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Between 8 to 14 calcium-calmodulin (Ca2+/CaM) dependent protein kinase-II (CaMKII) subunits form a complex that modulates synaptic activity. In living cells, the autoinhibited holoenzyme is organized as catalytic-domain pairs distributed around a central oligomerization-domain core. The functional significance of catalytic-domain pairing is not known. In a provocative model, catalytic-domain pairing was hypothesized to prevent ATP access to catalytic sites. If correct, kinase-activity would require catalytic-domain pair separation. Simultaneous homo-FRET and fluorescence correlation spectroscopy was used to detect structural changes correlated with kinase activation under physiological conditions. Saturating Ca2+/CaM triggered Threonine-286 autophosphorylation and a large increase in CaMKII holoenzyme hydrodynamic volume without any appreciable change in catalytic-domain pair proximity or subunit stoichiometry. An alternative hypothesis is that two appropriately positioned Threonine-286 interaction-sites (T-sites), each located on the catalytic-domain of a pair, are required for holoenzyme interactions with target proteins. Addition of a T-site ligand, in the presence of Ca2+/CaM, elicited a large decrease in catalytic-domain homo-FRET, which was blocked by mutating the T-site (I205K). Apparently catalytic-domain pairing is altered to allow T-site interactions.

The development of implantable glucose sensors for use in diabetes treatment has been pursued for decades. However, enzyme-based glucose sensors often fail in vivo. In our previous work, we engineered a novel glucose indicator protein (GIP) that can sense glucose without relying on any enzymes and cofactors. Nevertheless, this GIP is unsuitable for blood glucose monitoring due to its low dissociation constant. Here, we report a novel approach to creating a new GIP that can be used to monitor blood glucose level. By disrupting pi–pi stacking around GIP's glucose binding site through site-directed mutagenesis, we showed that GIP's dissociation constant can be manipulated from 0.026 mM to 7.86 mM. This approach yielded four GIP mutants. We showed that one of the mutants can be used to detect glucose from 0 to 32 mM, while another mutant can be employed to visualize intracellular glucose (0–200 μM) within living cells through FRET imaging microscopy measurement.

Monitoring changes in molecular conformation is essential for studying protein interactions within and between complexes. Current methods such as FRET or FCS can only reveal limited aspects of these changes, which in many cases is insufficient for interpretation. Fluorescent Polarization and Fluctuation Analysis (FPFA), a time-correlated single-photon counting technique that combines homo-FRET and FCS was developed to address this problem. In FPFA, changes in mass and/or shape of a protein complex, the number of fluorescent subunits per complex, as well as subunit proximity (1 - 10 nm), are simultaneously detected. Here we have extended FPFA by incorporating simultaneous hetero-FRET analysis so that additional structural changes can be monitored concurrently. This multi-modal approach was then used to observe changes in CaMKII holoenzyme in response to activation and subsequent interactions with NR2B. Revised FPFA revealed that catalytic-domain pairing is the fundamental structural motif of the holoenzyme organization. A genetically engineered dimeric-CaMKII functioned like native holoenzyme (8-14 subunits) in terms of activity, T286 autophosphorylation, structural changes triggered by calcium-calmodulin, and those associated with interaction with NR2B.

Calcium/Calmodulin (CaM) dependent protein kinase (CaMKII) is an important regulator of learning and memory, and it is thought to play a key role in sensing neuronal calcium oscillations. In cells, CaMKIIα primarily forms dodecamers comprised of two stacked rings of 6 subunits, each of which has 3 domains: an N-terminal catalytic domain, a C-terminal oligomerization domain, and between them, a regulatory domain. This regulatory domain activates the enzyme upon binding CaM and harbors a T286 autophosphorylation site that is associated with calcium independent persistent activation. To form the holoenzyme, the oligomerization domain of each subunit interacts with three other oligomerization domains: two laterally to form a ring of association domains, and one transversely to stabilize the double ring structure. Projecting outwards from the rings are catalytic domains, which form pairs, as indicated by homo-FRET analysis. Assemblies comprised of 8-14 subunits are also thought to exist, but it is not known if the number of subunits in the holoenzyme alters the activity or behavior of the catalytic domain pairs. To study the impact of subunit stoichiometry on catalytic domain pairing and T286 autophosphorylation, we generated mutants by altering oligomerization domain residues at the lateral interaction sites. Using fluorescence polarization and fluctuation analysis (FPFA) we simultaneously monitored homo-FRET between Venus-tagged catalytic domains, lateral diffusion of the holoenzyme, and the number of subunits present in these altered assemblies. We find that when the holoenzyme has fewer than 8 subunits, the holoenzyme diffusion time changes and homo-FRET between catalytic domains begins to attenuate. Finally, T286 autophosphorylation in a dimeric mutant holoenzyme is approximately half of that observed in the native holoenzyme. The structural implication of these changes will be discussed.

Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1-10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9 ± 1.2 subunit, but values ranged from 10-14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2 ± 1.3 subunits in the holoenzyme with values ranging from 9-12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved.

The in vivo high-throughput screening (HTS) of human immunodeficiency virus (HIV) protease inhibitors is a significant challenge because of the lack of reliable assays that allow the visualization of HIV targets within living cells. In this study, we developed a new molecular probe that utilizes the principles of Forster resonance energy transfer (FRET) to visualize HIV-1 protease inhibition within living cells. The probe is constructed by linking two fluorescent proteins: AcGFP1 (a mutant green fluorescent protein) and mCherry (a red fluorescent protein) with an HIV-1 protease cleavable p2/p7 peptide. The cleavage of the linker peptide by HIV-1 protease leads to separation of AcGFP1 from mCherry, quenching FRET between AcGFP1 and mCherry. Conversely, the addition of a protease inhibitor prevents the cleavage of the linker peptide by the protease, allowing FRET from AcGFP1 to mCherry. Thus, HIV-1 protease inhibition can be determined by measuring the FRET signal's change generated from the probe. Both in vitro and in vivo studies demonstrated the feasibility of applying the probe for quantitative analyses of HIV-1 protease inhibition. By cotransfecting HIV-1 protease and the probe expression plasmids into 293T cells, we showed that the inhibition of HIV-1 protease by inhibitors can be visualized or quantitatively determined within living cells through ratiometric FRET microscopy imaging measurement. It is expected that this new probe will allow high-content screening (HCS) of new anti-HIV drugs.

Semiconductor nanoparticles, also known as quantum dots (QDs) exhibit size-tunable, strong and narrow band-edge luminescence. Due to their unique optical, electronics properties and dimensional similarities with biological molecules, QD could allow an integration of nanotechnology and biology, leading to major advances in clinical diagnostics. Lateral flow strip assays based on gold and latex nanoparticles are commonly used in the detection of various biomolecules, but these methods are usually either qualitative or semi-quantitative in nature. QD based lateral flow strip assay can be used for quantitative detection of multiple biomolecules in a single strip. They have the potential in developing rapid, robust, portable multiplex quantitative detection system for many biomolecules including avian influenza virus. We have been successful in developing high quantum yield (as high as 80%) and narrow size distribution (FWMH is less than 25 nm) water soluble CdSe/ZnS QDs. Such QDs were covalently conjugated with specific proteins/antibodies and were employed in the development of later-flow strip assays. To investigate the suitability of the QD based detection system, different levels of Anti-Hemagglutinin antibodies (anti HA, an antibody which specifically binds to the avian influenza viral protein) were deposited on the specially designed lateral flow membranes. Protein G, which can specifically bind to the antibodies, were covalently conjugated with QDs (Emission Maximum 580 nm) and applied to the strip. The Protein G and anti-HA reaction occurred in less than 5 minutes as observed by exciting the strip under a UV lamp. The photoluminescence from the strips were quantitated using a hand-held optical fiber detection system.

