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 χ 1 and χ 2 are 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. Over the last three decades, the field of structural biology has made a tremendous progress. This progress should be mainly credited to high-resolution X-ray crystallography and, to a lesser extent, solution-state NMR. More recently, cryo-EM microscopy became a major source of medium-resolution data. Furthermore, solid-state NMR emerged as a valuable addition to the repertoire of structure-solving techniques. However, there are many important protein systems, which defy conventional structure-solving strategies. X-ray diffractometry is contingent on one's ability to obtain a crystalline sample. NMR spectroscopy is limited by the size of the system. Generally, all techniques, including cryo-EM microscopy, tend to have difficulties with those systems that are highly inhomogeneous and/or highly dynamic. Many of the most important cellular systems fall in this category, e.g. chromatin, nuclear pore complex, cilia, etc. Some of the aberrant protein assemblies also have these characteristics, e.g. the so-called protofibrils, neurofibrillary tangles, etc. For those more challenging samples, valuable structural information can often be obtained by means of ESR spectroscopy. Applications of ESR spectroscopy to protein samples usually rely on the popular spin-labeling reagent, (1-oxyl-2,2,5,5-tetramethylpyrrolinyl-3-methyl)-methanethiosulfonate (abbre...

Beyond structure: Deciphering site‐specific dynamics in proteins from double histidine‐based EPR measurements

“…The general autocorrelation function of a function is defined as: where τ represents the lag time and the angular brackets indicate the average value of the function. In our case, the function describes how the averaged orientation of across each frame of the MD simulation correlates with the initial orientation 77,78 . When τ is equal to t , the autocorrelation value is 1 since the averaged orientation is exactly the same as the initial orientation.…”

“…Subsequently, GB1 has been used extensively to develop site‐directed Cu(II) labeling using the dHis motif 37,48,52,53 . In addition, information on the structure and dynamics of R1‐labeled GB1 is also available 28,29,54 . As shown in Figure 1, GB1 contains 56 amino acids over four β sheets and one α helix 55 .…”

“…across each frame of the MD simulation correlates with the initial orientation. 77,78 When τ is equal to t, the autocorrelation value is 1 since the averaged orientation is exactly the same as the initial orientation. As the MD simulation continues, the averaged orientation of…”

“…38,48 Autocorrelation times were processed using python scripts provided by Prof. Nikolai Skrynnikov and Sergei Izmailov, similar to pyxmolpp2. 77,82 The autocorrelation functions were fit to a biexponential decay. Errors were obtained by calculating the range of τ R values which resulted in a minimum RMSD.…”

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site‐Specific Elucidation of Hidden Dynamics in Enzymes

“…Note that earlier MD and EPR distance results support that this site, which is at the end of the β‐sheet, is more dynamic [18] . Furthermore, previous measurements based on R1 were unable to conclusively quantify such subtle changes in backbone dynamics between the sites [10d, 19] …”