Identification of stability domains in a multidomain membrane protein

ERNESTO A. ROMAN; JOSÉ MARTÍN ARGUELLO; FRANCISCO LUIS GONZALEZ FLECHA

Los Cocos, Córdoba, Argentina.

Congreso; Reunion Anual de la Sociedad Argentina de Biofísica; 2009

Sociedad Argentina de Biofisica

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> 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.