Parallel Atomic Force Microscopy and NMR Spectroscopy To Investigate Self-Assembled Protein−Nucleotide Aggregates

Abstract

The concurrent use of atomic force microscopy (AFM) and NMR spectroscopy should enable one to link the structural information from the former to chemical information obtained with the latter. In this instance, the self-assembly of type I collagen into segmental long spacing (SLS) collagen crystallites is examined, and the first concrete evidence for the incorporation of the nucleotide ATP into these crystallites is presented. The type I collagen monomer contains no phosphorus, unlike the triphosphate group of the ATP, thus making 31 P NMR spectroscopy a highly specific probe for the location and interaction of ATP. The changes in the 31 P NMR spectral attributes upon self-assembly of SLS collagen crystallites under various conditions are interpreted with relation to the crystallite morphology observed with AFM. The formation of crystallites is found to correlate with decreased mobility of all three 31 P nuclei of ATP, as evinced by appreciable line broadening in the solution-state spectra. This is attributable to ATP incorporation into the crystallite structure. Analysis of the resonance frequencies for each of the 31 P nuclei of ATP indicates that the R- and A-phosphates have decreased electron density relative to the ‚-phosphate upon incorporation, implying direct charge-charge binding, with binding probabilities greater for the R- and A-phosphate groups than for the ‚-phosphate group. Three possible scaffolding roles of the ATP are proposed with respect to the structure of the SLS collagen crystallite based upon the evidence presented.