IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Identification of stability domains in a multidomain membrane protein
Autor/es:
ERNESTO A. ROMAN; JOSÉ MARTÍN ARGUELLO; FRANCISCO LUIS GONZALEZ FLECHA
Lugar:
Los Cocos, Córdoba, Argentina.
Reunión:
Congreso; Reunion Anual de la Sociedad Argentina de Biofísica; 2009
Institución organizadora:
Sociedad Argentina de Biofisica
Resumen:
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Folding and stability are critical for proteins to work
properly. However, forces that guide proteins through folding process for
either globular and membrane proteins are not well understood.
In previous meetings we reported structural characterization
of CopA, a PIB ATPase from the thermophilic organism Archaeoglobus fulgidus.
CopA has far UV CD spectra compatible with a major α-helical protein. Moreover,
near UV CD spectra and Trp fluorescence confirmed that CopA was properly
folded. When incubated with guanidinium hydrochloride (Gnd), CopA reversibly
unfolds following an apparent two-state process with an abnormally low ΔG.
Calculation of this parameter could be impaired since in these multidomain
proteins each domain may unfold independently. Thus, obtaining information of
specific domains from spectroscopic signals is difficult. Moreover, in this
case, the fact that CopA has 3 Trp residues difficults the calculation of the
stability of each domain of full length CopA, using Trp fluorescence determinations.
Here, we described the spectroscopic characterization of the
ATP binding domain (ATPBD) of CopA, isolated and purified as previously
described by Tsuda T et al. This protein was properly folded after purification
as evidenced by CD and Trp fluorescence, and reversibly unfolded upon
incubation with Gnd. Far UV CD spectra evidenced a completely loss of secondary
structure. When evaluated molar ellipticity at 222 nm it can be seen that the
denaturation process is completed through by at least a three state-model. Moreover,
Trp fluorescence showed that the Trp residue is largely exposed to the solvent
in its native state, and subsequently totally exposed when protein is
denatured. This process is also reversible and seems to be compatible with a
two-state process. However, the analysis of the center of spectral mass
suggests that unfolding of this domain is completed through at least a three
state model. These results confirm that CopA unfolding is a multistate process
and points out the importance of characterizing each domain to precisely
evaluate its stability. Moreover, it shows that even in single domains
unfolding profiles should be carefully analyzed.