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. Stiuso
  • G. Colonna
  • R. Ragone
  • M. Caraglia
  • J. W. B. Hershey
  • S. Beninati
  • Alberto Abbruzzese
Full Papers

DOI: 10.1007/BF01318888

Cite this article as:
Stiuso, P., Colonna, G., Ragone, R. et al. Amino Acids (1999) 16: 91. doi:10.1007/BF01318888

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 acidseIF-5AHypusineProtein foldingPosttranslational modification

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

Copyright information

© Springer-Verlag 1999

Authors and Affiliations

  • P. Stiuso
    • 1
  • G. Colonna
    • 1
    • 4
  • R. Ragone
    • 1
  • M. Caraglia
    • 1
  • J. W. B. Hershey
    • 2
  • S. Beninati
    • 3
  • Alberto Abbruzzese
    • 1
  1. 1.Department of Biochemistry and BiophysicsSecond University of NaplesNaplesItaly
  2. 2.Department of Biological Chemistry, School of MedicineUniversity of CaliforniaDavisUSA
  3. 3.Department of BiologySecond University of RomeItaly
  4. 4.Research Center of Computational and Biotechnological Sciences (CRISCEB)Second University of NaplesItaly