Automated Fragmentation Quantum Mechanical Calculation of 15N and 13C Chemical Shifts in a Membrane Protein.

In this work, we developed an accurate and cost-effective automated fragmentation quantum mechanics/molecular mechanics (AF-QM/MM) method to calculate the chemical shifts of 15N and 13C of membrane proteins. The convergence of the AF-QM/MM method was tested using Krokinobacter eikastus rhodopsin 2 as a test case. When the distance threshold of the QM region is equal to or larger than 4.0 Å, the results of the AF-QM/MM calculations are close to convergence. In addition, the effects of selected density functionals, basis sets, and local chemical environment of target atoms on the chemical shift calculations were systematically investigated. Our results demonstrate that the predicted chemical shifts are more accurate when important environmental factors including cross-protomer interactions, lipid molecules, and solvent molecules are taken into consideration, especially for the 15N chemical shift prediction. Furthermore, with the presence of sodium ions in the environment, the chemical shift of residues, retinal, and retinal Schiff base are affected, which is consistent with the results of the solid-state nuclear magnetic resonance (NMR) experiment. Upon comparing the performance of various density functionals (namely, B3LYP, B3PW91, M06-2X, M06-L, mPW1PW91, OB95, and OPBE), the results show that mPW1PW91 is a suitable functional for the 15N and 13C chemical shift prediction of the membrane proteins. Meanwhile, we find that the improved accuracy of the 13Cß chemical shift calculations can be achieved by the employment of the triple-ζ basis set. However, the employment of the triple-ζ basis set does not improve the accuracy of the 15N and 13Cα chemical shift calculations nor does the addition of a diffuse function improve the overall prediction accuracy of the chemical shifts. Our study also underscores that the AF-QM/MM method has significant advantages in predicting the chemical shifts of key ligands and nonstandard residues in membrane proteins than most widely used empirical models; therefore, it could be an accurate computational tool for chemical shift calculations on various types of biological systems.

Structural and functional consequences of the H180A mutation of the light-driven sodium pump KR2.

Krokinobacter eikastus rhodopsin 2 (KR2) is a light-driven pentameric sodium pump. Its ability to translocate cations other than protons and to create an electrochemical potential makes it an attractive optogenetic tool. Tailoring its ion-pumping characteristics by mutations is therefore of great interest. In addition, understanding the functional and structural consequences of certain mutations helps to derive a functional mechanism of ion selectivity and transfer of KR2. Based on solid-state NMR spectroscopy, we report an extensive chemical shift resonance assignment of KR2 within lipid bilayers. This data set was then used to probe site-resolved allosteric effects of sodium binding, which revealed multiple responsive sites including the Schiff base nitrogen and the NDQ motif. Based on this data set, the consequences of the H180A mutation are probed. The mutant is silenced in the presence of sodium while in its absence proton pumping is observed. Our data reveal specific long-range effects along the sodium transfer pathway. These experiments are complemented by time-resolved optical spectroscopy. Our data suggest a model in which sodium uptake by the mutant can still take place, while sodium release and backflow control are disturbed.

Solid-State NMR Spectroscopy on Microbial Rhodopsins.

Microbial rhodopsins represent the most abundant phototrophic systems known today. A similar molecular architecture with seven transmembrane helices and a retinal cofactor linked to a lysine in helix 7 enables a wide range of functions including ion pumping, light-controlled ion channel gating, or sensing. Deciphering their molecular mechanisms therefore requires a combined consideration of structural, functional, and spectroscopic data in order to identify key factors determining their function. Important insight can be gained by solid-state NMR spectroscopy by which the large homo-oligomeric rhodopsin complexes can be studied directly within lipid bilayers. This chapter describes the methodological background and the necessary sample preparation requirements for the study of photointermediates, for the analysis of protonation states, H-bonding and chromophore conformations, for 3D structure determination, and for probing oligomer interfaces of microbial rhodopsins. The use of data extracted from these NMR experiments is discussed in the context of complementary biophysical methods.

