SMQ-V043 N-001_ligas_size.pdf - Journal of the Mexican Chemical ...

26 Rev. Soc. Quím. Méx. Vol. 43, Núm. 1 (1999) V. Renugopalakrishnan et al.
single phosphorylated Ser l6 residue. A comparison of the
amino acid sequence, i.e. the primary structure, of bovine
amelogenin [5], with amelogenins from different species is
shown in Fig. 2.
Amelogenin structure function studies have been the
focus of research at our Harvard Laboratory since 1984 [6].
Amelogein mutants and synthetic fragments have potential
clinical use in fighting Amelogenisis imperfecta (AI), a dental
disease prevalent in Mexico.
3. Commonly Occurring Secondary Structural
Motifs in Proteins
When the structural studies began in 1984, its primary structure
was not known. Early structural studies of amelogenin
were frustrating since the CD (Circular Dichroism) spectral
features were not reminscent of proteins containing α-helical
and/or β-sheet patterns [7]. The primary structure of calf
amelogenin was derived by Edman degradation gas phase
sequencing [5] and later from its cDNA sequence. The agreement
between the two methods are excellent. Under normal
circumstances, amelogenin would have been labelled a protein
with a “random” structure e.g. lacking recognizable order. The
intrepretation of its spectral features took an unexpected and
surprising turn when its primary structure became availabIe
[5] and it was found to contain unusual tandem repeats, especially
starting from residues Gln 112 and extending upto Leu l38
and the repetitive tandem template being a tripeptide
sequence, (Gin-Pro-X) and such contiguous triplets occurred
scattered throughout its primary structure. Amelognin is not
the only protein in nature to contain such tandem repeats,
albeit, the triplet template occurring nine times consecutively
suggested that repeating polypeptide segment is probably
composed of a helical array of repetitive β-turns, hairpin or U-
turns wound around a common helical axis. Similar tandem
repeats, have also been found to occur in elastin, RNA
Polymerase II and it remains to be determined whether tandem
repeats serve important structural and hence functional
roles. The tandem repeats in elastin, [8, 9], have been ascribed
a role in its elastomeric property, like in connective tissues in
lungs, where the increase in volume during respiratory cycle
requires a protein framework that can quite easily expand and
contract while the tandem repeats in other proteins may have
other important biological functions
In the case of amelogenin, the major functional role is
triggering Ca ++ ion accumulation in the process of building
hydroxyapatite e.g. octacalcium phosphate crystals in enamel.
The well organizad mineral associated with enamel has been
strikingly demonstrated by recent atomic force microscopic
studies of normal and Amelogenesis imperfecta afflicted
human tooth enamel from our laboratories [10]. When one
examines the amelogenin primary structure, it is rather surprising
that such a primary structural entity will ever be implicated
in a hydroxyapatite build up process on a bewildering
time scale e.g. the entire process of mineral accumulation is
complete in the very early phase of mammalian tooth development
and amelogenin is rapidly degraded enzymatically after
initiating mineralízation of enamel.
4. Time-Resolved Picosecond Fluorescence
Studies of Bovine Amelogenin
We have inferred from the three different decay times, τ,
observed from pico-second fluorescene spectroscopic studies
of bovine amelogenin in solution [11], that either amelogenin
manifests three conformers equilibrating rapidly in aqueous
solution or the three Trp residues present in its primary structure
(Fig. 2) are on the surface of amelogenin, contributing to
the three different decay times, τ.
5. Circular Dichroism (CD) Spectroscopic
Studies of Bovine Amelogenin
Initial CD studies were indicative of a pattern similar to “random
coil” proteins, [12] CD spectroscopy provides qualitative
information on the secondary structure of a protein. CD bands
arise from n – π and π – π* transitions in the electronic structure
of a peptide moiety. CD spectral features of multiplet β-
turn structures remain obscure even today. Furthermore, CD
patterns of a β-sheet and β-turn protein with a large β-turn
contribution are not available in the literature with the exception
of tandem repeating segments in cereal storage proteins,
tropoelastin, and RNAse polymerase II. The CD spectra of
mouse amelogenin, a 179 residue hydrophobic protein a
recombinant manifests a temperature sensitive CD spectra in
10 mM phosphate buffer which resembles the CD spectra of
tropoelastin polypeptides [11].
6. Infrared Red (IR) and Raman Spectroscopic
Studies of Bovine Amelogenin
Neverthless, CD studies were contradicted by FT-IR studies
[12], and laser Raman studies from our Harvard laboratory
[13], which were suggestive of a β-turn and low β-sheet segments
with a low α-helical content. Amide I mode usually
occurs between 1600 and 1690 cm –1 and is largely C=O
stretching frequency, amide II mode consists of C–N stretching
and NH bending, occurring between 1480 and 1575 cm –1 ,
and amide III mode, occurring between 1229 and 1301 cm –1 is
similar to amide II mode. Amide II mode is sometimes, not
always, absent in the Raman spectra of proteins. The amide I
region of the FT-IR spectra of amelogenin as a function of pH
[12] was subjected to Fourier self-deconvolution which
revealed the sub-structures contributing to the broad amide I
band. Fourier self-deconvolution of amide I band showed that
the conformation features present in amelogenín were dominated
by β-sheet and β-turn segments. Raman spectrum of
amelogenin in aqueous solution [l3] revealed a quadruplet