Affinity Calcium Binding Research Articles

The Neuronal Voltage-Dependent Sodium Channel Type II IQ Motif Lowers the Calcium Affinity of the C-Domain of Calmodulin

Calmodulin (CaM) is the primary calcium sensor in eukaryotes. Calcium binds cooperatively to pairs of EF-hand motifs in each domain (N and C). This allows CaM to regulate cellular processes via calcium-dependent interactions with a variety of proteins, including ion channels. One neuronal target is NaV1.2, voltage-dependent sodium channel type II, to which CaM binds via an IQ motif within the NaV1.2 C-terminal tail (residues 1901-1938) [Mori, M., et al. (2000) Biochemistry 39, 1316-1323]. Here we report on the use of circular dichroism, fluorescein emission, and fluorescence anisotropy to study the interaction between CaM and NaV1.2 at varying calcium concentrations. At 1 mM MgCl2, both full-length CaM (CaM1-148) and a C-domain fragment (CaM76-148) exhibit tight (nanomolar) calcium-independent binding to the NaV1.2 IQ motif, whereas an N-domain fragment of CaM (CaM1-80) binds weakly, regardless of calcium concentration. Equilibrium calcium titrations of CaM at several concentrations of the NaV1.2 IQ peptide showed that the peptide reduced the calcium affinity of the CaM C-domain sites (III and IV) without affecting the N-domain sites (I and II) significantly. This leads us to propose that the CaM C-domain mediates constitutive binding to the NaV1.2 peptide, but that interaction then distorts calcium-binding sites III and IV, thereby reducing their affinity for calcium. This contrasts with the CaM-binding domains of voltage-dependent Ca2+ channels, kinases, and phosphatases, which increase the calcium binding affinity of the C-domain of CaM.

H/D Exchange and Mass Spectrometry-Based Method for Biophysical Analysis of Multidomain Proteins at the Domain Level

A protocol was developed to characterize the domain-specific thermodynamic stabilities of multidomain proteins using SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange). The protocol incorporates a protease digestion step into the conventional SUPREX protocol and enables folding free energy (DeltaGf) and cooperativity (m-value) measurements to be made on the individual domains of multidomain proteins in their native context (i.e., in the intact protein). Three multidomain protein systems (calmodulin, a Fyn construct, and transferrin) were used to validate the SUPREX-protease digestion protocol. The DeltaGf and m-value of each domain in the intact test proteins were measured in the absence and presence of ligands using the new protocol. Domain-specific thermodynamic parameters were obtained on each system; and the measured parameters were consistent with known biophysical properties of the test proteins. The known stabilization of the N-terminal domain of CaM in the context of the intact protein and the known binding affinity of a proline-rich peptide to the SH3 domain in the Fyn construct were successfully quantified using the new protocol. Qualitative information about the relative calcium binding affinities of the N- and C-terminal domains of CaM and about the relative iron binding affinities of the N- and C-terminal domains of transferrin was also obtained using the new protocol.

Abstract 1254: Novel Insights In Arrhytmogenesis Of Catecholaminergic Ventricular Tachycardia From The First Knock In Model Of Homozygous Calsequestrin Mutation

Cardiac Calsequestrin (CASQ2) is a high affinity low capacity calcium binding protein essential in the regulation of intracellular Ca storage and release. Mutations in CASQ2 have been linked to the recessive form of catecholaminergic polymorphic ventricular tachycardia (CPVTr). To elucidate the mechanisms of arrhythmogensis in CPVTr, we generated a knock-in mouse model carrier of the R33Q mutation identified in one of our patients (pts) with a severe CPVT phenotype. Continuous ECG recording (DSI implantable monitors) showed that homozygous (CASQ2 R33Q/R33Q ) mice develop bidirectional and polymorphic VT upon exposure to adrenergic triggers (noise, physical contact). CASQ2 R33Q/R33Q mice have no sign of structural cardiac abnormality (normal heart/body weight ratio, histology no fibrosis nor myofibrillar disarray). Calcium-binding affinity of CASQ2 R33Q/R33Q is identical to that of wild type (WT) CASQ2, its localization evaluated with confocal microscopy is superimposable to that of WT. Western blot analysis shows that CASQ2 R33Q/R33Q content in myocytes is reduced by 40% but Real Time PCR showed that levels of mRNA are not reduced suggesting an abnormal protein turn-over. Evaluation of electrophysiological properties of isolated CASQ2 R33Q/R33Q myocytes revealed that pacing (1–5 Hz) induces delayed afterdepolarizations (DADs; 18/32 cells 56%) and triggered activity (TA; 9/31 29%) addition of adrenalin (200 nM) enhances DADs and TA (DADs 15/16 cells 93%; TA 8/17 cells 47%) and also elicits (25% of myocytes) early after depolarizations (EADs) and EADs-mediated TA. Our data demonstrate that in analogy with our knock-in model of the dominant form of CPVT due to mutations in the cardiac ryanodine receptor (RyR2) the CASQ2 R33Q/R33Q mice 1) do not present signs of cardiomyopathy, 2) develop DADs and TA in vitro and bidirectional and polymorphic VT in vivo. At variance with RyR2 mice, CASQ2 R33Q/R33Q mice 1) present a more malignant phenotype, lower threshold for DADs, TA and VT; 2) EADs concur to arrhythmogenesis 3) overcoming with a knock-in the limitations of previous in vitro and in vivo models overexpressing mutant CASQ2 on the background of the WT protein we demonstrated that a reduction in CASQ2 is implicated in the arrhythmogenesis of recessive CPVT.

Modulation of the rate of cardiac muscle contraction by troponin C constructs with various calcium binding affinities

We investigated whether changing thin filament Ca(2+) sensitivity alters the rate of contraction, either during normal cross-bridge cycling or when cross-bridge cycling is increased by inorganic phosphate (P(i)). We increased or decreased Ca(2+) sensitivity of force production by incorporating into rat skinned cardiac trabeculae the troponin C (TnC) mutants V44QTnC(F27W) and F20QTnC(F27W). The rate of isometric contraction was assessed as the rate of force redevelopment (k(tr)) after a rapid release and restretch to the original length of the muscle. Both in the absence of added P(i) and in the presence of 2.5 mM added P(i) 1) Ca(2+) sensitivity of k(tr) was increased by V44QTnC(F27W) and decreased by F20QTnC(F27W) compared with control TnC(F27W); 2) k(tr) at submaximal Ca(2+) activation was significantly faster for V44QTnC(F27W) and slower for F20QTnC(F27W) compared with control TnC(F27W); 3) at maximum Ca(2+) activation, k(tr) values were similar for control TnC(F27W), V44QTnC(F27W), and F20QTnC(F27W); and 4) k(tr) exhibited a linear dependence on force that was indistinguishable for all TnCs. In the presence of 2.5 mM P(i), k(tr) was faster at all pCa values compared with the values for no added P(i) for TnC(F27W), V44QTnC(F27W), and F20QTnC(F27W). This study suggests that TnC Ca(2+) binding properties modulate the rate of cardiac muscle contraction at submaximal levels of Ca(2+) activation. This result has physiological relevance considering that, on a beat-to-beat basis, the heart contracts at submaximal Ca(2+) activation.

Potential influence of Asp in the Ca2+ coordination position 5 of parvalbumin on the calcium-binding affinity: A computational study

Parvalbumins (PV) are calcium-binding proteins, all sharing the common helix–loop–helix (EF-hand) motif. This motif contains a central twelve-residue Ca2+-binding loop with the flanking helices positioned roughly perpendicular to each other. The precise role of these coordination residues has been the subject of intense studies. In this work, we focus on the coordination position 5 in the CD Ca2+-binding site of silver hake parvalbumin isoform B (SHPV-B). The most common residue at site 5 of calcium-binding loop in canonical EF-hands is Asp [B.J. Marsden, G.S. Shaw, B.D. Sykes, Biochem. Cell Biol. 68 (1990) 587–601], but in the CD site of PV, this position is almost always serine (Ser). The substitution of Ser with Asp will add the 5th carboxylate residue in the CD coordination sphere. However, as predicted by the acid pair hypothesis, the Ca2+-binding affinity would be maximized in an EF-hand motif that has four carboxylate ligands paired along the ±x, and ±z-axes [R.E. Reid, R.S. Hodges, J. Theor. Biol. 84 (1980) 401–444]. Molecular dynamics simulations and free energy calculations were employed to investigate the influence of Ser to Asp mutation at position 5 on calcium-binding affinity. We found that the Asp variant exhibited remarkable stability during the entire molecular dynamics simulation, with not only the retention of the Ca2+-binding site, but also increased compactness in the coordination sphere. The S55D fragment also accommodated Ca2+ well. We conclude that the reason why Asp which is the most common residue at site 5 of calcium-binding loop in canonical EF-hands has never been identified at this position experimentally for PVs might be related to its physiological functions.

A Dynamic Model of Excitation-Contraction Coupling during Acidosis in Cardiac Ventricular Myocytes

Acidosis in cardiac myocytes is a major factor in the reduced inotropy that occurs in the ischemic heart. During acidosis, diastolic calcium concentration and the amplitude of the calcium transient increase, while the strength of contraction decreases. This has been attributed to the inhibition by protons of calcium uptake and release by the sarcoplasmic reticulum, to a rise of intracellular sodium caused by activation of sodium-hydrogen exchange, decreased calcium binding affinity to Troponin-C, and direct effects on the contractile machinery. The relative contributions and concerted action of these effects are, however, difficult to establish experimentally. We have developed a mathematical model to examine altered calcium-handling mechanisms during acidosis. Each of the alterations was incorporated into a dynamical model of pH regulation and excitation-contraction coupling to predict the time courses of key ionic species during acidosis, in particular intracellular pH, sodium and the calcium transient, and contraction. This modeling study suggests that the most significant effects are elevated sodium, inhibition of sodium-calcium exchange, and the direct interaction of protons with the contractile machinery; and shows how the experimental data on these contributions can be reconciled to understand the overall effects of acidosis in the beating heart.

Who's Got the Calcium?

The human cardiac voltage-gated sodium channel (hH1) is essential for normal heart function, and mutations in the alpha subunit of the channel cause deadly arrhythmias. Some of these mutations are in a region that confers critical regulation of channel function by calcium, but exactly how calcium regulates the channel has been unclear. The hH1 protein contains an EF-hand calcium-binding motif that may bind calcium directly and an IQ-type calmodulin-binding motif that could confer calcium-dependent changes through changes in calmodulin. Shah et al. explored interactions between these domains to help clarify the roles of these direct and indirect responses to calcium in channel function. The authors characterized the binding properties of various channel domains and mutants by monitoring 15N-3H heteronuclear single-quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectra. The EF-hand domain alone appeared to have a very low affinity for calcium (0.6 mM) that would be unlikely to have a physiological role. However, interaction of the IQ motif with the EF-hand domain enhanced calcium-binding affinity into the micromolar range. Contrary to expectations, a mutation in the EF-hand domain that causes arrhythmias in humans didn't alter calcium affinity but rather reduced affinity of the channel for binding calmodulin. On the other hand, a mutation in the IQ calmodulin-binding motif had little effect on affinity for calmodulin but reduced calcium-binding affinity. Also unusual was the finding that calcium-loaded calmodulin actually had lower affinity for an IQ motif-containing peptide than did calcium-free calmodulin. The authors propose that the effects of calcium can be explained as follows: In basal conditions, calmodulin is bound to the channel through the IQ motif. Increased concentrations of intracellular calcium weaken this interaction such that calmodulin is released, enabling the IQ motif to interact with the EF-hand domain, thus favoring calcium-dependent modulation of the channel through direct calcium binding. Further understanding of the intricate nature of these calcium-modulated domain interactions may reveal new therapeutic strategies to manage cardiac arrhythmias and other diseases associated with altered function of other members of this channel family in other cell types. V. N. Shah, T. L. Wingo, K. L. Weiss, C. K. Williams, J. R. Balser, W. J. Chazin, Calcium-dependent regulation of the voltage-gated sodium channel hH1: Intrinsic and extrinsic sensors use a common molecular switch. Proc. Natl. Acad. Sci. U.S.A. 103 , 3592-3597 (2006). [Abstract] [Full Text]

The Thrombospondin-1 N700S Polymorphism Does Not Alter von Willebrand Factor Multimer Size in Patients Suffering an Acute Myocardial Infarction.