Transient Near-UV Absorption of the Light-Driven Sodium Pump Krokinobacter eikastus Rhodopsin 2: A Spectroscopic Marker for Retinal Configuration.

We report a transient signature in the near-UV absorption of Krokinobacter eikastus rhodopsin 2 (KR2), which spans from the femtosecond up to the millisecond time scale. The signature rises with the all-trans to 13-cis isomerization of retinal and decays with the reisomerization to all-trans in the late photocycle, making it a promising marker band for retinal configuration. Hybrid quantum mechanics/molecular mechanics simulations show that the near-UV absorption signal corresponds to an S0 → S3 and/or an S0 → S5 transition, which is present in all photointermediates. These transitions exhibit a negligible spectral shift by the altering protein environment, in contrast to the main absorption band. This is rationalized by the extension of the transition densities that omits the Schiff base nitrogen. Further characterization and first steps into possible optogenetic applications were performed with near-UV quenching experiments of an induced photostationary state, yielding an ultrafast regeneration of the parent state of KR2.

Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR.

The functional mechanism of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) raises fundamental questions since the transfer of cations must differ from the better-known principles of rhodopsin-based proton pumps. Addressing these questions must involve a better understanding of its photointermediates. Here, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance spectroscopy on cryo-trapped photointermediates shows that the K-state with 13-cis retinal directly interconverts into the subsequent L-state with distinct retinal carbon chemical shift differences and an increased out-of-plane twist around the C14-C15 bond. The retinal converts back into an all-trans conformation in the O-intermediate, which is the key state for sodium transport. However, retinal carbon and Schiff base nitrogen chemical shifts differ from those observed in the KR2 dark state all-trans conformation, indicating a perturbation through the nearby bound sodium ion. Our findings are supplemented by optical and infrared spectroscopy and are discussed in the context of known three-dimensional structures.

Time-resolved IR spectroscopy reveals mechanistic details of ion transport in the sodium pump Krokinobacter eikastus rhodopsin 2.

We report a comparative study on the structural dynamics of the light-driven sodium pump Krokinobacter eikastus rhodopsin 2 wild type under sodium and proton pumping conditions by means of time-resolved IR spectroscopy. The kinetics of KR2 under sodium pumping conditions exhibits a sequential character, whereas the kinetics of KR2 under proton pumping conditions involves several equilibrium states. The sodium translocation itself is characterized by major conformational changes of the protein backbone, such as distortions of the α-helices and probably of the ECL1 domain, indicated by distinct marker bands in the amide I region. Carbonyl stretch modes of specific amino acid residues helped to elucidate structural changes in the retinal Schiff base moiety, including the protonation and deprotonation of D116, which is crucial for a deeper understanding of the mechanistic features in the photocycle of KR2.

Solid-state NMR analysis of the sodium pump Krokinobacter rhodopsin 2 and its H30A mutant.

Krokinobacter eikastus rhodopsin 2 (KR2) is a pentameric, light-driven ion pump, which selectively transports sodium or protons. The mechanism of ion selectivity and transfer is unknown. By using conventional as well as dynamic nuclear polarization (DNP)-enhanced solid-state NMR, we were able to analyse the retinal polyene chain between positions C10 and C15 as well as the Schiff base nitrogen in the KR2 resting state. In addition, 50% of the KR2 13C and 15N resonances could be assigned by multidimensional high-field solid-state NMR experiments. Assigned residues include part of the NDQ motif as well as sodium binding sites. Based on these data, the structural effects of the H30A mutation, which seems to shift the ion selectivity of KR2 primarily to Na+, could be analysed. Our data show that it causes long-range effects within the retinal binding pocket and at the extracellular Na+ binding site, which can be explained by perturbations of interactions across the protomer interfaces within the KR2 complex. This study is complemented by data from time-resolved optical spectroscopy.