Functional and Early Folding Residues are separated in proteins to increase evolvability and robustness

The three-dimensional structure of proteins captures evolutionary ancestry, and serves as starting point to understand the origin of diseases. Proteins adopt their structure autonomously by the process of protein folding. Over the last decades, the folding process of several proteins has been studied with temporal and spatial resolution which allowed the identification of so-called Early Folding Residues (EFR) in the folding process. These structurally relevant residues become affected early in the folding process and initiate the formation of secondary structure elements and guide their assembly.Using a dataset of 30 proteins and 3,337 residues provided by the Start2Fold database, discriminative features of EFR were identified by a systematical characterization. Therefore, proteins were represented as graphs in order to analyze topological descriptors of EFR. They constitute crucial connectors of protein regions which are distant at sequence level. Especially, these residues exhibit a high number of non-covalent contacts such as hydrogen bonds and hydrophobic interactions. This tendency also manifest as energetically stable local regions in a knowledge-based potential. Conclusively, these features are not only characteristic for EFR but also differ significantly with respect to functional residues. This unveils a split between structurally and functionally relevant residues in proteins which can drastically improve their evolvability and robustness.The characteristics of EFR cannot be attributed to trivial features such as the accessible surface area. Thus, the presented features are novel descriptors for EFR of the folding process. Potentially, these features can be used to design classifiers to predict EFR from structure or to implement structure quality assessment programs. The shown division of labor between functional and EFR has implications for the prediction of mutation effects as well as protein design and can provide insights into the evolution of proteins. Finally, EFR allow to further the understanding of the protein folding process due to their pivotal role.Author summaryProteins are chains of amino acids which adopt a three-dimensional structure and are then able to catalyze chemical reactions or propagate signals in organisms. Without external influence, most proteins fold into their correct structure, and a small number of Early Folding Residues (EFR) have been shown to become affected at the very start of the process. We demonstrated that these residues are located in energetically stable local conformations. EFR are in contact to many other residues of a protein and act as hubs between sequentially distant regions of a proteins. These distinct characteristics can give insights into what causes certain residues to initiate and guide the folding process. Furthermore, it can help our understanding regarding diseases such as Alzheimer’s or amyotrophic lateral sclerosis which are the result of protein folding gone wrong. We further found that the structurally relevant EFR are almost exclusively non-functional. Proteins separate structure and function, which increases evolvability and robustness and gives guidance for the artificial design of proteins.