Exploring the potential of the monobody scaffold: effects of loop elongation on the stability of a fibronectin type III domain.
The tenth fibronectin type III domain of human fibronectin (FNfn10) is a small, monomeric beta-sandwich protein, similar to the immunoglobulins. We have developed small antibody mimics, 'monobodies', using FNfn10 as a scaffold. We initially altered two loops of FNfn10 that are structurally equivalent to two of the hypervariable loops of the immunoglobulin domain. In order to assess the possibility of utilizing other loops in FNfn10 for target binding, we determined the effects of the elongation of each loop on the conformational stability of FNfn10. We found that all six loops of FNfn10 allowed the introduction of four glycine residues while retaining the global fold. Insertions in the AB and FG loops exhibited very small degrees of destabilization, comparable to or less than predicted entropic penalties due to the elongation, suggesting the absence of stabilizing interactions in these loops in wild-type FNfn10. Insertions in the BC, CD and DE loops, respectively, resulted in modest destabilization. In contrast, the EF loop elongation was highly destabilizing, consistent with previous studies showing the presence of stabilizing interactions in this loop. These results suggest that all loops, except for the EF loop, can be used for engineering a binding site, thus demonstrating excellent properties of the monobody scaffold.
Batori
Vincent
V
Department of Biochemistry and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Koide
Akiko
A
Koide
Shohei
S
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry; D001665:Binding Sites; D019277:Entropy; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D005353:Fibronectins; D011994:Recombinant Proteins