Computer modeling of nitroxide spin labels on proteins

Abstract

Electron paramagnetic resonance (EPR) using site-directed spin-labeling (SDSL) can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of inter-label distances using the double electron-electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of inter-label distances. We describe an algorithm, PRONOX, for rapid computation of inter-label distances based on calculation of spin label conformer distributions at any site of a protein. The program incorporates features of the label distribution established experimentally, including weighting of favorable conformers of the label. Distances calculated by PRONOX were compared with new DEER distances for amphiphysin and annexin B12, and with published data for FCHo2 (F-BAR), endophilin, and α-synuclein; a total of 44 inter-label distances. The program reproduced these distances accurately (r2=0.94, slope=0.98). For 9 of the 11 distances for amphiphysin, PRONOX reproduced the experimental data to within 2.5 Å. The speed and accuracy of PRONOX suggests that the algorithm can be used for fitting to DEER data for determination of protein tertiary structure.

Fibril Structure of Human Islet Amyloid Polypeptide

“…This starting model was based on CD and FT-IR data showing that hIAPP fibrils consist of ␤-sheets (19,25); experimental data from fiber diffraction suggesting that the ␤-strands are perpendicular to the fibril axis and that each ␤-strand is about 5 Å apart from the neighboring strand (16); and site-directed spin labeling data suggesting that the ␤-strands are parallel and in-register (17). The starting structure was generated by the in-house programs FIBRIL and PRO-NOX (26), which were used to produce input files for AMBER10 (27).…”

Identification of Receptor Binding-induced Conformational Changes in Non-visual Arrestins

“…The upper reliable distance limit for each sample was determined based on the maximum data collection time (t) used in each of the DEER experiments according to the equation, d Ϸ 5(t/2) 1 ⁄ 3 (45) and is reflected in the x axis of each distance distribution plot. The expected distance between arrestin-2 pairs was predicted via the PRONOX program (46) by measuring the distances between rotameric configurations of the two spin labels attached to the highest resolution arrestin-2 crystal structure available (24) based on an experimentally derived library of allowed rotameric configurations. For comparison, free and receptor-bound state distances were determined for the free state crystal structure of arrestin-2 (PDB entry 1G4M) and the crystal structure of arrestin-2 in the presence of V2Rpp (PDB entry 4JQI), respectively.…”

Conformational dynamics and distribution of nitroxide spin labels

“…Recently, two alternative approaches with fine-grained sampling were introduced for modeling the conformational distribution of spin labels. In PRONOX spin label conformations are created by scanning a user-defined grid of the dihedral angles, without considering a potential energy function of the i [109]. However, dihedral angle values for 1 and 2 that are frequently observed in crystal structures (Section 4.3) can be favored as an option.…”

“…In a first series of tests, mean square deviations and mean deviations between predicted and experimental distances were determined for 104 pairs of labeling sites in 8 proteins with the three available software packages MMM [41], PRONOX [109], and MtsslWizard [107] (Tables 2,3). For ten pairs of sites, predictions by all three programs deviated from the experimental results to an extent that was hardly consistent with the assumption that the protein structure remains unchanged in solution.…”

“…Predictions were made with the ambient temperature rotamer library of MMM (blue) [39][40][41], with PRONOX [109,110] in the low-clash mode (red) and with MtsslWizard [107,111] in the recommended mode (green). Distance distributions are normalized with respect to the maximum probability density.…”