An uncommon missense variant of thrombospondin-1 representing an asparagine (Asn-700) to serine substitution (Ser-700) has been identified as a potential genetic risk factor for myocardial infarction (MI). Thrombospondin-1 (TSP-1) is a glycoprotein stored in human platelet alpha granules and has been shown to regulate von Willebrand factor (VWF) multimer size. The TSP-1 domain responsible for VWF-reducing activity was localized to the calcium-binding C-terminal sequence at position Cys 974. Considering the proximity of the N700S TSP-1 polymorphism and the demonstration that the Ser-700 polymorphism alters the calcium binding affinity of this domain, we investigated whether altered VWF-reducing activity of Ser-700 polymorphism underlies the observed prothrombotic phenotype. Plasma samples were analyzed from a cohort of patients (N=31) who survived an acute myocardial infarction at age 45 or younger compared with age-matched controls (N=19). The relative percentage of high molecular weight VWF multimers did not differ significantly between individuals homozygous for either Ser-700 or Asn-700 (p=.20). Similarly, the relative percentage of high molecular weight multimers was 31.2% for the Ser-700 homozygous MI patients (N=21) versus 31.1% (p=.95) in the Asn-700 healthy controls (N=10). These findings were confirmed using the collagen binding assay to assess VWF activity relative to antigen concentrations. The mean ratio of CBA/Ag was .98 in the Ser-700 patients versus 1.0 in the Asn-700 control group (p=.22). Mean plasma thrombospondin-1 concentrations did not differ between the two groups. The VWF reducing capacity of TSP-1 was also assessed in vitro using recombinant TSP-1 N700S constructs containing the last EGF-like repeat, all type-3 repeats (calcium-binding domains), and C-terminal sequence. Incubation of Ser-700 peptide, Asn-700 peptide, or platelet purified full-length TSP-1 with ultra-large VWF (using either TTP plasma or recombinant ultra-large VWF) did not result in any significant reduction of VWF multimer size under static conditions. In conclusion, the association between coronary artery thrombosis and the TSP-1 Ser-700 polymorphism is not mediated through altered VWF reducing activity.

Expanding the range of free calcium regulation in biological solutions

Many biological systems use ethylene glycol bis (β-aminoethylether)- N, N, N′, N′-tetraacetic acid (EGTA) to regulate the free calcium concentration ([Ca 2+] free) in the presence of physiological levels of free Mg 2+ ([Mg 2+] free). Frequently, it is necessary to work at [Ca 2+] free beyond EGTA’s buffering capabilities. Therefore, we have developed methods to extend the buffering range by adding nitrilotriacetic acid (NTA) to solutions containing EGTA. This extension results from NTA having a lower K dCa ′ than EGTA. Such equilibria are solved by pCa Calculator, a computer program designed to aid in the study of Ca 2+-dependent physiological processes while accounting for the effects of pH, temperature, and ionic strength. With multiple chelators and pH buffers from which to choose, pCa Calculator calculates the total concentration of each species required to achieve specified free concentrations of Ca 2+, ATP, and Mg 2+. The program is intuitive, user-friendly, and flexible enough to fix or vary the [Mg-ATP 2−] and ionic strength. Moreover, it can account for increases in experimental volume from calcium addition. A comparative analysis is reported for testing solutions in the presence and absence of NTA by measuring the calcium binding affinity of fluorescent cardiac troponin C. These findings demonstrate that EGTA, when used in conjunction with NTA, improves and expands the regulation of free calcium in solution.

