Deciphering CaMKII Multimerization Using Fluorescence Correlation Spectroscopy and Homo-FRET Analysis

Sarkar, Priyabrata; Davis, Kaitlin; Puhl, Henry L.; Veetil, Jithesh V.; Nguyen, Tuan Anh; Vogel, Steven S.

doi:10.1016/j.bpj.2017.02.005

Cited by 19 publications

(17 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…For CaMKIIα‐30, we observe a high‐MW population corresponding to 10mer/12mer. Decameric CaMKII assemblies have been observed in solution‐based anisotropy measurements 30 and negative stain EM 31 . Since CaMKIIα‐30 has a disordered region and likely leads to a myriad of possible conformations for all kinase domains, this flexibility may affect the contrast used to calculate MW in the interferometric scattering microscopy method.…”

Section: Discussionmentioning

confidence: 99%

Characterization of CaMKIIα holoenzyme stability

et al. 2020

View full text Add to dashboard Cite

Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long-term memory formation and other Ca 2+ -dependent signaling cascades such as fertilization. Here, we investigated the stability of CaMKIIα using a combination of differential scanning calorimetry (DSC), X-ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability (apparent T m = 36 C), which is slightly stabilized by ATP/MgCl 2 binding (apparent T m = 40 C) and significantly stabilized by regulatory segment binding (apparent T m = 60 C). We crystallized the kinase domain of CaMKII bound to p-coumaric acid in the active site. This structure reveals solvent-exposed hydrophobic residues in the substrate-binding pocket, which are normally buried in the autoinhibited structure when the regulatory segment is present. This likely accounts for the large stabilization that we observe in DSC measurements comparing the kinase alone with the kinase plus regulatory segment. The hub domain alone is extremely stable (apparent T m~9 0 C), and the holoenzyme structure has multiple unfolding transitions ranging from~60 C to 100 C. Using MP, we compared a CaMKIIα holoenzyme with different variable linker regions and determined that the dissociation of both these holoenzymes occurs at a higher concentration (is less stable) compared with the hub domain alone. We conclude that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. These data support a model where domains within the holoenzyme interact. K E Y W O R D SCaMKII, differential scanning calorimetry, mass photometry, oligomer dissociation, thermal stability

show abstract

Section: Discussionmentioning

confidence: 99%

Characterization of CaMKIIα holoenzyme stability

et al. 2020

View full text Add to dashboard Cite

show abstract

“…Brightness analysis is a useful tool for measuring the stoichiometry of protein complexes 16 , 17 , 19 , 41 – 43 . Typically, the molecular brightness of an assembly of subunit, each tagged with a fluorophore, is divided by the molecular brightness of the fluorophore alone to determine how many fluorophore-tagged subunits are in the assembly.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to its utility for characterizing biosensors, auto-FPFA may also be useful for structure-function studies of large protein assemblies 17 – 19 , and mutagenesis studies 17 . For these types of studies, it is important to appreciate FPFA’s limitations.…”

Section: Discussionmentioning

confidence: 99%

Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors

Nguyen

Puhl

Pham

et al. 2018

Sci Rep

Self Cite

View full text Add to dashboard Cite

Genetically encoded biosensors function by linking structural change in a protein construct, typically tagged with one or more fluorescent proteins, to changes in a biological parameter of interest (such as calcium concentration, pH, phosphorylation-state, etc.). Typically, the structural change triggered by alterations in the bio-parameter is monitored as a change in either fluorescent intensity, or lifetime. Potentially, other photo-physical properties of fluorophores, such as fluorescence anisotropy, molecular brightness, concentration, and lateral and/or rotational diffusion could also be used. Furthermore, while it is likely that multiple photo-physical attributes of a biosensor might be altered as a function of the bio-parameter, standard measurements monitor only a single photo-physical trait. This limits how biosensors are designed, as well as the accuracy and interpretation of biosensor measurements. Here we describe the design and construction of an automated multimodal-microscope. This system can autonomously analyze 96 samples in a micro-titer dish and for each sample simultaneously measure intensity (photon count), fluorescence lifetime, time-resolved anisotropy, molecular brightness, lateral diffusion time, and concentration. We characterize the accuracy and precision of this instrument, and then demonstrate its utility by characterizing three types of genetically encoded calcium sensors as well as a negative control.

show abstract

“…The linker length and AD mutations merit study of their role in Ca 2+ /CaM activation dynamics. Two distinct modes for conformational coupling have been proposed; lateral spread of the activated conformation across the holoenzyme subunits (8) or transverse paired dimers (9, 32). The latter may also mediate activation-triggered subunit exchange, driven by a strained, central AD hub (57) ( Supporting Information Figure S3 ).…”

Section: Discussionmentioning

confidence: 99%

Conformational coupling by trans-phosphorylation in calcium calmodulin dependent kinase II

Pandini

Schulman

Khan

2019

Preprint

View full text Add to dashboard Cite

31The calcium calmodulin dependent protein kinase II (CaMKII) is a dodecameric holoenzyme 32 important for encoding memory. Its activation, triggered by binding of calcium calmodulin, persists 33 autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phos-34 phorylates the regulatory domain peptide of a donor kinase forming a chained dimer as a first stage 35 of autonomous activation. Protein dynamics simulations examined the conformational changes trig-36 gered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human 37 mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were 38 characterized by network centrality and community analysis. Mutual information related collective 39 motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD 40 conformational ensembles revealed the dynamic architecture of Inactive kinase domains was co-opted 41 in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured 42 peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determi-43 nants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. 44Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-45 lobe core. The relays were organized around a hydrophobic patch between the captured peptide and 46a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt 47 Author Summary 58Protein kinases play central roles in intracellular signalling. Auto-phosphorylation by bound 59 nucleotide typically precedes phosphate transfer to multiple substrates. Protein conformational 60 changes are central to kinase function, altering binding affinities to change cellular location and shunt 61 from one signal pathway to another. In the brain, the multi-subunit kinase, CaMKII is activated by cal-62 cium calmodulin upon calcium jumps produced by synaptic stimulation. Auto-transphosphorylation of 63 a regulatory peptide enables the kinase to remain activated and mediate long-term behavioural effects 64 after return to basal calcium levels. A database of mutated residues responsible for these effects is 65 difficult to reconcile solely with impaired nucleotide or substrate binding. Therefore, we have compu-66 tationally generated interaction networks to map the conformational plasticity of the kinase domains 67where most mutations localize. The network generated from the atomic structure of a phosphorylated 68 dimer resolves protein sectors based on their collective motions. The sectors link nucleotide and sub-69 strate binding sites in self-reinforcing relays between regulatory peptides. The self-reinforcement is 70 strengthened by phosphorylation consistent with the reported positive cooperativity of kinase activity 71 with calcium-calmodulin concentration. The network gives a better match with the mu...

show abstract

Deciphering CaMKII Multimerization Using Fluorescence Correlation Spectroscopy and Homo-FRET Analysis

Cited by 19 publications

References 46 publications

Characterization of CaMKIIα holoenzyme stability

Characterization of CaMKIIα holoenzyme stability

Auto-FPFA: An Automated Microscope for Characterizing Genetically Encoded Biosensors

Conformational coupling by trans-phosphorylation in calcium calmodulin dependent kinase II

Contact Info

Product

Resources

About