(A ) CD spectra of the complexin (CPX) accessory domain (CPX26 48) and the CPX minimal functional domain (CPX26 83).
We proceeded to conduct a structure-guided mutational analysis of Rpo26 and thereby delineated a minimal functional domain, Rpo26- 78-155), capable of rpo26 ∆ complementation and tgs1 ∆ cs suppression.
CD spectra of peptides corresponding to the CPX accessory domain alone (residues 26 48) or the minimal functional domain (residues 26 83) were recorded in PBS using Chirascan CD spectrometer (Applied Photophysics, UK) at 25°C from the range of 260 nm–198 nm at 1 nm bandwidth.
We note that in these experiments as well as those reported in Kümmel et al. we titrated full-length CPX (residues 1 134) into the blocked SNAREΔ60, and not the minimal functional domain (residues 26 83) as we had implied ("we used a complexin construct comprising both the central and accessory helices [residues 26 83]" [ Kümmel et al., 2011]), and we apologize for this reporting error.
We have now additionally carried out the ITC experiments with the minimal functional domain (CPX26 83) and find that this truncated version also binds to the blocked SNAREΔ60, albeit with slightly weaker affinity (Kd = 23.9 ± 0.1 µM) compared with full-length CPX.
By testing truncated versions of InvMce1, we determined that Mce1(1 178) comprises a minimal functional triphosphatase domain.
We refine the margins of the minimal functional Rpo26 domain, identify essential side chains by alanine scanning, and interpret the mutational effects by reference to the crystal structure of Rpo26 in RNA polymerase II.
The lambda displayed libraries proved to be very useful for particular applications, such as determination of the minimal binding or functional domain within a single gene product [ 11- 14] or for screening with monoclonal antibodies cDNA libraries constructed from small viral genome [ 15].
The Giardia Dicer likely represents a minimal functional Dicer protein and the additional domains present in mammalian Dicer may serve to allow more complex regulation of Dicer activity.
Based on comparative genome analysis across multiple organisms, protein families have been identified containing domains for which minimal functional annotation is available.
