Ultrafast Photoinduced Deactivation Dynamics of Proteorhodopsin

We report femtosecond time-resolved absorption change measurements of the photoinduced deactivation dynamics of a microbial rhodopsin in the ultraviolet–visible and mid-infrared range. The blue light quenching process is recorded in green proteorhodopsin’s (GPR) primary proton donor mutant E108Q from the deprotonated 13-<i>cis</i> photointermediate. The return of GPR to the dark state occurs in two steps, starting with the photoinduced 13-<i>cis</i> to all-<i>trans</i> reisomerization of the retinal. The subsequent Schiff base reprotonation via the primary proton acceptor (D97) occurs on a nanosecond time scale. This step is two orders of magnitude faster than that in bacteriorhodopsin, potentially because of the very high p<i>K</i>_A of the GPR primary proton acceptor

Dynamic Nuclear Polarization-Enhanced Solid-State NMR of a ¹³C-Labeled Signal Peptide Bound to Lipid-Reconstituted Sec Translocon

Dynamic nuclear polarization (DNP) has made it possible to record 2D double-quantum-filtered (DQF) solid-state NMR (ssNMR) spectra of a signal peptide bound to a lipid-reconstituted SecYEG translocon complex. The small quantity of peptide in the sample (∼40 nmol) normally prohibits multidimensional ssNMR experiments. Such small amounts are not the exception, because for samples involving membrane proteins, most of the limited sample space is occupied by lipids. As a consequence, a conventional 2D DQF ssNMR spectrum with the sample used here would require many weeks if not months of measurement time. With the help of DNP, however, we were able to acquire such a 2D spectrum within 20 h. This development opens up new possibilities for membrane protein studies, particularly in the exploitation of high-resolution spectroscopy and the assignment of individual amino acid signals, in this case for a signal peptide bound to the translocon complex

Detecting Substrates Bound to the Secondary Multidrug Efflux Pump EmrE by DNP-Enhanced Solid-State NMR

Escherichia coli EmrE, a homodimeric multidrug antiporter, has been suggested to offer a convenient paradigm for secondary transporters due to its small size. It contains four transmembrane helices and forms a functional dimer. We have probed the specific binding of substrates TPP⁺ and MTP⁺ to EmrE reconstituted into 1,2-dimyrist­oyl-<i>sn</i>-glycero-3-phospho­choline liposomes by ³¹P MAS NMR. Our NMR data show that both substrates occupy the same binding pocket but also indicate some degree of heterogeneity of the bound ligand population, reflecting the promiscuous nature of ligand binding by multidrug efflux pumps. Direct interaction between ¹³C-labeled TPP⁺ and key residues within the EmrE dimer has been probed by through-space ¹³C–¹³C correlation spectroscopy. This was made possible by the use of solid-state NMR enhanced by dynamic nuclear polarization (DNP) through which a 19-fold signal enhancement was achieved. Our data provide clear evidence for the long assumed direct interaction between substrates such as TPP⁺ and the essential residue E14 in transmembrane helix 1. Our work also demonstrates the power of DNP-enhanced solid-state NMR at low temperatures for the study for secondary transporters, which are highly challenging for conventional NMR detection

Antigenic Peptide Recognition on the Human ABC Transporter TAP Resolved by DNP-Enhanced Solid-State NMR Spectroscopy

The human transporter associated with antigen processing (TAP) is a 150 kDa heterodimeric ABC transport complex that selects peptides for export into the endoplasmic reticulum and subsequent loading onto major histocompatibility complex class I molecules to trigger adaptive immune responses against virally or malignantly transformed cells. To date, no atomic-resolution information on peptide–TAP interactions has been obtained, hampering a mechanistic understanding of the early steps of substrate translocation catalyzed by TAP. Here, we developed a mild method to concentrate an unstable membrane protein complex and combined this effort with dynamic nuclear polarization enhanced magic angle spinning solid-state NMR to study this challenging membrane protein–substrate complex. We were able to determine the atomic-resolution backbone conformation of an antigenic peptide bound to human TAP. Our NMR data also provide unparalleled insights into the nature of the interactions between the side chains of the antigen peptide and TAP. By combining NMR data and molecular modeling, the location of the peptide binding cavity has been identified, revealing a complex scenario of peptide–TAP recognition. Our findings reveal a structural and chemical basis of substrate selection rules, which define the crucial function of this ABC transporter in human immunity and health. This work is the first NMR study of a eukaryotic transporter protein and presents the power of solid-state NMR in this growing field

