Structure Relaxation Approximation (SRA) for Elucidation of Protein Structures from Ion Mobility Measurements (II). Protein Complexes

Abstract

Characterizing structures of protein complexes and their diseaserelated aberrations is essential to understanding molecular mechanisms of many biological processes. Electrospray ionization coupled with hybrid ion mobility/ mass spectrometry (ESI-IM/MS) methods offer sufficient sensitivity, sample throughput, and dynamic range to enable systematic structural characterization of proteomes. However, because ESI-IM/MS characterizes ionized protein systems in the gas phase, it generally remains unclear to what extent the protein ions characterized by IM/MS have retained their solution structures. Here, we discuss the first application of our computational structure relaxation approximation et al. J. Phys. Chem. B 2019, 123 (13), 2756−2769 to assign structures of protein complexes in the range from ∼16 to ∼60 kDa from their "native" IM/MS spectra. Our analysis shows that the computed IM/MS spectra agree with the experimental spectra within the errors of the methods. The structure relaxation approximation (SRA) indicates that native backbone contacts appear largely retained in the absence of solvent for the investigated protein complexes and charge states. Native contacts between polypeptide chains of the protein complex appear to be retained to a comparable extent as contacts within a folded polypeptide chain. Our computations also indicate that the hallmark "compaction" often observed for protein systems in native IM/MS measurements appears to be a poor indicator of the extent to which native residue−residue interactions are lost in the absence of solvent. Further, the SRA indicates that structural reorganization of the protein systems in IM/MS measurements appears driven largely by remodeling of the protein surface that increases its hydrophobic content by approximately 10%. For the systems studied here, this remodeling of the protein surface appears to occur mainly by structural reorganization of surface-associated hydrophilic amino acid residues not associated with β-strand secondary structure elements. Properties related to the internal protein structure, as assessed by void volume or packing density, appear unaffected by remodeling of the surface. Taken together, the structural reorganization of the protein surface appears to be generic in nature and to sufficiently stabilize protein structures to render them metastable on the time scale of IM/MS measurements.

Elucidating Structures of Protein Complexes by Collision-Induced Dissociation at Elevated Gas Pressures

“…To this end, we electrosprayed the protein complexes from native solution conditions and recorded their cross-sections in TIMS-1. As described in more detail elsewhere, comparison of the recorded cross-sections of the protein complexes to prior literature ,, and cross-sections calculated for their crystal structures by our PSA method − (Figure S1, Supporting Information) indicates that the precursor protein complexes retained their native-like structures.…”

“…In a dimer-of-dimers complex, the strong interface (a) connects two monomeric subunits into a strongly bound dimer, while two weak interfaces (b and c) arrange the dimer subunits into a tetramer (Figure D) . The X-ray structures reveal a lower number of salt bridges and hydrogen bonds in the b and c interfaces of streptavidin compared to those of neutravidin. ,, Further, our structure relaxation approximation (SRA) calculations indicate that fewer of these native contacts are retained in the interfaces b and c for streptavidin (∼45%) than for neutravidin (∼75%) in the absence of solvent . Thus, it is expected that cleaving the b and c interfaces in streptavidin require less energy than in neutravidin, resulting in a higher abundance of dimers for streptavidin.…”

“…Hence, the native neutravidin dimer is significantly stronger bound than the streptavidin dimer. Our SRA calculations indicate that these differences in their binding strengths are maintained upon removal of solvent because the native contacts of the dimer units within the intact, native-like tetrameric complexes are equally retained for both streptavidin and neutravidin (∼85%) . Thus, these considerations explain the apparent higher dissociation barrier and higher stability of the neutravidin dimers against further dissociation into monomers.…”

“…When “soft” IM/MS measurements are carried out under native conditions, long-standing efforts have established that even small monomeric proteins can be kinetically trapped in conformations close to their native structures that are metastable on the time scale of IM/MS experiments. − There is also ample evidence that protein complexes largely retain their native structures. ,,,− Indeed, computational modeling indicates that protein complexes retain up to ∼80% to 90% of their native contacts, with loss of native contacts occurring mainly in flexible regions by self-solvation of amino acid side chains. ,, However, much less is known about what structures are adopted by the subunits of protein complexes produced in situ in a mass spectrometer upon dissociation of the precursor protein complex and how they relate to the native structure of the protein complex.…”

“…It is also noteworthy that our experimental results obtained from CID of protein complexes in tandem-TIMS/MS are consistent with the computational findings via our SRA method. For instance, the higher stability of neutravidin dimers toward monomer fragmentation compared to those of streptavidin was predicted by the SRA calculations . It follows that detailed structural information on protein subunits could potentially be extrapolated from SRA calculations.…”