Characterization of physical properties of two isoforms of translation elongation factor 1A

Two tissue-specific isoforms of mammalian translation elongation factor 1A, eEF1A1 and eEF1A2, are 98% similar. However, some of their functions in the organism are different which in some cases could lead to the induction of carcinogenesis. We supposed that slight difference of primary sequences may cause significant differences of spatial structures of eEF1A isoforms affecting, in its turn, the ability of one or another isoform to interact with protein partners. The differential scanning microcalorimetry and circular dichroism in “near” and “far” UV regions were used to determine that potentially oncogenic eEF1A2 isoform possesses a more compact spatial organization than eEF1A1, and the differences are revealed at the levels of both secondary and tertiary structure of proteins.

Introduction.eEF1A is believed to be a multifunctional protein, the canonical functions of which are to promote efficient binding of aminoacyl-tRNA to 80S ribosome and to provide correct codon-anticodon interaction in ribosomal A site [1].Two tissue-specific isoforms of eEF1A have been identified, namely, eEF1A2, expressed only in neural and muscular tissues, and eEF1A1, discovered in all the other tissues [2,3].The reason of two isoforms presence is not yet understood.The appearance of eEF1A2 in non-specific tissue has been found to relate to the induction of carcinogenesis [4][5][6][7][8].It remains unknown in what way eEF1A2, 98% similar to eEF1A1, which is specific for this kind of tissue, may be related to the formation of tumours in the latter.We supposed that even slight aminoacid substitutions in eEF1A2 are capable of causing some divergence in spatial structures of this protein globule, which may result in the modification of its known functions and appearance of some new ones.To check our assumption we have performed the comparative analysis of molecular dynamics of both isoforms, and found out the possibility of differences in the secondary structure and dynamics of these protein globules [9,10].Current work presents experimental data on the comparison of eEF1A1 and eEF1A2 using differential scanning microcalorimetry and circular dichroism (CD) in "near" and "far" UV regions.
Materials and Methods.Isolation and characterization of eEF1A.eEF1A1 was isolated from rabbit liver using the combination of ion-exchange chromatography and gel-filtration, as described in [11], with slight modifications.
eEF1A2 was isolated from rabbit muscles using the same scheme, but for the first stage of purificationchromatography on Sephacril S-400, which did not effect the preparation purity.
The activity of eEF1A was determined in the reaction of GDP/[ 3 H]GDP exchange [12].Differential scanning microcalorimetry.Calorimetric measurements were conducted in precision scanning microcalorimeter SCAL-1 (SCAL Co. Ltd., Pushchino, Russia) in glass cells (0.3 ml) with the rate of 1.0 Ê per 1 min under excessive pressure of two atmospheres [13].The dialysis of all protein samples against corresponding buffer was conducted before measurements.Concentrations of proteins used were in the range of 2.3-2.5 mg/ml.Thermodynamic analysis of the profile of the excess of heat capacity was carried out according to [14].
CD in "far" and "near" UV regions.The secondary structure of proteins was investigated by CD spectroscopy using spectropolarimeter JASCO-600 (Japan) at wavelengths of 190-250 nm ("far" UV range) and 250-310 nm ("near" UV range).Molar ellipticity was calculated using the following equation: where Ñ -concentration of protein (mg/ml), Loptical path length of cuvette (mm), [q] exp -measured ellipticity (degrees) and Ì res -mean molecular mass of peptide residue (Da), calculated from its aminoacid sequence.Measurement of "far" UV was carried out in 0.1 mm cuvette, and measurement of near CD was conducted in 1 mm cuvette.Concentration of proteins was 1 mg/ml.
Results and Discussion.Differential scanning microcalorimetry.
Differential scanning microcalorimetry allows obtaining and comparing thermodynamic parameters, characteristic for heat-induced conformational changes of proteins [14,15].
Computer analysis of molecular dynamics of eEF1A isoforms testified to the possibility of conformational changes in eEF1A1 molecule due to interdomain interactions, while eEF1A2 is specific for a more closed conformation [9,10].Therefore, one can expect the comparison of the thermodynamic parameters of the two isoforms of eEF1A to be informative to reveal the difference in thermal stability and enthalpy of denaturation.Since the stability of protein molecule is associated with its structure, the analysis of thermodynamic characteristics of the isoforms may evidence either to structural similarity or difference in their structures, and characterize relative compactness of these proteins.
Tem per a ture dependences of the ex ces sive par tial heat ca pac ity are pre sented in Fig. 1.Al most two-fold dif fer ence of de na tur ation heat of two isoforms is observed.For eEF1A1 DH to tal is 580.0Kj\mol, while for eEF1A2 DH to tal is 910.0Kj/mol.Com par i son of the melt ing curves also shows that melt ing of the eEF1A1 mol e cule starts ear lier than that of eEF1A2.The maxima of tran si tion are 55.5°C for eEF1A1 and 62.7°C for eEF1A2.Half-width tem per a ture of the tran si tion (DT) equals 11.0°Ñ for eEF1A1, and 8.1°Ñ for eEF1A2.Thus, eEF1A isoforms pos sess dif fer ent spa tial structure.Sig nif i cant in crease in enthalpy of de na tur ation and de crease in half-width tran si tion at eEF1A2 melting tes tify to a more com pact con for ma tion of eEF1A2, which has been ear lier as sumed us ing the data of molec u lar dy nam ics [9,10].
"Near" UV CD.CD spectrum in near UV region (250-350 nm), presenting signals of such chromophores as aromatic amino acids and disulphide bonds, may provide useful information about the tertiary structure of a protein.
Signals in 250-270 nm region are specific for phenylalanine residues, 270-290 nm region -for tyrosine, and the ones in 280-300 nm region are inherent to tryptophan.Disulphide bonds provide weak signals along the entire near UV spectrum [16,17].
Taking into account that both eEF1A1 and eEF1A2 are 98% similar, have the same positions in the primary structures, and contain almost equal amount of aromatic amino acids (there is only one substitution of Phe for Ser in eEF1A2; overall amount of amino acids in eEF1A1 (eEF1A2) is Trp -5(5), Tyr -12(12), Phe -14(13)), the mentioned method could be useful to test the identity or difference of tertiary structures of the isoforms.
Near UV CD spec tra of eEF1A1 and eEF1A2 are shown in Fig. 2. The dif fer ence in CD sig nals was observed in the en tire spec trum of near UV re gion.We could not ex clude the fact that al ter ations in the Phe-spe -cific re gion of CD spec trum might re sult par tially from the dif fer ence in 393 rd po si tion of pri mary struc tures of the isoforms (Phe393 in eEF1A1 or Ser393 sub sti tu tion in eEF1A2).How ever, the dif fer ence in the rest of the spec trum is the di rect con se quence of changes in the tertiary struc ture of these pro teins.There fore, the data of near UV CD tes tify in fa vour of ev i dent dif fer ences in ter tiary struc tures of the isoforms.
"Far" UV CD.It proved to be im por tant to de termine whether lo cal dif fer ences of spa tial struc tures of eEF1A isoforms are ac com pa nied by changes in the second ary struc ture of these pro teins.The lat ter can be inves ti gated us ing CD spec tros copy in far UV re gion (190-250 nm).Pep tide bonds be come chromo phores at these wave lengths, and sig nal ap pears if the bonds are in reg u larly folded sur round ing.The spe cific form and size of CD spec trum are mainly con di tioned by a-he li cal struc tures in pro tein mol e cule [18].
Far UV CD spec tra of eEF1A1 and eEF1A2 are shown in Fig. 3.The in crease in the mo lar el lip tici ty peak at 210 nm was ob served for eEF1A2 as com pared to eEF1A1.Since this wave length is spe cific for a-he lical struc tures, the in cre ment of CD sig nal ev i dences to par tial loss of a-he li cal struc tures in the sec ond ary struc ture of eEF1A2.These data cor re late with the results of our mo lec u lar dy nam ics sim u la tion on par tial un wind ing of a-he lix Lys36-Glu48 of eEF1A2 [9,10].

