Conformational Changes Of Calmodulin Research Articles

Exploration of Ca⁺² Binding Affinity to DEAD-Box Helicase through Computational Approaches

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an occasional catastrophic fatal autosomal dominant or recessive inherited disease that affects an estimated ≈1-5000/10000 people including children, adolescents and young adults, which may cause syncope, abrupt cardiac death during exercise and emotional state. Calmodulin (CALM) functions as a messenger protein of intracellular Ca+2 signaling in cardiomyocytes that transmits complex Ca+2 ions to the proteins involved in cardiac contraction, and its activation is also facilitated by the binding of Ca+2 ions. CALM structure contains 4 EF-hands, each EF-hand holds a single Ca+2 ion (designated as, CA149, CA150, CA151 and CA152). In this study, we performed detailed in_silico analysis of normal and mutated (ASN53ILE) CALM structures to characterize their Ca+2 binding abilities. In CALM-ASN53ILE-Pep-IQ complex, we observed a binding shift of P68(Pep-IQ) as compared to CALM-WT. The root mean square deviation was in the range of 0.4-1 nm for all the systems, while root mean square fluctuation values were in the range of 0.3-0.6 nm for bound versus unbound proteins. Hydrogen-bond profiling was significantly different between CALM-WT and CALM-ASN53ILE over the course of simulation. We observed an introduction of β1 and β2-segment between α1- α2 and α3- α4 along with the movement of C-terminal approximately to 1800 in the apo-CALM-ASN53ILE. Thus, we propose that, ASN53ILE has a pathological impact in the progression of CPVT due to structural and conformational changes in CALM and its binding affinity towards P68(Pep-IQ). The current study may constitute a valuable starting point for CPVT therapeutics through the involvement of CALM-ASN53ILE for designing novel inhibitors to cope with neuropathological disorder.

Single-Molecule Electrical and Spectroscopic Profiling Protein Allostery Using a Gold Plasmonic Nanopore.

Direct structural and dynamic characterization of protein conformers in solution is highly desirable but currently impractical. Herein, we developed a single molecule gold plasmonic nanopore system for observation of protein allostery, enabling us to monitor translocation dynamics and conformation transition of proteins by ion current detection and SERS spectrum measurement, respectively. Allosteric transition of calmodulin (CaM) was elaborately probed by the nanopore system. Two conformers of CaM were well-resolved at a single-molecule level using both the ion current blockage signal and the SERS spectra. The collected SERS spectra provided structural evidence to confirm the interaction between CaM and the gold plasmonic nanopore, which was responsible for the different translocation behaviors of the two conformers. SERS spectra revealed the amino acid residues involved in the conformational change of CaM upon calcium binding. The results demonstrated that the excellent spectral characterization furnishes a single-molecule nanopore technique with an advanced capability of direct structure analysis.

Tracking Conformational Changes in Calmodulin in vitro, in Cell Extract, and in Cells by Electron Paramagnetic Resonance Distance Measurements.

It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron-electron resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide-DOTA-Gd(III) in the N- and C-terminal domains, we observed broad and varied interdomain distance distributions. The in vitro distance distributions of apo-CaM and holo-CaM in the presence and absence of the IQ target peptide can be described by combinations of closed, open, and collapsed conformations. In cell extract, apo- and holo-CaM bind to target proteins in a similar way as apo- and holo-CaM bind to IQ peptide in vitro. In HeLa cells, however, in the presence or absence of elevated in-cell Ca2+ levels CaM unexpectedly produced more open conformations and very broad distance distributions indicative of many different interactions with in-cell components. These results show-case the importance of in-cell analyses of protein structures.

