Early Folding Events, Local Interactions, and Conservation of Protein Backbone Rigidity

Abstract

Protein folding is in its early stages largely determined by the protein sequence and complex local interactions between amino acids, resulting in lower energy conformations that provide the context for further folding into the native state. We compiled a comprehensive data set of early folding residues based on pulsed labeling hydrogen deuterium exchange experiments. These early folding residues have corresponding higher backbone rigidity as predicted by DynaMine from sequence, an effect also present when accounting for the secondary structures in the folded protein. We then show that the amino acids involved in early folding events are not more conserved than others, but rather, early folding fragments and the secondary structure elements they are part of show a clear trend toward conserving a rigid backbone. We therefore propose that backbone rigidity is a fundamental physical feature conserved by proteins that can provide important insights into their folding mechanisms and stability.

Figure 1

Conceptual overview of how the approaches used in this work relate to protein folding.

Figure 2

Boxplots showing the distribution per amino acid residue of the original predicted backbone rigidity divided into normal and early folding classes. The number of amino acids in each distribution is indicated at the top of each graph, whereas the significance of the difference between the distributions is reported under the amino acid three-letter code. To see this figure in color, go online.

Figure 3

Boxplots showing the distribution of the average predicted backbone rigidity per secondary structure element as observed in the native fold, divided by whether they do or do not contain early folding residues (A) and the distributions of the length of these secondary structure elements (B). The significance of the difference between the distributions is indicated under the secondary structure element class. To see this figure in color, go online.

Figure 4

Boxplots showing the distribution of the median of the predicted backbone rigidity values per MSA column in the HHBLITS_lowSeqId set, with the data divided into normal and early folding classes, by the amino acid as observed in the target sequence for that MSA column. The number of MSA columns in each distribution is indicated at the top of each graph, whereas the significance of the difference between the distributions is reported under the amino acid three-letter code. To see this figure in color, go online.

Figure 5

The structural, dynamics and evolutionary properties of sperm whale apo-Mb (A) and horse ferricytochrome c (B) are shown as a function of their residue positions on the left, whereas the corresponding three-dimensional structures are on the right (PDB: 1MBC and 1HRC, respectively; structures are only available for the proteins together with their heme cofactors (black), and for cyt c the wild-type protein is depicted). In both panels, early folding residues are marked with green shading on the graphs and with green stick representations within the three-dimensional structures, with their residue positions and types indicated. A red line depicts the per-residue DynaMine-predicted backbone rigidity of the protein. The medians of predicted values in the corresponding HHBLITS_lowSeqId alignment columns are shown as a black line, whereas their first and third quartiles of the distribution are marked in dark gray, their minima and maxima with lighter gray. The blue shading between the dark gray quartile lines represents the sequence entropy for each alignment position, with darker blue indicating lower entropy (high evolutionary conservation). The secondary structure elements assigned by the POLYVIEW server are also provided, with early folding helices shown as green cylinders and others as gray cylinders. To see this figure in color, go online.