UC Santa Barbara

Intrinsically disordered proteins (IDPs) do not adopt a stable three-dimensional structure, thus posing a challenge to common characterization techniques that have found success with folded proteins. While IDP conformational characterization has been explored with small angle X-ray scattering (SAXS) and fluorescence resonance energy transfer (FRET), there is a demand for alternative experimental techniques that can provide new perspectives in this field.

Beyond biological IDPs, quantification of rapidly fluctuating conformational states is also crucial for the design and development of synthetic sequence-defined polymers. Inspired by the ability of proteins to assemble structures with high specificity, a class of polymeric materials emerged whose sequences can be tailored to achieve the same level of complexity. Among these, polypeptoids are the most promising due to their precise synthesis and chemical modifiability. The rational design of polypeptoid materials calls for a fundamental understanding of how their sequences influence their conformational behaviors.

We approach the conformational study of these two polymer systems through entropic elasticity measurements using magnetic tweezers. Force-extension measurements reveal insights into polymer structure at a broad range of length scales, from the local stiffness described by the persistence length to long-range interactions quantified by the Flory exponent. As these sequence-defined polymers are typically too short for magnetic tweezers experiments, we also explore ligation schemes to form long tether constructs. Here, we describe the investigation of the conformations of a model IDP, the neurofilament low molecular weight tail region (NFLt). Despite the high charge content, we find that NFLt is more compact than expected due to local hydrophobic attractions. Our results, specifically Flory exponent measurements in denaturant concentrations, are in remarkable agreement with previous results on other IDPs using SAXS or FRET. We thus demonstrate a novel framework to characterize the conformational ensembles of IDPs through entropic elasticity analysis.

We also investigate the effects of charge spacing on polypeptoid conformation through simple sequence designs and find conflicting observations on the electrostatic influence. Whereas the local flexibility is unperturbed, the solubility behavior in increasing ionic strength is dependent on the charge spacing. We attribute this unique conformational behavior to the chemical structure of polypeptoids, whose charge groups are on side chains. Our results contribute novel insights into the sequence-conformation relationship, as well as raising new questions on polyelectrolyte physics.

Through the studies on NFLt and charged polypeptoids, we establish a previously unexplored approach of using magnetic tweezers experiments and entropic elasticity analysis to quantify the conformations of sequence-defined polymers. For IDPs, magnetic tweezers can complement existing techniques (SAXS and FRET) and advance the general knowledge of protein conformational disorder. For polypeptoids, this framework can be applied for the investigation of other sequence effects, such as ampholyticity or hydrophobicity.