A Small-Angle X-ray Scattering Study of α-helical Bundle-Forming Peptide–Polymer Conjugates in Solution: Chain Conformations

Abstract

As a new family of soft materials, peptide/protein–polymer conjugates can lead to a wide range of potential biological and nonbiological applications. The performance of these materials depends on the protein structure and phase behavior arising from a balance between the enthalpic interactions of the components and surrounding media as well as the entropic contribution associated with polymer chain deformation. There is a great need to perform structural studies in solution that systematically investigate the polymer chain conformation upon linkage to a peptide or protein so as to evaluate how polymers affect the protein structure of the biomolecule and, consequently, its functionality. Combinations of a range of factors including low contrast, weak scattering signals in dilute solutions as well as difficulties in separating the component scattering contributions, pose significant challenges to structural characterization. Here we present a synchrotron small-angle X-ray scattering (SAXS) study of two model helix bundle forming peptide–polymer conjugates and show that with analytical modeling of the scattering intensity detailed structural information on both peptide structure and polymer conformation can be extracted. The peptide–poly(ethylene glycol) (PEG) conjugates are based on peptides that self-associate to form well-defined 3- or 4-helix bundles and the PEG chain is covalently linked either to the end or the side of the peptide (i.e. end- or side-conjugation). Using a simplified analytical geometrical body form factor model, where the peptide–polymer bundles are modeled as parallel cylinders with attached Gaussian chains, a quantitative description of the scattering behavior can be reached. On the basis of the simplified structural model, the protein tertiary structures, i.e., the α-helix bundle, remains largely intact and maintains its oligomeric state but exhibits slight swelling in solution with respect to the crystal structure. The PEG chain conformation appears to slightly depend on the conjugate architecture. In terms of the chain dimension represented by Rg, the end-conjugated PEG exhibit similar value as compared to free PEG for the molecular weight studied (2 kDa). For the side-conjugates our simple scattering model seems to indicate a systematically slightly lower values for Rg, i.e., a slight compression, in particular for the highest molecular weight (5 kDa). However, considering the limitations of the model and experimental uncertainties, further investigations, such as neutron scattering, is needed to illustrate detailed chain conformation. The present studies can be extended to other peptide–polymer or protein–polymer hybrid systems to extract information on both protein structure and polymer chain conformation. This work will thus provide valuable guidance to understand their structure and phase behavior using X-ray and neutron scattering.