Molecular Structure of the Surface-Immobilized Super Uranyl Binding Protein

Abstract

Recently, a super uranyl binding protein (SUP) was developed, which exhibits excellent sensitivity/selectivity to bind uranyl ions. It can be immobilized onto a surface in sensing devices to detect uranyl ions. Here, sum frequency generation (SFG) vibrational spectroscopy was applied to probe the interfacial structures of surface-immobilized SUP. The collected SFG spectra were compared to the calculated orientation-dependent SUP SFG spectra using a one-excitonic Hamiltonian approach based on the SUP crystal structures to deduce the most likely surface-immobilized SUP orientation(s). Furthermore, discrete molecular dynamics (DMD) simulation was applied to refine the surface-immobilized SUP conformations and orientations. The immobilized SUP structures calculated from DMD simulations confirmed the SUP orientations obtained from SFG data analyzed based on the crystal structures and were then used for a new round of SFG orientation analysis to more accurately determine the interfacial orientations and conformations of immobilized SUP before and after uranyl ion binding, providing an in-depth understanding of molecular interactions between SUP and the surface and the effect of uranyl ion binding on the SUP interfacial structures. We believe that the developed method of combining SFG measurements, DMD simulation, and Hamiltonian data analysis approach is widely applicable to study biomolecules at solid/liquid interfaces.

Probing protein aggregation at buried interfaces: distinguishing between adsorbed protein monomers, dimers, and a monomer–dimer mixture in situ

“…The combined computer simulation and SFG studies can be used to identify the most likely protein conformation and orientation, as we demonstrated previously. 60 Such a combined study can also verify the conclusions obtained from the simulation, and provide more detailed and more reliable structural information from SFG studies. Although this research only shows the capability of SFG to study BSA monomers and dimers, we believe that SFG is able to examine protein aggregates with more complicated structures such as trimer, tetramers etc.…”

“…, ssp and ppp) spectra of BSA as a function of BSA orientation. A schematic to show the Hamiltonian method has been published previously 60 and a revised schematic is included in the ESI (Section S2). † For BSA with a certain orientation, the Hamiltonian method started with a data matrix with diagonal elements as the peak centers of the uncoupled amide I signal of each amino acid ( e.g.…”

“… 57–60 We have shown in our previous publication that such a Hamiltonian data analysis approach can be used along with SFG results to deduce interfacial protein orientation and conformation. 60 Using the Hamiltonian method, we can calculate the SFG spectra of adsorbed BSA as a function of orientation. The calculated BSA orientation dependent SFG spectra of the BSA monomer and dimer are markedly different.…”

Probing Orientations and Conformations of Peptides and Proteins at Buried Interfaces

“…The protein structure obtained from MD simulations should be used to calculate SFG signal strength ratio to determine protein orientation (as well as the conformation) at interfaces. 43 In addition to SFG, we also applied attenuated total reflectance (ATR)-FTIR spectroscopy as a supplemental tool to study interfacial peptides/proteins. 37,41 ATR-FTIR measures different orientation parameters compared to SFG, thus providing additional independent measurements to SFG.…”

“…41 Recently, we adopted a Hamiltonian approach to calculate SFG spectra to compare to the experimentally measured data to determine interfacial protein orientation and conforma-tion. 43 Differing from our earlier SFG data analysis method for protein orientation determination, which used the SFG signals contributed from the protein α-helical components, 40,41 here the Hamiltonian approach calculates SFG spectra using all the amino acids in various secondary structures in the protein. 43 First, a Hamiltonian matrix was constructed with the uncoupled amide I stretching peak center (e.g., 1640 cm −1 ) as the diagonal elements.…”