Interaction of NaV1.2 IQ Motif with Disease-Causing Mutants of Calmodulin

Calmodulin (CaM) binds constitutively to an IQ motif (residues 1901-1927, NaV1.2IQp) in the cytosolic C-terminal tail of the a-subunit of the neuronal voltage-gated sodium channel NaV1.2. Solution structures of CaM C-domain (CaMC) bound to NaV1.2IQp showed calcium binding reverses the orientation of (Ca2+)2-CaMC (2M5E.pdb) relative to apo CaMC (2KXW.pdb). Our structure of full-length apo human CaM bound to human NaV1.2IQp showed that only CaMc interacts with NaV1.2IQp. Thermodynamic and NMR analysis of the CaM-NaV1.2IQp complex showed no evidence for CaMN-NaV1.2IQp interactions, and indicated nearly identical affinity for calcium binding to the 4 EF-hand sites of CaM. Residues in NaV1.2IQp had different chemical environments when bound to apo and (Ca2+)4-CaM, consistent with the calcium-trigged change observed in the (Ca2+)2-CaMC-NaV1.2IQp interface (2M5E.pdb). Apo human CaM carrying cardiomyopathy-causing mutations [N53I(N54I), D95V(D96V), A102V(A103V), E104A(E105A), D129G(D130G), and F141L(F142L)] all bound tightly to NaV1.2IQp. However, mutations in CaMC inhibited Ca2+-mediated conformational changes of CaM bound to NaV1.2IQp. This suggests that high cytosolic Ca2+ levels would not be sufficient to trigger reversal of the orientation of these CaMC mutants. The effects of other recently described disease-causing CaM mutations are being investigated. NIH R01 GM57001 (MAS), NIH T32 NS045549 (RM), UI CCOM FUTURE in Biomedicine (AMK)

Nile red-doped fluorescent semiconducting polymer dots as a highly sensitive hydrophobicity probe: protein conformational changes detection and plasma membrane imaging

One-pot synthesis of a photostable, hydrophobic and green fluorescent probe remains a challenge to develop luminescent biomaterials as well as biomedia technology. Semiconducting polymer dots (PDs) hold a great promise as fluorescence nanoprobes due to their photostability, biocompatibility and high quantum yield. Here, the synthesis and characterization of Nile red-doped PFBT PDs as a highly sensitive hydrophobicity probe has been reported. Nile red, a phenoxazone dye, fluoresces intensely in organic solvents and hydrophobic lipids and in varying color; however its fluorescence is totally quenched in water. First, Nile red-doped poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-(2,1′,3)-thiadiazole)] (PFBT) PDs were synthesized through a nano-precipitation technique. Energy transfer from PFBT to Nile red happens through FRET mechanism. The PDs exhibited a red fluorescence with a peak centered at 620 nm (photo-excited at 460 nm). In aqueous media, the fluorescence strongly quenched, but by increasing the hydrophobicity the fluorescence of PDs significantly enhances as the polarity of the medium decreases. This can happen by adding organic solvents or nonpolar species to the medium. The Nile red@PFBT PDs have remarkable specification over Nile red dye. Compared to Nile red, this probe is soluble in water, more photostable, and more brightness due to light-harvesting ability of PDs and less wavelength shift in different medium polarity. We used this probe to investigate polarity of different organic solvents, to study surface hydrophobicity changes of BSA and also Ca2+ determination due to conformational changes of calmodulin. Also we used this probe for imaging of lipid bilayers and cellular membrane.

Development and Characterization of a Model Post‐Translationsally Modified Protein Library

From plants to humans, calmodulin lysine methyltransferase (CaM KMT) is a highly conserved protein responsible for the trimethylation of Lys‐115 in calmodulin (CaM). Recent findings suggest that CaM trimethylation is important in a wide range of metabolic, cellular, and developmental functions. Furthermore, we used CaM KMT as a protein engineering tool to post‐translationally modify a high‐quality protein library of the CaM central linker, using a coexpression system in E. coli. We showed that trimethylation differentially altered the conformational changes of CaM associated with the binding of calcium, CaM's thermal stability, and binding specificity towards CaM‐peptide target sequences. However, the substrate specificity of CaM KMT is unknown. A total of 40 conservative and non‐conservative amino acid substitutions were designed and introduced 3 residue positions (positions P(−3) to P(+3)) flanking the target Lys‐115 trimethylation site. In vitro trimethylation assays, using bacterially expressed and purified CaM KMT with purified mutants, identified positions and putative target sequences for CaM KMT. These results suggest alternate in vivo substrates and cellular roles for CaM KMT. Furthermore, these results provide further design information towards producing an unbiased and targeted post‐translationally modified library of novel sequences, thereby providing an additional tool for designing and generating proteins with stringent protein‐target specificities.Support or Funding InformationNSF KY ESPCoR 0IA‐1355438 Bucks for Brains‐ University of Kentucky Office of Undergraduate ResearchThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Probing Conformational and Functional Substates of Calmodulin by High-Pressure FTIR

