Two Sides of the Coin: Ezrin/Radixin/Moesin and Merlin Control Membrane Structure and Contact Inhibition.

The merlin-ERM (ezrin, radixin, moesin) family of proteins plays a central role in linking the cellular membranes to the cortical actin cytoskeleton. Merlin regulates contact inhibition and is an integral part of cell-cell junctions, while ERM proteins, ezrin, radixin and moesin, assist in the formation and maintenance of specialized plasma membrane structures and membrane vesicle structures. These two protein families share a common evolutionary history, having arisen and separated via gene duplication near the origin of metazoa. During approximately 0.5 billion years of evolution, the merlin and ERM family proteins have maintained both sequence and structural conservation to an extraordinary level. Comparing crystal structures of merlin-ERM proteins and their complexes, a picture emerges of the merlin-ERM proteins acting as switchable interaction hubs, assembling protein complexes on cellular membranes and linking them to the actin cytoskeleton. Given the high level of structural conservation between the merlin and ERM family proteins we speculate that they may function together.

Mutation in a flexible linker modulates binding affinity for modular complexes.

Tandem beta zippers are modular complexes formed between repeated linear motifs and tandemly arrayed domains of partner proteins in which ß-strands form upon binding. Studies of such complexes, formed by LIM domain proteins and linear motifs in their intrinsically disordered partners, revealed spacer regions between the linear motifs that are relatively flexible but may affect the overall orientation of the binding modules. We demonstrate that mutation of a solvent exposed side chain in the spacer region of an LHX4-ISL2 complex has no significant effect on the structure of the complex, but decreases binding affinity, apparently by increasing flexibility of the linker.

Coherent phenomena in photosynthetic light harvesting: part two-observations in biological systems.

Considerable debate surrounds the question of whether or not quantum mechanics plays a significant, non-trivial role in photosynthetic light harvesting. Many have proposed that quantum superpositions and/or quantum transport phenomena may be responsible for the efficiency and robustness of energy transport present in biological systems. The critical experimental observations comprise the observation of coherent oscillations or "quantum beats" via femtosecond laser spectroscopy, which have been observed in many different light harvesting systems. Part Two of this review aims to provide an overview of experimental observations of energy transfer in the most studied light harvesting systems. Length scales, derived from crystallographic studies, are combined with energy and time scales of the beats observed via spectroscopy. A consensus is emerging that most long-lived (hundreds of femtoseconds) coherent phenomena are of vibrational or vibronic origin, where the latter may result in coherent excitation transport within a protein complex. In contrast, energy transport between proteins is likely to be incoherent in nature. The question of whether evolution has selected for these non-trivial quantum phenomena may be an unanswerable question, as dense packings of chromophores will lead to strong coupling and hence non-trivial quantum phenomena. As such, one cannot discern whether evolution has optimised light harvesting systems for high chromophore density or for the ensuing quantum effects as these are inextricably linked and cannot be switched off.

Coherent phenomena in photosynthetic light harvesting: part one-theory and spectroscopy.

The role of non-trivial quantum mechanical effects in biology has been the subject of intense scrutiny over the past decade. Much of the focus on potential "quantum biology" has been on energy transfer processes in photosynthetic light harvesting systems. Ultrafast laser spectroscopy of several light harvesting proteins has uncovered coherent oscillations dubbed "quantum beats" that persist for hundreds of femtoseconds and are putative signatures for quantum transport phenomena. This review describes the language and basic quantum mechanical phenomena that underpin quantum transport in open systems such as light harvesting and photosynthetic proteins, including the photosystem reaction centre. Coherent effects are discussed in detail, separating various meanings of the term, from delocalized excitations, or excitons, to entangled states and coherent transport. In particular, we focus on the time, energy and length scales of energy transport processes, as these are critical in understanding whether or not coherent processes are important. The role played by the protein in maintaining chromophore systems is analysed. Finally, the spectroscopic techniques that are used to probe energy transfer dynamics and that have uncovered the quantum beats are described with reference to coherent phenomena in light harvesting.

Disparate binding kinetics by an intrinsically disordered domain enables temporal regulation of transcriptional complex formation.

Intrinsically disordered regions are highly represented among mammalian transcription factors, where they often contribute to the formation of multiprotein complexes that regulate gene expression. An example of this occurs with LIM-homeodomain (LIM-HD) proteins in the developing spinal cord. The LIM-HD protein LHX3 and the LIM-HD cofactor LDB1 form a binary complex that gives rise to interneurons, whereas in adjacent cell populations, LHX3 and LDB1 form a rearranged ternary complex with the LIM-HD protein ISL1, resulting in motor neurons. The protein-protein interactions within these complexes are mediated by ordered LIM domains in the LIM-HD proteins and intrinsically disordered LIM interaction domains (LIDs) in LDB1 and ISL1; however, little is known about how the strength or rates of binding contribute to complex assemblies. We have measured the interactions of LIM:LID complexes using FRET-based protein-protein interaction studies and EMSAs and used these data to model population distributions of complexes. The protein-protein interactions within the ternary complexes are much weaker than those in the binary complex, yet surprisingly slow LDB1:ISL1 dissociation kinetics and a substantial increase in DNA binding affinity promote formation of the ternary complex over the binary complex in motor neurons. We have used mutational and protein engineering approaches to show that allostery and modular binding by tandem LIM domains contribute to the LDB1LID binding kinetics. The data indicate that a single intrinsically disordered region can achieve highly disparate binding kinetics, which may provide a mechanism to regulate the timing of transcriptional complex assembly.

A Quantitative Fluorescence-Based Assay for Assessing LIM Domain-Peptide Interactions.

We have developed Förster resonance energy transfer (FRET)-based experiments for measuring the binding affinity, off-rates, and inferred on-rates for interactions between a family of transcriptional regulators and their intrinsically disordered binding partners. It was difficult to evaluate these interactions previously, as the transcriptional regulators are obligate binding proteins that aggregate in the absence of a binding partner. The assays rely on fusion constructs where binding domains are linked by a flexible tether containing a specific protease site, with fluorescent proteins at either end that display FRET when the complex is formed. Loss of FRET is monitored after cutting the tether followed by dilution or competition with a non-fluorescent peptide. These methods allowed a wide range of binding affinities (10-9 -10-5 m) to be determined. Our data indicate that interactions of closely related proteins can have surprisingly different binding properties.