How Forgiving Can Proteins Be? The Biophysical Characterization of De Novo Proteins from a Combinatorial Library with Novel Topology

Abstract

The field of \textit{de novo} protein design carries immense potential for creating novel proteins guided by the principles of nature. Despite the challenges in designing proteins, recent research has leveraged the driving forces of protein folding, particularly the burial of hydrophobic residues, to computationally design new complex topologies using binary patterning.$^{1}$ This work studies the structural tolerance of \textit{de novo} proteins to mutations within their hydrophobic cores by investigating variants from a combinatorial library. Differential scanning calorimetry (DSC), a technique that measures the melting temperature and enthalpy of a protein unfolding event, demonstrated high thermal stability of two mutant proteins. Hydrogen-deuterium exchange measured by mass spectrometry (HDX-MS) analyzed the conformational behavior of these mutants. While the mutants had varying degrees of isotopic exchange, only a fraction of the available hydrogens underwent exchange in the three most promising mutants. A consistent trend can be drawn from the results of both techniques: the most thermodynamically stable mutant of DSC incorporated the fewest deuterium atoms in HDX-MS experiments, suggesting a correlation between thermal and conformational stability. These findings demonstrate that \textit{de novo} proteins of a combinatorial library can be successfully designed with novel backbone structures, tolerating variations within their hydrophobic cores. This presents a promising avenue for true \textit{de novo} design, achieving complexity and stability approaching that of natural proteins.