In response to intracellular Ca2+-concentration changes, the highly dynamic and flexible Ca2+-sensing protein calmodulin (CaM) interacts with more than 300 diverse target proteins that are involved in numerous signaling pathways in eukaryotic cells. This unique promiscuous target binding behavior and the underlying functional versatility of CaM is a result of its structural flexibility. CaM spans multiple conformational substates in solution providing adaptable binding surfaces for different target proteins. K-Ras4B, known as a key regulator in the ERK pathway, was identified as specific binding partner of CaM, causing the dissociation of the lipidated K-Ras4B from the plasma membrane and thus reducing its activity. However, the interaction of K-Ras4B and CaM has not been elucidated in detail until now. In order to evaluate the conformational space of CaM and shed more light on the mechanism of subsequent target recognition and protein function, identification and characterization of functionally relevant conformational substates is mandatory. Applying pressure in combination with spectroscopies such as FTIR spectroscopy enables to populate and probe otherwise transient low-lying excited conformational substates of CaM close in energy to ground state, but with a smaller partial volume. Evidence has grown that these states are functionally relevant, for instance in recognition and ligand binding events. The pressure-induced conformational changes of CaM were studied in its Ca2+-free and Ca2+-bound state and in the presence of the C-terminal K-Ras4B peptide. We show that not only Ca2+-binding, but also the presence of the target peptide has a drastic effect on the conformational dynamics of the protein.

Protein species-specific characterization of conformational change induced by multisite phosphorylation

Phosphorylation is a central mechanism for regulating the structure and function of proteins in the cell, but accurate characterization of a specific protein phospho-species is challenging due to the difficulty of separating it from other species, as well as the limitations of the traditional structural methods. By using selective top-down ETD, we were able to identify six specific phospho-species of calmodulin (CaM). Phosphorylation of CaM at four sites by CK2 was found to follow a sequential order, with Ser81 as the first, Thr79 as the second, and Ser101 or Thr117 as the third. By combining top-down ETD with hydrogen/deuterium exchange, the impact of phosphorylation on CaM's structure was elucidated in a species-specific manner. A negligible structural effect was observed for mono-phosphorylation at Ser81, or di-phosphorylation at Ser81-Thr79, or tri-phosphorylation at Ser81-Thr79-Ser101 or Ser81-Thr79-Thr117. However, it was found that a significant phosphorylation-induced conformational change in CaM was caused by simultaneous phosphorylation at Ser101 and Thr117. The dramatically increased deuterium incorporation for residues between 102 and 119 strongly suggests that the structure of this region has been greatly changed.

QCM-D Monitoring of Binding-Induced Conformational Change of Calmodulin

Understanding conformational changes are important when studying a protein such as calmodulin (CaM), which activates various target enzymes and regulates numerous physiological functions. CaM is a highly flexible protein that can transitorily adopt various conformations. A quartz crystal microbalance with dissipation (QCM-D) sensor was used to study binding-induced conformational changes of surface-immobilized CaM. Structural changes of CaM were evaluated using the Voigt’s viscoelastic model with frequency (ΔF) and dissipation change (ΔD). When Apo-CaM layer was incubated in 0.1 mM Ca2+ solution, the layer decreased by approximately 0.56 nm, due to the release of coupled water molecules and conformational change. The application of CaM itself also caused a significantly more compact layer, supporting previous findings that CaM dimerization forms a collapsed structure that exposes a hydrophobic tunnel. The binding characteristics of CaM with peptides derived from proteins in a signal transduction pathway also demonstrated diverse biophysical properties of the CaM complexes. Each peptide showed a unique ΔF/ΔD pattern indicating versatility of CaM configuration to favorably adjust to each target molecule. The study demonstrates that the QCM-D sensor is capable of simultaneously studying binding affinity and plasticity of protein configuration for target binding. The CaM data obtained on hydrated protein layer thickness is complementary to configuration measurements of a single CaM molecule.

Calcium-dependent conformational transition of calmodulin determined by Fourier transform infrared spectroscopy

The Ca2+-induced conformational changes in calmodulin (CaM) were monitored by Fourier transform infrared spectroscopy (FT-IR) at different molar ratios of Ca2+ to CaM. The results show that these changes occur in two distinctive transitions. The first transition involves significant changes in the overall secondary structure with a small gain in solvent accessibility, and is completed after the second Ca2+ binds to both EF-hands of its C-terminal domain. The second transition is accompanied by CaM folding into a tighter, less hydrogen-exchangeable structure, and is completed by the addition of the fourth Ca2+ to have four Ca2+ per molecule. Particularly, α-helices in CaM–nCa2+(n=0, 1, 2) are less stable than those in CaM–nCa2+(n=3, 4).

