3- instead of 4-helix formation in a de novo designed protein in solution revealed by small-angle X-ray scattering

De novo design and chemical synthesis of proteins and their mimics are central approaches for understanding protein folding and accessing proteins with novel functions. We have previously described carbohydrates as templates for the assembly of artificial proteins, so-called carboproteins. Here, we describe the preparation and structural studies of three alpha-helical bundle carboproteins, which were assembled from three different carbohydrate templates and one amphiphilic hexadecapeptide sequence. This heptad repeat peptide sequence has been reported to lead to 4-alpha-helix formation. The low resolution solution structures of the three carboproteins were analyzed by means of small-angle X-ray scattering (SAXS) and synchrotron radiation circular dichroism (SRCD). The ab initio SAXS data analysis revealed that all three carboproteins adopted an unexpected 3+1-helix folding topology in solution, while the free peptide formed a 3-helix bundle. This finding is consistent with the calculated alpha-helicities based on the SRCD data, which are 72 and 68 % for two of the carboproteins. The choice of template did not affect the overall folding topology (that is for the 3+1 helix bundle) the template did have a noticeable impact on the solution structure. This was particularly evident when comparing 4-helix carboprotein monomers with the 2x2-helix carboprotein dimer as the latter adopted a more compact conformation. Furthermore, the clear conformational differences observed between the two 4-helix (3+1) carboproteins based on D-altropyranoside and D-galactopyranoside support the notion that folding is affected by the template, and subtle variations in template distance-geometry design may be exploited to control the solution fold. In addition, the SRCD data show that template assembly significantly increases thermostability