A Structure-free Method for Quantifying Conformational Flexibility in proteins

Abstract

All proteins sample a range of conformations at physiologic temperatures and this inherent flexibility enables them to carry out their prescribed functions. A comprehensive understanding of protein function therefore entails a characterization of protein flexibility. Here we describe a novel approach for quantifying a protein's flexibility in solution using small-angle X-ray scattering (SAXS) data. The method calculates an effective entropy that quantifies the diversity of radii of gyration that a protein can adopt in solution and does not require the explicit generation of structural ensembles to garner insights into protein flexibility. Application of this structure-free approach to over 200 experimental datasets demonstrates that the methodology can quantify a protein's disorder as well as the effects of ligand binding on protein flexibility. Such quantitative descriptions of protein flexibility form the basis of a rigorous taxonomy for the description and classification of protein structure.

Figure 1. Results from calculations on simulated systems.

Alignments of structures in each conformational ensemble are shown on the left. Simulated SAXS profiles and calculated RgD are also shown.

Figure 2. Dimensionless Kratky plots.

Dotted lines are drawn at and (qR_g)²I(q)/I(0) = 1.104. Folded proteins have a local maximum where the two lines intersect. (a) Disordered spectrum: C-terminal region of the Bromodomain adjacent to zinc finger protein domain 2B; Partially folded spectrum: Splicing factor U2 Auxiliary Factor 65 KD (U2AF65), residues 148–475; Folded spectrum: Chymotrypsinogen A. (b–e) Dimensionless Kratky plots of 226 proteins from the BIOISIS and SASBDB databases organized into quartiles based on their entropy values. The entropy values are divided into four quartiles for the purpose of illustration. The plots are colored such that lower entropies are blue and higher entropies are red.

Figure 3. Dimensionless Kratky plots (top row), calculated RgD entropy values (insets in top row), and Porod Debye plots (bottom row) for MnmE: E. coli MnmE in isolation (black) and bound to GppNHp (green), and GDP-AlFx (purple); wtTIA-1: The alterative splicing factor wtTIA-1 RRM123 in the absence of RNA (black) and bound to 11-nucleotide AU-rich segment taken from the 3′-untranslated region of tnf-α (green); RPA-DBC: The DNA-binding core of heterotrimeric Replication protein A in the absence (black) and presence (green) of a 30 nucleotide ssDNA substrate; U2AF65: U2 auxiliary factor residues 148-475, in the absence (black) and presence of RNA (green); C3b: Complement fragment C3b in the unbound (black) state and bound to the extracellular fibrinogen binding protein (Efb) from S. aureus (green).

Since we work with normalized Intensity profiles (that are divided by I(0)) the y-axis of each Porod-Debye plots is divided by I(0).