Probing the ATP Hydrolysis Cycle of the ABC Multidrug Transporter LmrA by Pulsed EPR Spectroscopy

Members of the ATP binding cassette (ABC) transporter superfamily translocate various types of molecules across the membrane at the expense of ATP. This requires cycling through a number of catalytic states. Here, we report conformational changes throughout the catalytic cycle of LmrA, a homodimeric multidrug ABC transporter from <i>L. lactis.</i> Using site-directed spin labeling and pulsed electron–electron double resonance (PELDOR/DEER) spectroscopy, we have probed the reorientation of the nucleotide binding domains and transmembrane helix 6 which is of particular relevance to drug binding and part of the dimerization interface. Our data show that LmrA samples a very large conformational space in its apo state, which is significantly reduced upon nucleotide binding. ATP binding but not hydrolysis is required to trigger this conformational change, which results in a relatively fixed orientation of both the nucleotide binding domains and transmembrane helices 6. This orientation is maintained throughout the ATP hydrolysis cycle until the protein cycles back to its apo state. Our data present strong evidence that switching between two dynamically and structurally distinct states is required for substrate translocation

Photocycle and Vectorial Proton Transfer in a Rhodopsin from the Eukaryote <i>Oxyrrhis marina</i>

Retinylidene photoreceptors are ubiquitously present in marine protists as first documented by the identification of green proteorhodopsin (GPR). We present a detailed investigation of a rhodopsin from the protist <i>Oxyrrhis marina</i> (OR1) with respect to its spectroscopic properties and to its vectorial proton transport. Despite its homology to GPR, OR1’s features differ markedly in its pH dependence. Protonation of the proton acceptor starts at pH below 4 and is sensitive to the ionic conditions. The mutation of a conserved histidine H62 did not influence the p<i>K</i>_a value in a similar manner as in other proteorhodopsins where the charged histidine interacts with the proton acceptor forming the so-called His-Asp cluster. Mutational and pH-induced effects were further reflected in the temporal behavior upon light excitation ranging from femtoseconds to seconds. The primary photodynamics exhibits a high sensitivity to the environment of the proton acceptor D100 that are correlated to the different initial states. The mutation of the H62 does not affect photoisomerization at neutral pH. This is in agreement with NMR data indicating the absence of the His-Asp cluster. The subsequent steps in the photocycle revealed protonation reactions at the Schiff base coupled to proton pumping even at low pH. The main electrogenic steps are associated with the reprotonation of the Schiff base and internal proton donor. Hence, OR1 shows a different theme of the His-Asp organization where the low p<i>K</i>_a of the proton acceptor is not dominated by this interaction, but by other electrostatic factors

Host–Guest Complexes as Water-Soluble High-Performance DNP Polarizing Agents

Dynamic nuclear polarization (DNP) enhances the sensitivity of solid-state NMR (SSNMR) spectroscopy by orders of magnitude and, therefore, opens possibilities for novel applications from biology to materials science. This multitude of opportunities implicates a need for high-performance polarizing agents, which integrate specific physical and chemical features tailored for various applications. Here, we demonstrate that for the biradical bTbK in complex with captisol (CAP), a β-cyclodextrin derivative, host–guest assembling offers a new and easily accessible approach for the development of new polarizing agents. In contrast to bTbK, the CAP-bTbK complex is water-soluble and shows significantly improved DNP performance compared to the commonly used DNP agent TOTAPOL. Furthermore, NMR and EPR data reveal improved electron and nuclear spin relaxation properties for bTbK within the host molecule. The numerous possibilities to functionalize host molecules will permit designing novel radical complexes targeting diverse applications