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Amino Acids

, Volume 16, Issue 1, pp 91-106

Structural organization of the human eukaryotic initiation factor 5A precursor and its site-directed variant Lys50 → Arg

  • P. StiusoAffiliated withDepartment of Biochemistry and Biophysics, Second University of Naples
  • , G. ColonnaAffiliated withDepartment of Biochemistry and Biophysics, Second University of NaplesResearch Center of Computational and Biotechnological Sciences (CRISCEB), Second University of Naples
  • , R. RagoneAffiliated withDepartment of Biochemistry and Biophysics, Second University of Naples
  • , M. CaragliaAffiliated withDepartment of Biochemistry and Biophysics, Second University of Naples
  • , J. W. B. HersheyAffiliated withDepartment of Biological Chemistry, School of Medicine, University of California
  • , S. BeninatiAffiliated withDepartment of Biology, Second University of Rome
  • , Alberto AbbruzzeseAffiliated withDepartment of Biochemistry and Biophysics, Second University of Naples

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Summary

The molecular properties of the human eukaryotic initiation factor 5A precursor and its site directed Lys50 → Arg variant have been investigated and compared. Structure perturbation methods were used to gain information about the protein architecture in solution. Intrinsic and extrinsic spectroscopic probes strategically located in the protein matrix detected the independent unfolding of two molecular regions. Three cystemes out of four were titrated in the native protein and the peculiar presence of a tyrosinate band at neutral pH was detected. At alkaline pH only two tyrosines out of three were titratable in the native protein, with an apparent pK of about 9.9. Native protein and its Lys50 → Arg variant reacted in a similar fashion to guanidine and to pH variation, but differently to thermal stress. The complex thermal unfolding of both proteins indicated the presence of intermediates. Spectroscopic data showed that these intermediates are differently structured. Consequently, the two proteins seem to have different unfolding pathways.

Keywords

Amino acids eIF-5A Hypusine Protein folding Posttranslational modification