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

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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.

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Abbreviations

AEDANS:

acetyl-N′-(8-sulpho-l-naphthyl) ethylene-diamine

CD:

circular dichroism

DTNB:

5,5′-dithiobis(2-nitrobenzoic acid)

ε :

molar extinction coefficient

Δε :

molar extinction difference

eIF-5A:

eukaryotic initiation factor 5A, namely the hypusine-containing protein

eIF-5A precursor [or ec-eIF-5A(Lys)]:

eukaryotic initiation factor 5A precursor, i.e., the unmodified precursor form of eIF-5A produced inEscherichia coli by expression of human eIF-5AcDNA containing Lys in position 50

GdnHCI:

guanidinium chloride

I-AEDANS:

N-iodo-AEDANS

N-AcCys-AEDANS:

N-acetylcysteine-AEDANS, Mr, relative molecular mass

ODU:

optical density unit

RMS:

root mean square

TrisHCl:

Tris (hydroxymethyl)amino-methane hydrochloride

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Second University of Naples, Via Costantinopoli 16, I-80138, Naples, Italy

    P. Stiuso, G. Colonna, R. Ragone, M. Caraglia &  Alberto Abbruzzese

  2. Department of Biological Chemistry, School of Medicine, University of California, Davis, USA

    J. W. B. Hershey

  3. Department of Biology, Second University of Rome, Italy

    S. Beninati

  4. Research Center of Computational and Biotechnological Sciences (CRISCEB), Second University of Naples, Italy

    G. Colonna

Authors
  1. P. Stiuso
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  2. G. Colonna
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  3. R. Ragone
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  4. M. Caraglia
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  5. J. W. B. Hershey
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  6. S. Beninati
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  7. Alberto Abbruzzese
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Stiuso, P., Colonna, G., Ragone, R. et al. Structural organization of the human eukaryotic initiation factor 5A precursor and its site-directed variant Lys50 → Arg. Amino Acids 16, 91–106 (1999). https://doi.org/10.1007/BF01318888

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  • DOI: https://doi.org/10.1007/BF01318888

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