Luis Galiano

Drug Pressure Selected Mutations in HIV-1 Protease Alter Flap Conformations

The flap conformations of two drug-resistant HIV-1 protease constructs were characterized by molecular dynamic (MD) simulations and distance measurements with pulsed electron paramagnetic resonance (EPR) spectroscopy. MD simulations accurately regenerate the experimentally determined distance profiles and provide structural interpretations of the EPR data. The combined analyses show that the average conformation of the flaps, the range of flap opening and closing, and the flexibility of the flaps differ markedly in HIV-1PR as multiple mutations arise in response to antiviral therapy, providing structural insights into the mechanism of inhibitor resistance.

Solute effects on spin labels at an aqueous-exposed site in the flap region of HIV-1 protease.

The effects of solutes on spin-label mobility and protein conformation have been investigated with X-band continuous-wave and pulsed electron paramagnetic resonance (EPR) spectroscopy for spin labels attached to an aqueous-exposed site in the beta-hairpin flap region of HIV-1 protease. Specifically, we examined the effects of glycerol, sucrose, PEG3000, and Ficoll400 for four commonly used nitroxide spin labels and found that the largest perturbations to the EPR line shapes occur for solutions containing PEG3000 and glycerol. From comparisons of the spectral line shapes and distance distribution profiles of spin-labeled HIV-1 protease with and without inhibitor, it was concluded that solutes such as glycerol and PEG3000 alter the line shapes of the spin label in the beta-hairpin flaps of HIV-1 PR by modulation of spin-label mobility through changes in preferential interactions with the solutes. It is noteworthy that the high osmolality of the 40% glycerol solution did not alter the conformation of the flaps as determined from pulsed EPR distance measurements.

Characterization of the disordered‐to‐α‐helical transition of IA3 by SDSL‐EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy coupled with site‐directed spin labeling (SDSL) is a valuable tool for characterizing the mobility and conformational changes of proteins but has seldom been applied to intrinsically disordered proteins (IDPs). Here, IA3 is used as a model system demonstrating SDSL‐EPR characterization of conformational changes in small IDP systems. IA3 has 68 amino acids, is unstructured in solution, and becomes α‐helical upon addition of the secondary structural stabilizer 2,2,2‐trifluoroethanol (TFE). Two single cysteine substitutions, one in the N‐terminus (S14C) and one in the C‐terminus (N58C), were generated and labeled with three different nitroxide spin labels. The resultant EPR line shapes of each of the labels were compared and each reported changes in mobility upon addition of TFE. Specifically, the spectral line shape parameters h(+1)/h(0), the local tumbling volume (VL), and the percent change of the h(−1) intensity were utilized to quantitatively monitor TFE‐induced conformational changes. The values of h(+1)/h(0) as a function of TFE titration varied in a sigmoidal manner and were fit to a two‐state Boltzmann model that provided values for the midpoint of the transition, thus, reporting on the global conformational change of IA3. The other parameters provide site‐specific information and show that S14C‐SL undergoes a conformational change resulting in more restricted motion than N58C‐SL, which is consistent with previously published results obtained by studies using NMR and circular dichroism spectroscopy indicating a higher degree of α‐helical propensity of the N‐terminal segment of IA3. Overall, the results provide a framework for data analyzes that can be used to study induced unstructured‐to‐helical conformations in IDPs by SDSL.

Pulsed EPR Distance Measurements in Soluble Proteins by Site‐Directed Spin Labeling (SDSL)

The resurgence of pulsed electron paramagnetic resonance (EPR) in structural biology centers on recent improvements in distance measurements using the double electron‐electron resonance (DEER) technique. This unit focuses on EPR‐based distance measurements by site‐directed spin labeling (SDSL) of engineered cysteine residues in soluble proteins, with HIV‐1 protease used as a model. To elucidate conformational changes in proteins, experimental protocols were optimized and existing data analysis programs were employed to derive distance‐distribution profiles. Experimental considerations, sample preparation, and error analysis for artifact suppression are also outlined herein. Curr. Protoc. Protein Sci. 74:17.17.1‐17.17.29.

Characterizing Solution Surface Loop Conformational Flexibility of the GM2 Activator Protein

GM2AP has a β-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL.