In situ observation of conformational dynamics and protein ligand-substrate interactions in outer-membrane proteins with DEER/PELDOR spectroscopy.

Observation of structure and conformational dynamics of membrane proteins at high resolution in their native environments is challenging because of the lack of suitable techniques. We have developed an approach for high-precision distance measurements in the nanometer range for outer-membrane proteins (OMPs) in intact Escherichia coli and native membranes. OMPs in Gram-negative bacteria rarely have reactive cysteines. This enables in situ labeling of engineered cysteines with a methanethiosulfonate spin label (MTSL) with minimal background signals. Following overexpression of the target protein, spin labeling is performed with E. coli or isolated outer membranes (OMs) under selective conditions. The interspin distances are measured in situ, using pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. The residual background signals, which are problematic for in situ structural biology, contribute specifically to the intermolecular part of the signal and can be selectively removed to extract the desired interspin distance distribution. The initial cloning stage can take 5-7 d, and the subsequent protein expression, OM isolation, spin labeling, PELDOR experiment, and data analysis typically take 4-5 d. The described protocol provides a general strategy for observing protein ligand-substrate interactions, oligomerization, and conformational dynamics of OMPs in their native OM and intact E. coli.

Structural basis for functional interactions in dimers of SLC26 transporters.

The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.

Phosphoramidite building blocks with protected nitroxides for the synthesis of spin-labeled DNA and RNA.

TEMPO spin labels protected with 2-nitrobenzyloxymethyl groups were attached to the amino residues of three different nucleosides: deoxycytidine, deoxyadenosine, and adenosine. The corresponding phosphoramidites could be incorporated by unmodified standard procedures into four different self-complementary DNA and two RNA oligonucleotides. After photochemical removal of the protective group, elimination of formic aldehyde and spontaneous air oxidation, the nitroxide radicals were regenerated in high yield. The resulting spin-labeled palindromic duplexes could be directly investigated by PELDOR spectroscopy without further purification steps. Spin-spin distances measured by PELDOR correspond well to the values obtained from molecular models.

A Cytidine Phosphoramidite with Protected Nitroxide Spin Label: Synthesis of a Full-Length TAR RNA and Investigation by In-Line Probing and EPR Spectroscopy.

EPR studies on RNA are complicated by three major obstacles related to the chemical nature of nitroxide spin labels: Decomposition while oligonucleotides are chemically synthesized, further decay during enzymatic strand ligation, and undetected changes in conformational equilibria due to the steric demand of the label. Herein possible solutions for all three problems are presented: A 2-nitrobenzyloxymethyl protective group for nitroxides that is stable under all conditions of chemical RNA synthesis and can be removed photochemically. By careful selection of ligation sites and splint oligonucleotides, high yields were achieved in the assembly of a full-length HIV-1 TAR RNA labeled with two protected nitroxide groups. PELDOR measurements on spin-labeled TAR in the absence and presence of arginine amide indicated arrest of interhelical motions on ligand binding. Finally, even minor changes in conformation due to the presence of spin labels are detected with high sensitivity by in-line probing.

A combined optical and EPR spectroscopy study: azobenzene-based biradicals as reversible molecular photoswitches.

Azobenzene compounds are known as versatile examples for photoswitchable systems because of their isomeric cis- and trans-configurations. The switching between these isomers can be reversibly controlled by light excitation. In this study we characterize two members of this class by joining the azobenzene moiety with each two paramagnetic nitroxide spin labels. Two different linkers were chosen to tune the molecular properties. The combined approach using optical and EPR spectroscopy proved the reversibility of photoexcitation and high fatigue resistance. Furthermore, depending on the nature of the linker, PELDOR distance measurements monitored clearly the photo-induced structural changes of the azobenzene unit. Thus, a powerful concept is presented resulting from the combination of these two complementary spectroscopic techniques.

Hydration layer coupling and cooperativity in phase behavior of stimulus responsive peptide polymers.

It is shown that hydrophilic (backbone) and hydrophobic (side chain) hydration layers of elastin-like polypeptides (ELPs), a class of stimulus responsive peptide polymers that exhibit lower critical solution temperature (LCST) phase transition behavior, can exist in a coupled and decoupled state. The decoupled hydration state consists of hydrophobic and hydrophilic hydration layers that respond independently to temperature, while the coupled hydration state is characterized by a common, cooperative dehydration of both hydration layers. It is further shown that the primary sequence of an ELP can be tuned to exhibit either of the hydration layer coupling modes. Charged side chains lead to decoupling, while strongly hydrophobic side chains trigger stronger interaction between hydrophilic and hydrophobic hydration, leading to coupling of both layers. Further, for aprotic residues this coupling is fostered by decreasing bulkiness of hydrophobic side chains due to larger hydration numbers and water molecules mediating coupling between side chain and backbone hydration shells. For coupled hydration shells, the LCST phase transition characterized by spin probing continuous wave electron paramagnetic resonance spectroscopy is reminiscent of a first-order process even on nanoscopic length scales. In contrast, analogous synthetic polymers exhibit nanoscale phase transitions over a broad temperature range, indicating that their nanoscale phase behavior is not of first order. Hence, our results indicate that ELPs are the first identified class of polymers that exhibit a first-order inverse phase transition on nanoscopic length scales. These results may also provide insights into the role of hydration layers in governing the structure-function relationship of intrinsically disordered proteins.