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There fore, the com par a tive anal y sis of spa tial structures of two tis sue-spe cific eEF1A isoforms, us ing biophys i cal meth ods, has been car ried out for the first time.One of the isoforms is in volved in ovar ian can cer and can cer of lungs in hu mans [4,5,7,8].The struc ture of eEF1A2 has been shown as more com pact and char acter ized by par tial un wind ing of a-he lix Lys36-Glu48.Some dif fer ences have been also discovered in the tertiary struc tures of pro tein isoforms.These specificities of spa tial or ga ni za tion may re sult in the ex po sure of func tional sites in eEF1A2, ca pa ble of bind ing sig nal mol e cules, which may serve as one of the reasons for oncogenicity of this isoform.
Current work is among the first studies, proving that 98% similar isoforms of homologous proteins may have different spatial conformations.Thus, not only the changes in the primary structure, revealed e.g. in the appearance of new domains, or in elimination or appearance of new sites of post-translational modifications, but also the changes in their spatial conformation are important for the functional divergence of the protein isoforms.These data will allow extending the direction of searching for multiple families of various isoproteins.

Fig. 2
Fig.2 CD spectra of eEF1A isoforms in near UV region: 1 -eEF1A1; 2 -eEF1A2 Wave lengths, nm The authors are grateful to the Fund of fundamental researches of the Ministry of Science and Education of Ukraine, INTAS and Wellcome Trust for their financial support.The authors are also grateful to B. S. Melnyk for the measurements of CD spectra.