The autoinhibited calcium/calmodulin-dependent protein kinases-II (CaMKII) assembles to form a holoenzyme structure with multiple catalytic domain pairs radiating out from a central core composed of oligomerization domains. Activation of CaMKII upon binding calcium2+/calmodulin (CaM) is known to initiate a biochemical cascade resulting in CaMKII translocation to synaptic spines and the induction of long-term potentiation. Little is known about the structural changes in the intact CaMKII holoenzyme responsible for this activation. Fluorescence polarization and fluctuation analysis (FPFA) microscopy, a method that combines time-resolved anisotropy and fluorescence correlation spectroscopy (FCS), was used to monitor Venus-CaMKIIα (where a Venus fluorescent-protein is tagged onto the N-terminus of CaMKIIα) before and after activation with CaM. FPFA simultaneously monitors brightness (a measure of the number of subunits in the holoenzyme), homo-FRET (a catalytic domain proximity detector) and diffusion time (a parameter sensitive to the mass and hydrodynamic volume of the holoenzyme). Together these measurements can potentially detect an array of different covert structural changes in the CaMKII holoenzyme triggered by CaM. In homogenates prepared from HEK cells expressing Venus-CaMKIIα, the addition of CaM triggered T286 autophosphorylation, an indicator of kinase activation. FPFA revealed a large increase in the holoenzyme diffusion time. Because changes in holoenzyme subunit stoichiometry were not observed, the change in diffusion time most likely indicates a large change in hydrodynamic volume, such as catalytic domain pair extension. This change was not observed in Venus-CaMKIIα-TT305/6DD, a mutant that cannot bind calmodulin. Holoenzyme activation by CaM did trigger a small, dose-dependent biphasic change in anisotropy (homo-FRET between Venus-tagged catalytic domains), but its magnitude was too small to be interpreted as catalytic domain un-pairing. Interpretation of these experiments will be discussed.

CaMKII is a multimeric enzyme that regulates long-term potentiation in the hippocampus. The assembly and organization of 8-14 subunits in a holoenzyme is thought to be required for transduction of calcium spike frequencies, cooperative binding to calmodulin, persistent activation by T286 autophosphorylation, and translocation into synaptic spines. CaMKII assembles into a holoenzyme by virtue of its unique C-terminal association domain (AD). ADs form a central hub-like structure from which regulatory and catalytic domains project. Each AD interacts tightly with three other subunits, two laterally and one transversely, to form a stable core composed of two stacked rings. FRET and analytical centrifugation has indicated that individual catalytic domains can form pairs by virtue of low affinity binding sites. But catalytic domains of the CaMKII mutant lacking AD do not form pairs in physiological conditions. We hypothesize that catalytic domain pairing is the fundamental structure that allows positive cooperativity and T286 autophosphorylation. Furthermore we postulate that high affinity AD core interactions lead to a high local concentration of catalytic domains that allows low-affinity catalytic domain pairing. To test this hypothesis, we mutated the lateral surface of the CaMKIIα AD. When expressed in HEK293 cells, the mutation generated a paired dimeric CaMKIIα. In support of the hypothesis, our study shows that the dimeric enzyme has the same affinity for calmodulin, a similar Hill coefficient for enzymatic activity, and T286 autophosphorylation comparable to that observed in the wild type holoenzyme. In contrast, the monomeric mutant CaMKII lacking AD has lower affinity, no cooperativity, and reduced T286 autophosphorylation. While catalytic domain pairing was observed in the dimeric mutant, biophysical analysis suggests that the nature of the pairing might be altered.

The calcium-calmodulin dependent protein kinase-II (CaMKII) is a multimeric kinase involved in memory and synaptic modulation. In the auto-inhibited state, a CaMKII holoenzyme is composed of catalytic domain pairs that are distributed around a central core of association domains. Upon binding calcium/calmodulin (Ca2+/CaM) the kinase is activated. Ca2+/CaM binding is also thought to trigger a conformational change that exposes the T interaction site on the catalytic domain, The T-site allows the kinase to interact with several synaptic proteins, including NMDA receptors. In the context of the intact holoenzyme, little is known about the conformational changes initiated by Ca2+/CaM binding to the regulatory domain or by subsequent T-site interactions. We have used Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid microscopy technique to assess these changes. FPFA simultaneously measures Forster resonance energy transfer (FRET, a 1-10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and diffusion time (an attribute sensitive to the mass and shape of a protein complex). In homogenates, activation with Ca2+/CaM triggered catalytic-domain pair extension (as indicated by diffusion time) without catalytic-domain pair separation (as assayed by homo-FRET). CaMKIINtide inhibitor peptide, a T-site ligand, triggered catalytic-domain pair separation, but only when co-incubated with Ca2+/CaM. Furthermore, a T-site mutant, CaMKII (I205K), which cannot bind to NMDA receptors or translocate to spines, exhibited an increase in FRET (catalytic-domain dimerization) upon activated by Ca2+/CaM, and also prevented CaMKIINtide triggered catalytic-domain pair separation. These observations suggest that catalytic-domains remain paired in the activated holoenzyme by virtue of T-site regulated interactions. Catalytic-domain pairs only separate if exogenous T-site ligands can effectively compete for the sites holding catalytic domains together.