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αB-crystallin is a highly dynamic, polydisperse small heat-shock protein that can form oligomers ranging in mass from 200 to 800 kDa. Here we use a multifaceted mass spectrometry approach to assess the role of the C-terminal tail in the self-assembly of αB-crystallin. Titration experiments allow us to monitor the binding of peptides representing the C-terminus to the αB-crystallin core domain, and observe individual affinities to both monomeric and dimeric forms. Notably, we find that binding the second peptide equivalent to the core domain dimer is considerably more difficult than the first, suggesting a role of the C-terminus in regulating assembly. This finding motivates us to examine the effect of point mutations in the C-terminus in the full-length protein, by quantifying the changes in oligomeric distribution and corresponding subunit exchange rates. Our results combine to demonstrate that alterations in the C-terminal tail have a significant impact on the thermodynamics and kinetics of αB-crystallin. Remarkably, we find that there is energy compensation between the inter- and intra-dimer interfaces: when one interaction is weakened, the other is strengthened. This allosteric communication between binding sites on αB-crystallin is likely important for its role in binding target proteins.

The activation of the dodecameric Ca2+/calmodulin dependent kinase II (CaMKII) holoenzyme is critical for memory formation. We now report that CaMKII has a remarkable property, which is that activation of the holoenzyme triggers the exchange of subunits between holoenzymes, including unactivated ones, enabling the calcium-independent phosphorylation of new subunits. We show, using a single-molecule TIRF microscopy technique, that the exchange process is triggered by the activation of CaMKII, and that exchange is modulated by phosphorylation of two residues in the calmodulin-binding segment, Thr 305 and Thr 306. Based on these results, and on the analysis of molecular dynamics simulations, we suggest that the phosphorylated regulatory segment of CaMKII interacts with the central hub of the holoenzyme and weakens its integrity, thereby promoting exchange. Our results have implications for an earlier idea that subunit exchange in CaMKII may have relevance for information storage resulting from brief coincident stimuli during neuronal signaling. How do fleeting signals passing through the neurons of our brains become memories that can last for years or even decades? An enzyme called CaMKII is known to have an important role in the formation of memories. CaMKII adds phosphate groups to proteins—a process that is called phosphorylation—and is itself activated when calcium levels increase inside the neurons where the enzyme is found. Individual CaMKII proteins bind together in groups of 12 to form a ‘holoenzyme’. When one of the 12 subunits is activated by calcium, it can phosphorylate the other subunits in the same holoenzyme. Once this happens, the activation of CaMKII can continue after the initial rise in calcium has ceased, and this effect is thought to be involved in the formation of long-term memories. 30 years ago, Francis Crick—famous for his role in the discovery of the double helix—proposed that memory formation might involve ‘memory-storage molecules’ passing an activated state to unactivated molecules, and John Lisman later suggested that CaMKII could fulfil this role by swapping subunits of holoenzymes between activated and unactivated ones. Now, Stratton, Lee et al. have tested whether CaMKII can exchange subunits by using advanced microscopy to track single molecules of CaMKII labelled with fluorescent markers. This revealed that activation can cause CaMKII subunits repeatedly to mix between holoenzymes—and this only happens once a first holoenzyme has been activated. Subunits of CaMKII join together via a central ‘hub’ region, but when a subunit is activated, the phosphorylated segment may interact with the hub. This weakens the connections between the subunits, thereby making it easier for subunits to exchange between holoenzymes. This process provides a mechanism by which a level of activated CaMKII can be maintained, even if some subunits become degraded and long after the disappearance of the initial activation signal.

Single-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli . Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNA-binding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT) 70 , one molecule of (dT) 35 , or two molecules of (dT) 35 ] was found to prevent SSB subunit exchange. Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry.

Activation triggers the exchange of subunits in Ca2+/calmodulin-dependent protein kinase II (CaMKII), an oligomeric enzyme that is critical for learning, memory, and cardiac function. The mechanism by which subunit exchange occurs remains elusive. We show that the human CaMKII holoenzyme exists in dodecameric and tetradecameric forms, and that the calmodulin (CaM)-binding element of CaMKII can bind to the hub of the holoenzyme and destabilize it to release dimers. The structures of CaMKII from two distantly diverged organisms suggest that the CaM-binding element of activated CaMKII acts as a wedge by docking at intersubunit interfaces in the hub. This converts the hub into a spiral form that can release or gain CaMKII dimers. Our data reveal a three-way competition for the CaM-binding element, whereby phosphorylation biases it towards the hub interface, away from the kinase domain and calmodulin, thus unlocking the ability of activated CaMKII holoenzymes to exchange dimers with unactivated ones. How does memory outlast the lifetime of the molecules that encode it? One enzyme that is found in neurons and has been suggested to help long-term memories to form is called CaMKII. Each CaMKII assembly is typically composed of 12 to 14 protein subunits associated in a ring and can exist in either an “unactivated” or “activated” state. In 2014, researchers showed that CaMKII assemblies can exchange subunits with each other. Importantly, an active CaMKII can mix with an unactivated CaMKII and share its activation state. CaMKII may use this mechanism to spread information to the next generation of proteins – thereby allowing activation to outlast the lifespan of the initially activated proteins. However the molecular mechanism that underlies this process was not clear. Now, Bhattacharyya et al. – including some of the researchers involved in the 2014 work – address two questions about this mechanism. How do subunits exchange between CaMKII assemblies? And how does the activation of CaMKII initiate subunit exchange? A closed-ring hub ties the subunits of CaMKII together, similar to the organization of the segments in an orange. To undergo subunit exchange, the hub must open up to release and accept subunits. Bhattacharyya et al. have now uncovered an intrinsic flexibility in the hub that is triggered by a short peptide segment in CaMKII. This segment, which is exposed in activated CaMKII but not in the unactivated form, can crack open the hub ring by binding between the hub subunits, like a finger separating the segments of an orange. This allows the hub to flex and expand, and once open, the hub’s flexibility allows room for subunits to be released or accepted. Although this subunit exchange mechanism could be a powerful means for spreading the activated state throughout signaling pathways, the biological relevance of this phenomenon has not been clarified. However, the mechanistic framework provided by Bhattacharyya et al. may allow new experiments to be performed that test the consequences of subunit exchange in live cells and organisms. It could also enable investigations into the importance of subunit exchange in long-term memory.

