skip to main content

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • Structural and dynamic origins of ESR lineshapes in spin-labeled GB1 domain: the insights from spin dynamics simulations based on long MD trajectories
Title: Structural and dynamic origins of ESR lineshapes in spin-labeled GB1 domain: the insights from spin dynamics simulations based on long MD trajectories
Abstract

Site-directed spin labeling (SDSL) ESR is a valuable tool to probe protein systems that are not amenable to characterization by x-ray crystallography, NMR or EM. While general principles that govern the shape of SDSL ESR spectra are known, its precise relationship with protein structure and dynamics is still not fully understood. To address this problem, we designed seven variants of GB1 domain bearing R1 spin label and recorded the corresponding MD trajectories (combined length 180 μs). The MD data were subsequently used to calculate time evolution of the relevant spin density matrix and thus predict the ESR spectra. The simulated spectra proved to be in good agreement with the experiment. Further analysis confirmed that the spectral shape primarily reflects the degree of steric confinement of the R1 tag and, for the well-folded protein such as GB1, offers little information on local backbone dynamics. The rotameric preferences of R1 side chain are determined by the type of the secondary structure at the attachment site. The rotameric jumps involving dihedral angles χ1and χ2are sufficiently fast to directly influence the ESR lineshapes. However, the jumps involving multiple dihedral angles tend to occur in (anti)correlated manner, causing smaller-than-expected movements of the R1 proxyl ring. Of interest, ESR spectra of GB1 domain with solvent-exposed spin label can be accurately reproduced by means of Redfield theory. In particular, the asymmetric character of the spectra is attributable to Redfield-type cross-correlations. We envisage that the current MD-based, experimentally validated approach should lead to a more definitive, accurate picture of SDSL ESR experiments.

  more » « less
Award ID(s):
1613007
PAR ID:
10154055
Author(s) / Creator(s):
; ; ; ; ;
Publisher / Repository:
Nature Publishing Group
Date Published:
Journal Name:
Scientific Reports
Volume:
10
Issue:
1
ISSN:
2045-2322
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; ( , Protein Science)
    Abstract

    We present the structure of an engineered protein–protein interface between two beta barrel proteins, which is mediated by interactions between threonine (Thr) residues. This Thr zipper structure suggests that the protein interface is stabilized by close‐packing of the Thr residues, with only one intermonomer hydrogen bond (H‐bond) between two of the Thr residues. This Thr‐rich interface provides a unique opportunity to study the behavior of Thr in the context of many other Thr residues. In previous work, we have shown that the side chain (χ1) dihedral angles of interface and core Thr residues can be predicted with high accuracy using a hard sphere plus stereochemical constraint (HS) model. Here, we demonstrate that in the Thr‐rich local environment of the Thr zipper structure, we are able to predict theχ1dihedral angles of most of the Thr residues. Some, however, are not well predicted by the HS model. We therefore employed explicitly solvated molecular dynamics (MD) simulations to further investigate the side chain conformations of these residues. The MD simulations illustrate the role that transient H‐bonding to water, in combination with steric constraints, plays in determining the behavior of these Thr side chains.

    Broader Audience Statement: Protein–protein interactions are critical to life and the search for ways to disrupt adverse protein–protein interactions involved in disease is an ongoing area of drug discovery. We must better understand protein–protein interfaces, both to be able to disrupt existing ones and to engineer new ones for a variety of biotechnological applications. We have discovered and characterized an artificial Thr‐rich protein–protein interface. This novel interface demonstrates a heretofore unknown property of Thr‐rich surfaces: mediating protein–protein interactions.

     
    more » « less
  2. ; ( , ChemPlusChem)
    Abstract

    Electron paramagnetic resonance (EPR) has become a powerful probe of conformational heterogeneity and dynamics of biomolecules. In this Review, we discuss different computational modeling techniques that enrich the interpretation of EPR measurements of dynamics or distance restraints. A variety of spin labels are surveyed to provide a background for the discussion of modeling tools. Molecular dynamics (MD) simulations of models containing spin labels provide dynamical properties of biomolecules and their labels. These simulations can be used to predict EPR spectra, sample stable conformations and sample rotameric preferences of label sidechains. For molecular motions longer than milliseconds, enhanced sampling strategies and de novo prediction software incorporating or validated by EPR measurements are able to efficiently refine or predict protein conformations, respectively. To sample large‐amplitude conformational transition, a coarse‐grained or an atomistic weighted ensemble (WE) strategy can be guided with EPR insights. Looking forward, we anticipate an integrative strategy for efficient sampling of alternate conformations by de novo predictions, followed by validations by systematic EPR measurements and MD simulations. Continuous pathways between alternate states can be further sampled by WE‐MD including all intermediate states.

     
    more » « less
  3. ; ; ; ( , Protein Science)
    Abstract

    Site‐specific dynamics in proteins are at the heart of protein function. While electron paramagnetic resonance (EPR) has potential to measure dynamics in large protein complexes, the reliance on flexible nitroxide labels is limitating especially for the accurate measurement of site‐specific β‐sheet dynamics. Here, we employed EPR spectroscopy to measure site‐specific dynamics across the surface of a protein, GB1. Through the use of the double Histidine (dHis) motif, which enables labeling with a Cu(II) – nitrilotriacetic acid (NTA) complex, dynamics information was obtained for both α‐helical and β‐sheet sites. Spectral simulations of the resulting CW‐EPR report unique site‐specific fluctuations across the surface of GB1. Additionally, we performed molecular dynamics (MD) simulations to complement the EPR data. The dynamics observed from MD agree with the EPR results. Furthermore, we observe small changes ingǁvalues for different sites, which may be due to small differences in coordination geometry and/or local electrostatics of the site. Taken together, this work expands the utility of Cu(II)NTA‐based EPR measurements to probe information beyond distance constraints.

     
    more » « less
  4. ; ; ; ; ; ; ; ; ; ( , The Journal of Physical Chemistry B)
    We demonstrate a host-guest molecular recognition approach to advance double electron-electron resonance (DEER) distance measurements of spin-labeled proteins. We synthesized an iodoacetamide (IA) derivative of 2,6-diazaadamantane nitroxide (DZD) spin label that could be doubly incorporated into T4 Lysozyme (T4L) by site-directed spin labeling (SDSL) with efficiency up to 50% per cysteine. The rigidity of the fused ring structure and absence of mobile methyl groups increase the spin echo dephasing time (Tm) at temperatures above 80 K. This enables DEER measurements of distances >4 nm in DZD labeled-T4L in glycerol/water at temperatures up to 150 K, with increased sensitivity compared to common spin label such as MTSL. Addition of β-cyclodextrin (β-CD) reduces the rotational correlation time of the label, slightly increases Tm, and most importantly, narrows (and slightly lengthens) the inter-spin distance distributions. The distance distributions are in good agreement with simulated distance distributions obtained by rotamer libraries. These results provide a foundation for developing supramolecular recognition to facilitate long-distance DEER measurements at near physiological temperatures. 
    more » « less
  5. ; ; ; ; ( , Angewandte Chemie International Edition)
    Abstract

    Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis‐CuIINTA label to obtain site‐specific information on dynamics for both an α‐helix and β‐sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW‐EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary α‐helix in human glutathione transferase A1‐1 (hGSTA1‐1). We observe two dynamical modes for the helix. The addition of the inhibitor GS‐Met and GS‐Hex resulted in hGSTA1‐1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis‐based labelling approach to measure site‐specific dynamics using room temperature lineshape analysis.

     
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email