreased 435-fold, which was much more than expected from the change in E{sub m}. Examination of the temperature dependence of the ET rate (k{sub ET}) revealed that the mutation caused a 13.6-fold decrease in the electronic coupling (H{sub AB}) for the reaction. A similar decrease was predicted from a comparative analysis of the crystal structures of reduced M98L and native amicyanins. The most direct route of ET for this reaction is through the Met98 ligand. Inspection of the structures suggests that the major determinant of the large decrease in the experimentally determined values of H{sub AB} and k{sub ET} is the increased distance from the copper to the protein within the type 1 site of M98L amicyanin.« less
  • The mutation of the axial ligand of the type I copper protein amicyanin from Met to Lys results in a protein that is spectroscopically invisible and redox inactive. M98K amicyanin acts as a competitive inhibitor in the reaction of native amicyanin with methylamine dehydrogenase indicating that the M98K mutation has not affected the affinity for its natural electron donor. The crystal structure of M98K amicyanin reveals that its overall structure is very similar to native amicyanin but that the type I binding site is occupied by zinc. Anomalous difference Fourier maps calculated using the data collected around the absorption edgesmore » of copper and zinc confirm the presence of Zn{sup 2+} at the type I site. The Lys98 NZ donates a hydrogen bond to a well-ordered water molecule at the type I site which enhances the ability of Lys98 to provide a ligand for Zn{sup 2+}. Attempts to reconstitute M98K apoamicyanin with copper resulted in precipitation of the protein. The fact that the M98K mutation generated such a selective zinc-binding protein was surprising as ligation of zinc by Lys is rare and this ligand set is unique for zinc.« less
  • Cu X-ray absorption edge features of 19 Cu(I) and 40 Cu(II) model complexes have been systematically studied and correlated with oxidation state and geometry. Studies of Cu(I) model complexes with different coordination number reveal that an 8983-8984-eV peak (assigned as the Cu 1s ..-->.. 4p transition) can be correlated in energy, shape, and intensity with ligation and site geometry of the cuprous ion. These Cu(I) edge features have been qualitatively interpreted with ligand field concepts. Alternatively, no Cu(II) complex exhibits a peak below 8985.0 eV. The limited intensity observed in the 8983-8985-eV region for some Cu(II) complexes is associated withmore » the tail of an absorption peak at approx. 8986 eV which is affected by the covalency of the equatorial ligands. These models studies allow accurate calibration of a normalized difference edge procedure which is used for the quantitative determination of Cu(I) content in copper complexes of mixed oxidation state composition. This normalized difference edge analysis is then used to quantitatively determine the oxidation states of the copper sites in type 2 copper-depleted (T2D) and native forms of the multicopper oxidase, Rhus vernicifera laccase. The type 3 site of the T2D laccase is found to be fully reduced and stable to oxidation by O/sub 2/ or by 25-fold protein equivalents of ferricyanide, but it can be oxidized by reaction with peroxide. The increase in intensity of the 330-nm absorption feature which results from peroxide titration of T2D laccase is found to correlate linearly with the percent of oxidation of the binuclear copper site.« less
  • Highlights: Black-Right-Pointing-Pointer IQGAP1 interacts with Aurora-A through its RGCt domain. Black-Right-Pointing-Pointer Overexpression of IQGAP1 prevents ubiquitination of Aurora-A. Black-Right-Pointing-Pointer Overexpression of IQGAP1 enhances the protein stability of Aurora-A. Black-Right-Pointing-Pointer Overexpression of IQGAP1 promotes the kinase activity of Aurora-A. -- Abstract: IQGAP1, a ubiquitously expressed scaffold protein, has been identified in a wide range of organisms. It participates in multiple aspects of cellular events by binding to and regulating numerous interacting proteins. In our present study, we identified a new IQGAP1 binding protein named Aurora-A which is an oncogenic protein and overexpressed in various types of human tumors. In vitro analysismore » with GST-Aurora-A fusion proteins showed a physical interaction between Aurora-A and IQGAP1. Moreover, the binding also occurred in HeLa cells as endogenous Aurora-A co-immunoprecipitated with IQGAP1 from the cell lysates. Overexpression of IQGAP1 resulted in an elevation of both expression and activity of Aurora-A kinase. Endogenous IQGAP1 knockdown by siRNA promoted Aurora-A degradation whereas IQGAP1 overexpression enhanced the stability of Aurora-A. Additionally, we documented that the IQGAP1-induced cell proliferation was suppressed by knocking down Aurora-A expression. Taken together, our results showed an unidentified relationship between Aurora-A and IQGAP1, and provided a new insight into the molecular mechanism by which IQGAP1 played a regulatory role in cancer.« less