Polyampholytes are polymers containing both positively and negatively charged groups along their backbone. The presence of these charged residues enables the development of stimuli-responsive and multi-functional materials. Incorporating ionic groups into polymeric materials has been shown to improve thermal and mechanical properties, as well as provide anti-fouling and cryopreservation effects. Naturally occurring polyampholytes are found in intrinsically disordered proteins (IDPs), which constitute 25-30% of known functional eukaryotic proteins. IDPs play critical roles in disease due to their involvement in forming condensed phases in intracellular environments.

Recent studies by computational and experimental scientists indicate that charge sequence significantly influences the phase separation behavior of IDPs. To utilize charge sequence as a design parameter for polyampholyte materials and to enhance our understanding of the electrostatic contributions to peptide phase behavior, it is essential to understand how charge sequence affects polyampholyte conformation.

This thesis experimentally investigates the effects of charge sequence on polyampholyte conformation and phase behavior. Using Fmoc-based solid-phase peptide synthesis, we construct sequence-specific polyampholyte peptides with varying charge blockiness, from alternating to diblock arrangements.

Experiments are conducted in dilute concentrations to better assess single-chain structure and dynamics. In the first part of this thesis, we characterize the conformation of L-chiral polyampholyte peptides. In the second part, we synthesize atactic polypeptides by incorporating D-chiral residues to better isolate electrostatic interactions and reduce hydrogen bond interactions. We generally observe increased phase separation with increased blockiness.

Small angle scattering reveals that these polyampholytes exhibit random coil or self-avoiding walk conformations with minimal differences in size at small block lengths. We also observe microphase separations in mid-sized blocks of the atactic system, consistent with theoretical predictions. Lastly, we characterize the dynamics of polyampholyte solutions. All experiments are compared to solutions with added NaCl, where electrostatic interactions are screened. Comparing our experimental results with recent simulations and complexation experiments provides further insights into the thermodynamic driving forces behind our observations.