A Compact Intermediate State of Calmodulin in the Process of Target Binding

Calmodulin undergoes characteristic conformational changes by binding Ca(2+), which allows it to bind to more than 300 target proteins and regulate numerous intracellular processes in all eukaryotic cells. We measured the conformational changes of calmodulin upon Ca(2+) and mastoparan binding using the time-resolved small-angle X-ray scattering technique combined with flash photolysis of caged calcium. This measurement system covers the time range of 0.5-180 ms. Within 10 ms of the stepwise increase in Ca(2+) concentration, we identified a distinct compact conformational state with a drastically different molecular dimension. This process is too fast to study with a conventional stopped-flow apparatus. The compact conformational state was also observed without mastoparan, indicating that the calmodulin forms a compact globular conformation by itself upon Ca(2+) binding. This new conformational state of calmodulin seems to regulate Ca(2+) binding and conformational changes in the N-terminal domain. On the basis of this finding, an allosteric mechanism, which may have implications in intracellular signal transduction, is proposed.

Conformational changes of calmodulin on calcium and Peptide binding monitored by film bulk acoustic resonators.

Film bulk acoustic resonators (FBAR) are mass sensitive, label-free biosensors that allow monitoring of the interaction between biomolecules. In this paper we use the FBAR to measure the binding of calcium and the CaMKII peptide to calmodulin. Because the mass of the calcium is too small to be detected, the conformational change caused by the binding process is measured by monitoring the resonant frequency and the motional resistance of the FBAR. The resonant frequency is a measure for the amount of mass coupled to the sensor while the motional resistance is influenced by the viscoelastic properties of the adsorbent. The measured frequency shift during the calcium adsorptions was found to be strongly dependent on the surface concentration of the immobilized calmodulin, which indicates that the measured signal is significantly influenced by the amount of water inside the calmodulin layer. By plotting the measured motional resistance against the frequency shift, a mass adsorption can be distinguished from processes involving measurable conformational changes. With this method three serial processes were identified during the peptide binding. The results show that the FBAR is a promising technology for the label-free measurement of conformational changes.

Subtle pH differences trigger single residue motions for moderating conformations of calmodulin

This study reveals the essence of ligand recognition mechanisms by which calmodulin (CaM) controls a variety of Ca(2+) signaling processes. We study eight forms of calcium-loaded CaM each with distinct conformational states. Reducing the structure to two degrees of freedom conveniently describes main features of the conformational changes of CaM via simultaneous twist-bend motions of the two lobes. We utilize perturbation-response scanning (PRS) technique, coupled with molecular dynamics simulations. PRS is based on linear response theory, comprising sequential application of directed forces on selected residues followed by recording the resulting protein coordinates. We analyze directional preferences of the perturbations and resulting conformational changes. Manipulation of a single residue reproduces the structural change more effectively than that of single/pairs/triplets of collective modes of motion. Our findings also give information on how the flexible linker acts as a transducer of binding information to distant parts of the protein. Furthermore, by perturbing residue E31 located in one of the EF hand motifs in a specific direction, it is possible to induce conformational change relevant to five target structures. Independently, using four different pK(a) calculation strategies, we find this particular residue to be the charged residue (out of a total of 52), whose ionization state is most sensitive to subtle pH variations in the physiological range. It is plausible that at relatively low pH, CaM structure is less flexible. By gaining charged states at specific sites at a pH value around 7, such as E31 found in the present study, local conformational changes in the protein will lead to shifts in the energy landscape, paving the way to other conformational states. These findings are in accordance with Fluorescence Resonance Energy Transfer (FRET) measured shifts in conformational distributions towards more compact forms with decreased pH. They also corroborate mutational studies and proteolysis results which point to the significant role of E31 in CaM dynamics.

Mass spectrometry-based carboxyl footprinting of proteins: Method evaluation

Protein structure determines function in biology, and a variety of approaches have been employed to obtain structural information about proteins. Mass spectrometry-based protein footprinting is one fast-growing approach. One labeling-based footprinting approach is the use of a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and glycine ethyl ester (GEE) to modify solvent-accessible carboxyl groups on glutamate (E) and aspartate (D). This paper describes method development of carboxyl-group modification in protein footprinting. The modification protocol was evaluated by using the protein calmodulin as a model. Because carboxyl-group modification is a slow reaction relative to protein folding and unfolding, there is an issue that modifications at certain sites may induce protein unfolding and lead to additional modification at sites that are not solvent-accessible in the wild-type protein. We investigated this possibility by using hydrogen deuterium amide exchange (H/DX). The study demonstrated that application of carboxyl group modification in probing conformational changes in calmodulin induced by Ca2+ binding provides useful information that is not compromised by modification-induced protein unfolding.