Here, we report novel methods to measure rate constants for homodimer subunit exchange using double electron–electron resonance (DEER) electron paramagnetic resonance spectroscopy measurements and nuclear magnetic resonance spectroscopy based paramagnetic relaxation enhancement (PRE) measurements. The techniques were demonstrated using the homodimeric protein Dsy0195 from the strictly anaerobic bacterium Desulfitobacterium hafniense Y51. At specific times following mixing site-specific MTSL-labeled Dsy0195 with uniformly 15 N-labeled Dsy0195, the extent of exchange was determined either by monitoring the decrease of MTSL-labeled homodimer from the decay of the DEER modulation depth or by quantifying the increase of MTSL-labeled/ 15 N-labeled heterodimer using PREs. Repeated measurements at several time points following mixing enabled determination of the homodimer subunit dissociation rate constant, k −1 , which was 0.037 ± 0.005 min −1 derived from DEER experiments with a corresponding half-life time of 18.7 min. These numbers agreed with independent measurements obtained from PRE experiments. These methods can be broadly applied to protein–protein and protein-DNA complex studies.

The exchange of CaMKII enzymes between larger structures called holoenzymes may provide the molecular mechanism underlying the long-term stability of memories.The exchange of CaMKII enzymes between larger structures called holoenzymes may provide the molecular mechanism underlying the long-term stability of memories.

In human lenses, C-terminal cleavage of αA-crystallin at residues 172,168, and 162 have been reported. The effect of C-terminal truncation of αA-crystallin on subunit exchange and heterooligomer formation with αB-crystallin and homooligomer formation with native αA-crystallin is not known. We have conducted fluorescence resonance energy transfer studies which have shown that the rates of subunit exchange of αA₁-₁₇₂ and αA₁-₁₆₈ with αB-wt were two-fold lower than for αA-wt interacting with αB-wt. The subunit exchange rate between αA₁-₁₆₂ and αB-wt was six-fold lower. These data suggest that cleavage of the C-terminal residues could significantly affect heterooligomerization. On the other hand, the subunit exchange rates between αA-wt and the truncated αA-crystallins were either unchanged or only slightly decreased, which suggest that homooligomerization may not be significantly influenced by C-terminal truncation. The main conclusion from this study is that cleavage of C-terminal residues of αA-crystallin including the nine residues of the flexible tail is expected to significantly affect the formation of heteroaggregates. Reconstitution experiments showed that the presence of an intact C-terminus is essential for the formation of fully integrated heteroaggregates with equal proportion of αA and αB subunits.

The bacteriophage T4 AsiA protein is a multifunctional protein that simultaneously acts as both a repressor and activator of gene expression during the phage life cycle. These dual roles with opposing transcriptional consequences are achieved by modification of the host RNA polymerase in which AsiA binds to conserved region 4 (SR4) of σ 70 , altering the pathway of promoter selection by the holoenzyme. The mechanism by which AsiA flips this genetic switch has now been revealed, in part, from the three‐dimensional structure of AsiA and the elucidation of its interaction with SR4. The structure of AsiA is that of a novel homodimer in which each monomer is constructed as a seven‐helix bundle arranged in four overlapping helix—loop—helix elements. Identification of the protein interfaces for both the AsiA homodimer and the AsiA—σ 70 complex reveals that these interfaces are coincident. Thus, the AsiA interaction with σ 70 necessitates that the AsiA homodimer dissociate to form an AsiA—SR4 heterodimer, exchanging one protein subunit for another to alter promoter choice by RNA polymerase.

Escherichia coli glucosamine-6-phosphate synthase (GlmS) is a dimeric enzyme from the glutamine-dependent amidotransferases family, which catalyses the conversion of D-fructose-6-phosphate (Fru6P) and glutamine (Gln) into D-glucosamine-6-phosphate (GlcN6P) and glutamate, respectively. Extensive X-ray crystallography investigations have been reported, highlighting the importance of the dimeric association to form the sugar active site as well as significant conformational changes of the protein upon substrate and product binding. In the present work, an approach based on time-resolved noncovalent mass spectrometry has been developed to study the dynamics of GlmS subunit exchange. Using 14 N versus 15 N labeled proteins, the kinetics of GlmS subunit exchange was monitored with the wild-type enzyme in the presence of different substrates and products as well as with the protein bearing a key amino acid mutation specially designed to weaken the dimer interface. Determination of rate constants of subunit exchange revealed important modifications of the protein dynamics: while glutamine, glutamate, and K603A mutation accelerates subunit exchange, Fru6P and GlcN6P totally prevent it. These results are described in light of the available structural information, providing additional useful data for both the characterization of GlmS catalytic process and the design of new GlmS inhibitors. Finally, time-resolved noncovalent MS can be proposed as an additional biophysical technique for real-time monitoring of protein dynamics.

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage λ DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of λ DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes.