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

Revista de la Sociedad Química de México, Vol. 43, Núm. 1 (1999) 24-29
Revisión
An Integrated, Holistic Experimental and Theoretical Approach
Applied to the Derivation of the 3D Structure of Bovine Amelogenin
implicated in Amelogenesis imperfecta, a Molecular Disease
Characterized by a Single Point Mutation
V. Renugopalakrishnan l, 2 *, R. Garduño-Juárez 3 , Juan Carlos Hernández Guerrero 4 ,
Patricia N. Casillas Lavín 4 and K. Ilangovan 5
1 Harvard Medical School, Harvard University, 423 Av., Boston, MA 02115, USA, Fax +1.617.738.4960; 2 Instituto de Química,
Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Coyoacán, 04510 México DF, México. Fax
+52-5616-2217, email: renu@servidor.unam.mx; 3 Centro de Ciencias Físicas, Universidad Nacional Autónoma de México,
62210 Cuernavaca, Morelos, México; 4 Facultad de Odontología, Universidad Nacional Autónoma de México, 04510 México DF,
México; 5 Instituto de Ingeniería, Universidad Nacional Autónoma de México, 04510 México DF, México.
Resumen. La amelogenina, una fosfoproteína de 170 residuos, de
~19kDa, extracelular e hidrofóbica, con una estructura elipsoidal,
secretada por los ameloblastes dentales bovinos, contiene una estructura
primaria única comparada con otras proteínas de mamíferos de
estructura primaria conocida. Se caracteriza por una proporción alta
de Leu, His, Met, Gln, y Pro, y también contiene un residuo de Ser 16
fosforilado. Se revisan los estudios de ingeniería genética combinados
con investigación en biología estructural. La estructura tridimensional
de la amelogenina dental de bovinos ha sido el tema de investigación
del autor principal en la Escuela Médica de Harvard desde
1984, empleando aproximaciones teóricas y experimentales
integradas. Estas aproximaciones se describen mediante un diagrama
de flujo. Su estructura tridimensional contiene numerosos giros β-,
una estructura espiral β-, segmentos de hojas β-aislados, y tiene un
contenido bajo α-helicoidal. Los mutantes de la amelogenina y
algunos fragmentos sintéticos tienen potencial clínico en el tratamiento
de la Amelogenesis imperfecta, una enfermedad dental prevalente
en México.
Palabras clave: RMN 3D, IR-TF, Raman, DC, Flourescencia, Síntesis
de polipéptidos, Enfermedad molecular, Dinámica molecular, Canal de
iones, M179 resíduo recombinante, amelogenina, estructura secundaria.
Introduction
Abstract. Bovine Amelogenin, a 170-residue, ~19 kDa extracellular,
hydrophobic, phosphoprotein with an overall ellipsoidal shape,
secreted by ameloblasts from bovine tooth enamel, contains a primary
structure which is unique in comparison to other known primary
structures of mammalian proteins. lt is characterized by a large proportion
of Leu, His, Met, Gln, and Pro residues and also contains a
single phosphorylated Ser16 residue. Genetic engineering studies
combined with structural biological research are discussed in this
review. 3D structure of bovine tooth amelogenin has been the focus
of research by the principal author at Harvard Medical School since
1984 using an integrated experimental and theoretical approach. A
flow sheet describes the general strategy for such an integrated
approach. Its 3D structure contains numerous β-turns, a β-spiral
structure, isolated β-sheet segments, and is low in α-helical content.
Amelogenin mutants and synthetic fragments have potencial clinical
use in fighting amelogenesis imperfecta, a dental disease which is
prevalent in Mexico.
Key Words: 3D NMR, FT-IR, Raman, CD, Time-resolved
Fluorescence, Polypeptide synthesis, Molecular Disease, Molecular
Dynamics, Ion Channel, Ml79-a 179 residue recombinant mouse
amelogenin, Secondary Structure.
Structural biology of proteins has made significant advances
in the last two decades, propelled by multi-nuclear 2D and 3D
NMR spectroscopy [l], X-ray and Neutron diffraction Fourier
transform infrared (FT-IR) [2], laser Raman spectroscopy
(LRS), small angle neutron scattering.(SANS), time-resolved
flurorescence spectroscopy, molecular mechanics-dynamics
approach [3], solid-phase peptide synthesis and the capability
to produce monoclonally pure proteins by expression of
appropriate cDNA’s in a bacterial cell e.g. E. coli and site-specific
mutagenesis to synthesize mutant proteins. Structural
biology and molecular biology have therefore become interdependent.
Despite all these impressive technical developments,
the solution-phase structure of proteins with M r > 30
kDa by heteronuclear multi-dimensional NMR [1] continues
to pose formidable challenges especially in the assignment of
proton resonances. FT-IR and LRS have no limitations on the
M r of proteins which can be investigated using these two
methods. The development of 2D Heterospectral IR-Raman
[4], is a significant advance of immense value to protein structural
biology. In terms of the structural information content,
NMR spectroscopy rivals with X-ray and neutron diffraction.
In the case of membrane proteins and proteins that are stubborn
to crystallization due to their mobility of the glycosamino
glycan (GAG) and lipid side chains [1] e.g. glycoproteins, pro-