# Revealing Accessibility of Cryptic Protein Binding Sites within the Functional Collagen Fibril
## 4 Computational Studies of the Collagen Fibril
### SASA of Collagen I Fibril Reveals Exposure of Partner Binding Sites from the Fibril Surface  
In order to assess the site-specific accessibility at the collagen fibril surface, we performed SASA calculations with different probe sizes on all residues of the 3 \(\times\) 3 fibril SRU model (Figure 2A) using  
Figure 2: (**A**) A representative cross-section showing the 3 \(\times\) 3 SRU matrix used in the solvent accessible surface area (SASA) calculations. The model contains three SRUs in each the \(x\)- and \(y\)-dimensions, and the \(z\)-dimension extends to one D-period in length. Triple helical monomers are color-coded by D-segments as in Figure 1. Double-headed arrows indicate the three distinct layers in the x-dimension. (**B**) Correlation of SASA with binding site distance. The modified 5 Å SASA of 17 known binding sites with respect to the orientation proposed by [PERSON] et al. (“surface A”) is plotted vs. distance of the site from the surface. the Molecular Surface (MS) program [102; 103]. In these calculations, the SASA gives the surface area of an individual residue that is accessible to a probe of a certain radius. We initially calculated SASA with a 1.4 A probe, the radius of a water molecule, in order to measure the water accessibility within the fibril. Remarkably, there is uniformly high SASA, even into the D-segments of the inner layer, to water molecules throughout the 3 \(\times\) 3 fibril SRU model. This indicates that despite appearing densely packed, there is significant space for water within the triple helices, even within the core of the collagen fibril. In an attempt to model access of ligands to the binding sites in the fibril, we determined the SASA of the different D-segments, using a larger spherical probe with a 5 A radius, termed the 5 A SASA. As we are interested in determining binding site exposure relative to the surface A or surface B (see Figure 2A), we used a simple approach in which we subtract the 5 A SASA of SRUs of the inner layer from the 5 A SASA of the surface layer. This subtraction gives accessibility only from the fibril surface, when compared to the fibril core. This allows us to probe the accessibility of the residues that are exposed to the fibril surface. We applied the calculation to each residue of the middle SRUs along the \(y\)-axis of the 3 \(\times\) 3 fibril SRU model (Figure 2A) since they are surrounded by all of the neighbors that would be present in the full fibril in order to obtain a baseline for the 5 A SASA. As the triple helix is composed of three chains, we used the average 5 A SASA of the three chains at each position for the calculation. We have plotted the modified 5 A SASA of 17 known binding sites in collagen I with respect to their distance from the surface A, which exposes D4 and D5 (Figure 2B). We find that of these 17 sites, only five show a high modified 5 A SASA; the other sites appear to have minimal modified 5 A SASA suggesting that the majority of the collagen binding sites are not accessible from surface A. This underscores that although access to these binding sites by binding partners is required for a multitude of biological processes, their exposure within the context of the collagen fibril is minimal. We analyzed the modified 5 A SASA in residue-specific detail throughout the collagen fibril. For visualization purposes, we present SASA form the perspectives of both surfaces A and B. The residue-specific modified 5 A SASA for each D-segment relative to a particular surface is plotted in Figure 3A,B. Our SASA analysis reveals non-uniform SASA between the D-segments, as expected due to the quasihexagonal arrangement of the microfibril. In particular, high SASA is observed in the D-segment at the assumed surface (D4 and 5 in surface A and D1 in surface B), and the accessible surface area is significantly lower in D-segments deeper into the fibril.