Abstract:
:The gene-3-protein (G3P) of filamentous phage is essential for their propagation. It consists of three domains. The CT domain anchors G3P in the phage coat, the N2 domain binds to the F pilus of Escherichia coli and thus initiates infection, and the N1 domain continues by interacting with the TolA receptor. Phage are thus only infective when the three domains of G3P are tightly linked, and this requirement is exploited by Proside, an in vitro selection method for proteins with increased stability. In Proside, a repertoire of variants of the protein to be stabilized is inserted between the N2 and the CT domains of G3P. Stabilized variants can be selected because they resist cleavage by a protease and thus maintain the essential linkage between the domains. The method is limited by the proteolytic stability of G3P itself. We improved the stability of G3P by subjecting the phage without a guest protein to rounds of random in vivo mutagenesis and proteolytic Proside selections. Variants of G3P with one to four mutations were selected, and the temperature at which the corresponding phage became accessible for a protease increased in a stepwise manner from 40 degrees C to almost 60 degrees C. The N1-N2 fragments of wild-type gene-3-protein and of the four selected variants were purified and their stabilities towards thermal and denaturant-induced unfolding were determined. In the biphasic transitions of these proteins domain dissociation and unfolding of N2 occur in a concerted reaction in the first step, followed by the independent unfolding of domain N1 in the second step. N2 is thus less stable than N1, and it unfolds when the interactions with N1 are broken. The strongest stabilizations were caused by mutations in domain N2, in particular in its hinge subdomain, which provides many stabilizing interactions between the N1 and N2 domains. These results reveal how the individual domains and their assembly contribute to the overall stability of two-domain proteins and how mutations are optimally placed to improve the stability of such proteins.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Martin A,Schmid FXdoi
10.1016/s0022-2836(03)00359-0keywords:
subject
Has Abstractpub_date
2003-05-09 00:00:00pages
863-75issue
4eissn
0022-2836issn
1089-8638pii
S0022283603003590journal_volume
328pub_type
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