Atomic Force Microscopy Study of the Conformational Change in Immobilized Calmodulin

Maintaining the biological functionality of immobilized proteins is the key to the success of numerous protein-based biomedical devices. To that end, we studied the conformational change in calmodulin (CaM) immobilized on chemical patterns. 1-Cysteine-mutated calmodulin was immobilized on a mercapto-terminated surface through cysteine-Hg-mercapto coupling. Utilizing atomic force microscopy (AFM), the average height of immobilized calmodulin was determined to be 1.87 ± 0.19 nm. After incubation in EGTA solution, the average height of the protein changed to 2.26 ± 0.21 nm, indicating the conformational change of CaM to Apo-CaM. Immobilized CaM also demonstrated a conformational change upon the reaction with known calmodulin antagonist chlorpromazine (CPZ). After incubation in CPZ solution, the average height of CPZ-bound CaM increased to 2.32 ± 0.20 nm, demonstrating that immobilized CaM has a similar response to that in bulk solution. These results show that the immobilization of calmodulin on a solid support does not interfere with the ability of the protein to bind calcium and calmodulin antagonists. Our results demonstrate the feasibility of employing AFM to probe and understand protein conformational changes.

Trifluoperazine regulation of calmodulin binding to Fas: a computational study.

Death-inducing signaling complex (DISC) formation is a critical step in Fas-mediated signaling for apoptosis. Previous experiments have demonstrated that the calmodulin (CaM) antagonist, trifluoperazine (TFP) regulates CaM-Fas binding and affects Fas-mediated DISC formation. In this study, we investigated the anti-cooperative characteristics of TFP binding to CaM and the effect of TFP on the CaM-Fas interaction from both structural and thermodynamic perspectives using combined molecular dynamics simulations and binding free energy analyses. We studied the interactions of different numbers of TFP molecules with CaM and explored the effects of the resulting conformational changes in CaM on CaM-Fas binding. Results from these analyses showed that the number of TFP molecules bound to CaM directly influenced α-helix formation and hydrogen bond occupancy within the α-helices of CaM, contributing to the conformational and motion changes in CaM. These changes affected CaM binding to Fas, resulting in secondary structural changes in Fas and conformational and motion changes of Fas in CaM-Fas complexes, potentially perturbing the recruitment of Fas-associated death domain for DISC formation. The computational results from this study reveal the structural and molecular mechanisms that underlie the role of the CaM antagonist, TFP, in regulation of CaM-Fas binding and Fas-mediated DISC formation in a concentration-dependent manner.

Departure of Calmodulin from the IQ Domain of Ca V1.3 Channels During Calcium-Dependent Inactivation

Studies of calmodulin (CaM) regulation of CaV channels have long focused on an IQ domain in the carboxy-terminus of channels. Strong evidence indicates that Ca2+-free CaM (apoCaM) preassociates with the IQ domain before Ca2+ entry through channels (e.g., Liu et alNature 463:968), and that Ca2+/CaM has the potential to bind the IQ. Hence, Ca2+-dependent inactivation (CDI) of channels has been proposed to result from Ca2+-dependent conformational changes of CaM, all while bound to the IQ. Yet, analysis of Ca2+/CaM complexed with the IQ domain of CaV2.1 (Structure 16:607) hints that Ca2+/CaM departs from the IQ domain during channel regulation. To generalize this hypothesis, we here alanine scanned the entire IQ domain of CaV1.3, a prototype channel homologous to long-studied CaV1.2. Surprisingly, CDI was strongly diminished at only three residues: the signature isoleucine (I[0]), an alanine situated four residues upstream (A[-4]), and a downstream phenylalanine (F[4]). How do these mutations decrease CDI? If CDI were suppressed by inhibiting Ca2+/CaM binding to an IQ effector site, then Ca2+/CaM binding to this domain should exhibit commensurate disruption. However, FRET two-hybrid assays indicated no such decrease in binding. Alternatively, mutations could attenuate CDI by inhibiting apoCaM preassociation with the channel. Indeed, binding assays revealed strong mutational effects on apoCaM interaction with the IQ domain, consistent with the observed CDI deficits. Indeed, overexpression of wild-type CaM largely restored CDI of mutant channels, fitting with recovery of apoCaM preassociation via mass action. Thus, while the IQ domain appears to be important as a preassociation locus for apoCaM, CDI is likely triggered by the departure of Ca2+/CaM to interact with effector sites elsewhere on the channel (Ben Johny, Yang, and Yue, this meeting). This departure would fundamentally transform our understanding of CaM/channel regulation.

