Transient complexes of redox proteins: structural and dynamic details from NMR studies

Redox proteins participate in many metabolic routes, in particular those related to energy conversion. Protein-protein complexes of redox proteins are characterized by a weak affinity and a short lifetime. Twodimensional NMR spectroscopy has been applied to many redox protein complexes, providing a wealth of information about the process of complex formation, the nature of the interface and the dynamic properties of the complex. These studies have shown that some complexes are non-specific and exist as a dynamic ensemble of orientations while in other complexes the proteins assume a single orientation. The binding interface in these complexes consists of a small hydrophobic patch for specificity, surrounded by polar, uncharged residues that may enhance dissociation, and, in most complexes, a ring or patch of charged residues that enhances the association by electrostatic interactions. The entry and exit port of the electrons is located within the hydrophobic interaction site, ensuring rapid electron transfer from one redox centre to the next

Dynamics In The Transient Complex Of Plastocyanin-cytochrome F From Prochlorothrix Hollandica

The nature of transient protein complexes can range from a highly dynamic ensemble of orientations to a single well-defined state. This represents variation in the equilibrium between the encounter and final, functional state. The transient complex between plastocyanin (Pc) and cytochrome f (cyt of the cyanobacterium Prochlorothrix hollandica was characterized by NMR spectroscopy. Intermolecular pseudocontact shifts and chemical shift perturbations were used as restraints in docking calculations to determine the structure of the wild-type Pc-cytf complex. The orientation of Pc is similar to orientations found in Pc-cytf complexes from other sources. Electrostatics seems to play a modest role in complex formation. A large variability in the ensemble of lowest energy structures indicates a dynamic nature of the complex. Two unusual hydrophobic patch residues in Pc have been mutated to the residues found in other plastocyanins (Y12G/P14L). The binding constants are similar for the complexes of cytf with wild-type PC and mutant PC, but the chemical shift perturbations are smaller for the complex with mutant PC. Docking calculations for the Y12G/P14L Pc-cytf complex did not produce a converged ensemble of structures. Simulations of the dynamics were performed using the observed averaged NMR parameters as input. The results indicate a surprisingly large amplitude of mobility of Y12G/P14L Pc within the complex. It is concluded that the double mutation shifts the complex further from the well-defined toward the encounter state

A two-armed probe for in-cell DEER measurements on proteins

The application of double electron-electron resonance (DEER) with site-directed spin labeling (SDSL) to measure distances in proteins and protein complexes in living cells puts rigorous restraints on the spin-label. The linkage and paramagnetic centers need to resist the reducing conditions of the cell. Rigid attachment of the probe to the protein improves precision of the measured distances. Here, three two-armed GdIII complexes, GdIII-CLaNP13a/b/c were synthesized. Rather than the disulfide linkage of most other CLaNP molecules, a thioether linkage was used to avoid reductive dissociation of the linker. The doubly GdIII labeled N55C/V57C/K147C/T151C variants of T4Lysozyme were measured by 95 GHz DEER. The constructs were measured in vitro, in cell lysate and in Dictyostelium discoideum cells. Measured distances were 4.5 nm, consistent with results from paramagnetic NMR. A narrow distance distribution and typical modulation depth, also in cell, indicate complete and durable labeling and probe rigidity due to the dual attachment sites

Corrigendum: a two-armed probe for in-cell DEER measurements on proteins (vol 26, pg 17128, 2020)

CORRIGENDUM Q. Miao, E. Zurlo, D. de Bruin, J. A. J. Wondergem, S. P. Skinner, M. Timmer, A. Blok, D. Heinrich, M. Overhand, M. Huber,* M. Ubbink* 17128–17133 A Two-Armed Probe for In-Cell DEER Measurements on Proteins Chem. Eur. J., 2020, 26 DOI: 10.1002/chem.202002743 All authors have agreed that Dr. Simon P. Skinner has made a significant contribution to this work by performing experiments and analyzing data and that his name should have been included in the list of authors. The corrected list of authors therefore reads: Dr. Qing Miao, Dr. Enrico Zurlo, Donny de Bruin, Joeri A. J. Wondergem, Dr. Simon P. Skinner, Monika Timmer, Anneloes Blok, Prof. Dr. Doris Heinrich, Dr. Mark Overhand, Dr. Martina Huber, Prof. Dr. Marcellus Ubbink The relevant affiliations for Dr. Skinner are (1) Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, Einsteinweg 55, 2333, CC Leiden, The Netherlands and (2) School of Molecular and Cellular Biology and Astbury Centre, University of Leeds, Leeds LS2 9JT, UK. The Acknowledgement section should not contain the sentence “and Dr. Simon Skinner for CLaNP5 labeled T4lys NMR data.Macromolecular Biochemistr

Mapping the encounter state of a transient protein complex by PRE NMR spectroscopy

Many biomolecular interactions proceed via a short-lived encounter state, consisting of multiple, lowly-populated species invisible to most experimental techniques. Recent development of paramagnetic relaxation enhancement (PRE) nuclear magnetic resonance (NMR) spectroscopy has allowed to directly visualize such transient intermediates in a number of protein-protein and protein-DNA complexes. Here we present an analysis of the recently published PRE NMR data for a protein complex of yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP). First, we describe a simple, general method to map out the spatial and temporal distributions of binding geometries constituting the Cc-CcP encounter state. We show that the spatiotemporal mapping provides a reliable estimate of the experimental coverage and, at higher coverage levels, allows to delineate the conformational space sampled by the minor species. To further refine the encounter state, we performed PRE-based ensemble simulations. The generated solutions reproduce well the experimental data and lie within the allowed regions of the encounter maps, confirming the validity of the mapping approach. The refined encounter ensembles are distributed predominantly in a region encompassing the dominant form of the complex, providing experimental proof for the results of classical theoretical simulations

Methyl group assignment using pseudocontact shifts with PARAssign

Macromolecular Biochemistr