Selective assembly of a high stability AAB collagen heterotrimer

Abstract

How collagen is able to obtain control of helix composition and register is poorly understood yet is critical for determining the structure and properties of the most abundant protein in the human body. In humans there are 28 known types of collagen that can form homotrimeric (AAA) or heterotrimeric (AAB and ABC) compositions. Additionally, because of a single amino acid offset between peptide chains in the triple helix, distinct heterotrimers of different registers can be formed. In this communication we describe an AAB collagen heterotrimer with controlled composition and register. This is the first report of a collagen heterotrimer whose thermal stability is greater than that of any of its component parts and therefore is the dominant species in solution. The design concept is simple: combination of peptides who follow the canonical (X-Y-Gly)(n) amino acid repeat in a 2:1 ratio in which the more abundant peptide has a charge 1/2 and opposite of the other should result in the formation of an AAB heterotrimeric collagen helix. This will be the dominant species because it is neutral (zwitterionic) while homotrimers should be destabilized because of charge repulsion. Here we show by circular dichroism, differential scanning calorimetry, and NMR that, in a 2:1 mixture of the peptides (EOGPOG)(5) and (PRG)(10), the AAB heterotrimer is the dominant structure in solution and melts 10 degrees C higher in temperature than the next most stable species.

Figure 1

CD data for the first derivative of the melting curve for (EOGPOG)₅ homotrimer (black), non-annealed (blue) and annealed (red) samples of the 2:1 mixture of (EOGPOG)₅ and (PRG)₁₀.

Figure 2

(a) NOESY spectrum and molecular model highlighting the cross-peaks between the arginine and glutamic acid side-chains and the atoms giving rise to the NOEs, sequential and intra-residue peaks are not labeled for clarity (b) Alternate view of the model highlighting the hydrogen bonds between the guanidium groups and hydroxyproline backbone carbonyls using colored arrows.