3H1334 サブミリ秒時間分解能X線小角散乱を用いたカルシウム結合に伴うカルモジュリンの構造変化(3H 蛋白質_物性5,日本生物物理学会第49回年会)

3H1334 サブミリ秒時間分解能X線小角散乱を用いたカルシウム結合に伴うカルモジュリンの構造変化(3H 蛋白質_物性5,日本生物物理学会第49回年会)

A molecular-imprint nanosensor for ultrasensitive detection of proteins

Molecular imprinting is a technique for preparing polymer scaffolds that function as synthetic receptors. Imprinted polymers that can selectively bind organic compounds have proven useful in sensor development. Although creating synthetic molecular-imprinting polymers that recognize proteins remains challenging, nanodevices and nanomaterials show promise in this area. Here, we show that arrays of carbon-nanotube tips with an imprinted non-conducting polymer coating can recognize proteins with subpicogram per litre sensitivity using electrochemical impedance spectroscopy. We have developed molecular-imprinting sensors specific for human ferritin and human papillomavirus derived E7 protein. The molecular-imprinting-based nanosensor can also discriminate between Ca(2+)-induced conformational changes in calmodulin. This ultrasensitive, label-free electrochemical detection of proteins offers an alternative to biosensors based on biomolecule recognition.

Abstract 96: Conformational and thermodynamics analyses of the regulation of trifluoperazine on camodulin binding to Fas: Implications for cancer chemotherapy

Abstract Fas-mediated signaling pathway is an important mechanism for apoptosis in a variety of cells, including cholangiocarcinoma and other cancer cells. The formation of death inducing signaling complex (DISC) is a critical step for Fas-mediated signaling. Recent experimental studies showed that calmodulin (CaM) binds to Fas and regulates Fas-mediated DISC formation and the binding of CaM to Fas is inhibited by the CaM antagonist, trifluoperazine (TFP) (J Cell Biochem, 103:788, 2008). The present study sought to determine the structural basis and molecular mechanisms of TFP regulation of Fas-mediated DISC formation. We used a combination of molecular dynamics simulations and binding free energy analyses to investigate the effect of TFP on CaM-Fas binding. We studied the interactions of different number(s) of TFP(s) with CaM and the effect of the resulting conformational changes of CaM on CaM/Fas binding. We found that the binding affinity of one TFP with CaM was affected by the sequential binding of other TFP(s) to CaM, which was consistent with near-UC circular dichroism data (Biochemistry 37:15300, 1998). The number of TFP(s) bound to CaM was shown to affect the conformational and motion changes of CaM, and thus directly affect CaM binding to Fas. Consistent with previous experimental observations that CaM-Fas binding was affected by TFP concentrations (J Biol Chem, 280:29964, 2005), we demonstrated that one TFP bound to CaM inhibited CaM-Fas binding while the subsequent two TFPs or four TFPs bound to CaM resulted in the recovery of CaM-Fas binding (Table 1). Results also showed that conformational and motional changes of Fas by binding to CaM were directly related to the number of TFPs bound to CaM, which could further affect Fas recruitment of FADD to the DISC. This study provides structural and thermodynamics evidence elucidating the role of the CaM antagonist, TFP, in regulating CaM-Fas binding and Fas-mediated DISC formation and apoptosis.Table 1.The change of CaM-Fas binding affinity by the CaM antagonist, TFP. CaM_Fas_WTCaM_1TFP_FasCaM_2TFPs_FasCaM_4TFPs_FasΔG−7.11±4.67−1.32±0.99−6.96±3.47−6.42±1.06ΔΔG 5.79*0.150.69All values in this table were expressed in terms of kcal/mol. ΔG is the binding free energy for the complex, and ΔΔG is the relative binding free energies with respect to the CaM-Fas complex without CaM antagonist TFP(s). * Differences are statistically significant (Student's t-test, p<0.05) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 96.