Strategies to identify and suppress crosstalk signals in double electron–electron resonance (DEER) experiments with gadolinium^III and nitroxide spin-labeled compounds

Double electron–electron resonance (DEER) spectroscopy applied to orthogonally spin-labeled biomolecular complexes simplifies the assignment of intra- and intermolecular distances, thereby increasing the information content per sample. In fact, various spin labels can be addressed independently in DEER experiments due to spectroscopically nonoverlapping central transitions, distinct relaxation times, and/or transition moments; hence, they are referred to as spectroscopically orthogonal. Molecular complexes which are, for example, orthogonally spin-labeled with nitroxide (NO) and gadolinium (Gd) labels give access to three distinct DEER channels that are optimized to selectively probe NO–NO, NO–Gd, and Gd–Gd distances. Nevertheless, it has been previously recognized that crosstalk signals between individual DEER channels can occur, for example, when a Gd–Gd distance appears in a DEER channel optimized to detect NO–Gd distances. This is caused by residual spectral overlap between NO and Gd spins which, therefore, cannot be considered as perfectly orthogonal. Here, we present a systematic study on how to identify and suppress crosstalk signals that can appear in DEER experiments using mixtures of NO–NO, NO–Gd, and Gd–Gd molecular rulers characterized by distinct, nonoverlapping distance distributions. This study will help to correctly assign the distance peaks in homo- and heterocomplexes of biomolecules carrying not perfectly orthogonal spin labels.

New capsaicin analogs as molecular rulers to define the permissive conformation of the mouse TRPV1 ligand-binding pocket

The capsaicin receptor TRPV1 is an outstanding representative of ligand-gated ion channels in ligand selectivity and sensitivity. However, molecular interactions that stabilize the ligand-binding pocket in its permissive conformation, and how many permissive conformations the ligand-binding pocket may adopt, remain unclear. To answer these questions, we designed a pair of novel capsaicin analogs to increase or decrease the ligand size by about 1.5 Å without altering ligand chemistry. Together with capsaicin, these ligands form a set of molecular rulers for investigating ligand-induced conformational changes. Computational modeling and functional tests revealed that structurally these ligands alternate between drastically different binding poses but stabilize the ligand-binding pocket in nearly identical permissive conformations; functionally, they all yielded a stable open state despite varying potencies. Our study suggests the existence of an optimal ligand-binding pocket conformation for capsaicin-mediated TRPV1 activation gating, and reveals multiple ligand-channel interactions that stabilize this permissive conformation.

Orthogonally spin-labeled rulers help to identify crosstalk signals and improve DEER signal fidelity

DEER spectroscopy applied to orthogonally spin-labeled proteins is a versatile technique which allows simplifying the assignment of distances in complex spin systems and thereby increasing the information content that can be obtained per sample. In fact, orthogonal spin labels can be independently addressed in DEER experiments due to spectroscopically non-overlapping central transitions, distinct relaxation times and/or transition moments. Here we focus on molecular rulers orthogonally labeled with nitroxide (NO) and gadolinium (Gd) spins, which give access to three distinct DEER channels, probing NO-NO, NO-Gd and Gd-Gd distances. It has been previously suggested that crosstalk signals between individual DEER channels might occur, for example, between NO and Gd due to their inevitable spectral overlap. However, a systematic study to address these issues has not yet been carried out. Here, we perform a thorough three-channel DEER analysis on mixtures of NO-NO, NO-Gd and Gd-Gd molecular rulers characterized by distinct, non-overlapping distance distributions to study under which conditions crosstalk signals occur and how they can be identified or suppressed to improve signal fidelity. This study will help to improve the assignment of the correct distances in homo- and hetero-complexes of orthogonally spin-labeled proteins.

Accessing distributions of exchange and dipolar couplings in stiff molecular rulers with Cu(ii) centres

Novel approaches to quantitatively analyse distributed exchange couplings are described and tested on experimental data sets for stiff synthetic molecules.

Spatiotemporal control of mitotic exit during anaphase by an aurora B-Cdk1 crosstalk

According to the prevailing ‘clock’ model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the ‘ruler’ model. Here we found that Cdk1 remains active during anaphase due to ongoing APC/CCdc20- and APC/CCdh1-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular ‘rulers’ and ‘clocks’ licenses mitotic exit only after proper chromosome separation.