JBIC Journal of Biological Inorganic Chemistry

, Volume 10, Issue 2, pp 167–180

Modulation of zinc- and cobalt-binding affinities through changes in the stability of the zinc ribbon protein L36

  • Wenpeng Kou
  • Harsha S. Kolla
  • Alfonso Ortiz-Acevedo
  • Donovan C. Haines
  • Matthew Junker
  • Gregg R. Dieckmann
Original Article

Abstract

Cysteine-rich Zn(II)-binding sites in proteins serve two distinct functions: to template or stabilize specific protein folds, and to facilitate chemical reactions such as alkyl transfers. We are interested how the protein environment controls metal site properties, specifically, how naturally occurring tetrahedral Zn(II) sites are affected by the surrounding protein. We have studied the Co(II)- and Zn(II)-binding of a series of derivatives of L36, a small zinc ribbon protein containing a (Cys)3His metal coordination site. UV–vis spectroscopy was used to monitor metal binding by peptides at pH 6.0. For all derivatives, the following trends were observed: (1) Zn(II) binds tighter than Co(II), with an average KAZn/KACo of 2.8(±2.0)×103; (2) mutation of the metal-binding ligand His32 to Cys decreases the affinity of L36 derivatives for both metals; (3) a Tyr24 to Trp mutation in the β-sheet hydrophobic cluster increases KAZn and KACo; (4) mutation in the β-hairpin turn, His20 to Asn generating an Asn-Gly turn, also increases KAZn and KACo; (5) the combination of His20 to Asn and Tyr24 to Trp mutations also increases KAZn and KACo, but the increments versus C3H are less than those of the single mutations. Furthermore, circular dichroism, size-exclusion chromatography, and 1D and 2D 1H NMR experiments show that the mutations do not change the overall fold or association state of the proteins. L36, displaying Co(II)- and Zn(II)-binding sensitivity to various sequence mutations without undergoing a change in protein structure, can therefore serve as a useful model system for future structure/reactivity studies.

Keywords

Zinc ribbon Metal binding constants Total correlation spectroscopy Nuclear Overhauser enhancement spectroscopy Thiolate-rich zinc site 

Abbreviations

CD

Circular dichroism

DI

Deionized

DTNB

5,5′-Dithiobis(2-nitrobenzoic acid)

ESI-MS

Electrospray ionization mass spectrometry

Fmoc

9-Fluorenylmethoxycarbonyl

HEPES

N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid

HPLC

High performance liquid chromatography

ICP-MS

Inductively coupled plasma mass spectrometry

LFSE

Ligand field stabilization energy

LMCT

Ligand-to-metal charge transfer

NOESY

Nuclear Overhauser enhancement spectroscopy

SEC

Size-exclusion chromatography

TCEP·HCl

Tris(2-carboxyethyl)phosphine hydrochloride

TFA

Trifluoroacetic acid

TOCSY

Total correlation spectroscopy

Supplementary material

775_2005_625_ESM_supp.pdf (2.8 mb)
(PDF 2.9 MB)

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Copyright information

© SBIC 2005

Authors and Affiliations

  • Wenpeng Kou
    • 1
  • Harsha S. Kolla
    • 1
  • Alfonso Ortiz-Acevedo
    • 1
  • Donovan C. Haines
    • 1
  • Matthew Junker
    • 2
  • Gregg R. Dieckmann
    • 1
  1. 1.Department of ChemistryThe University of Texas at DallasRichardsonUSA
  2. 2.Department of Molecular and Cell BiologyThe University of Texas at DallasRichardsonUSA

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