The Sarcomeric Control of Energy Conversion

The Frank-Starling Law, Fenn Effect, and Suga's suggestions of cardiac muscle constant contractile efficiency establish the dependence of cardiac mechanics and energetics on the loading conditions. Consistent with these observations, this review suggests that the sarcomere control of contraction consists of two dominant feedbacks: (1) a cooperativity mechanism (positive feedback), whereby the number of force-generating cross-bridges (XBs) determines the affinity of calcium binding to the troponin regulatory protein; and (2) a mechanical (negative) feedback, whereby the filament shortening velocity affects the rate of XB turnover from the force to the non-force generating conformation. The study explains the roles of these feedbacks in providing the adaptive control of energy consumption by the loading conditions and validates the dependence of the cooperativity mechanism on the number of strong XBs. The cooperativity mechanism regulates XB recruitment. It explains the cardiac force-length calcium relationship, the related Frank-Starling Law of the heart, and the adaptive control of new XB recruitment and the associated adenosine triphosphate (ATP) consumption. The mechanical feedback explains the force-velocity relationship and the constant and high-contractile efficiency. These mechanisms were validated by testing the force responses to large amplitude (100 nm/sarcomere) sarcomere length (SL) oscillations, in intact tetanized trabeculae (utilizing 30 microM cyclopiazonic). The force responses to large-length oscillations lag behind the imposed oscillations at low extracellular calcium concentration ([Ca(2+)](0)) and slow frequencies (<4 Hz, 25 degrees C), yielding counterclockwise hystereses in the force-length plane. The force was higher during shortening than during lengthening. The area within these hystereses corresponds to the external work generated from new XB recruitment during each oscillation, and it is determined by the delay in the force response. Characterization of the delayed response and its dependence on the SL, force, and calcium allows identification of the regulation of XB recruitment. The direct dependence of the phase on force indicates that XB recruitment is determined directly by the force (i.e., the number of strong XBs) and indirectly by SL or calcium. The suggested feedbacks determine cardiac energetics: 1) the constant and high contractile efficiency is an intrinsic property of the single XB, due to the mechanical feedback; and 2) the XBs are the myocyte sensors that modulate XB recruitment in response to length and load changes through the cooperativity mechanism.

Calcium-Binding Properties of Wild-Type and EF-Hand Mutants of S100B in the Presence and Absence of a Peptide Derived from the C-Terminal Negative Regulatory Domain of p53

S100B is a dimeric Ca(2+)-binding protein that undergoes a 90 +/- 3 degrees rotation of helix 3 in the typical EF-hand domain (EF2) upon the addition of calcium. The large reorientation of this helix is a prerequisite for the interaction between each subunit of S100B and target proteins such as the tumor suppressor protein, p53. In this study, Tb(3+) was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate of Ca(2+) ion dissociation. In competition studies with Tb(3+), the dissociation rates of Ca(2+) (k(off)) from the EF2 domains of S100B in the absence and presence of the p53 peptide was determined to be 60 and 7 s(-)(1), respectively. These data are consistent with a previously reported result, which showed that that target peptide binding to S100B enhances its calcium-binding affinity [Rustandi et al. (1998) Biochemistry 37, 1951-1960]. The corresponding Ca(2+) association rate constants for S100B, k(on), for the EF2 domains in the absence and presence of the p53 peptide are 1.1 x 10(6) and 3.5 x 10(5) M(-)(1) s(-)(1), respectively. These two association rate constants are significantly below the diffusion control ( approximately 10(9) M(-)(1) s(-)(1)) and likely involve both Ca(2+) ion association and a Ca(2+)-dependent structural rearrangement, which is slightly different when the target peptide is present. EF-hand calcium-binding mutants of S100B were engineered at the -Z position (EF-hand 1, E31A; EF-hand 2, E72A; both EF-hands, E31A + E72A) and examined to further understand how specific residues contribute to calcium binding in S100B in the absence and presence of the p53 peptide.

Differential Effects of β1 and β2 Subunits on BK Channel Activity

High conductance, calcium- and voltage-activated potassium (BK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (β) subunits. β1 and β2 subunits increase apparent channel calcium sensitivity. The β1 subunit also decreases the voltage sensitivity of the channel and the β2 subunit produces an N-type inactivation of BK currents. We further characterized the effects of the β1 and β2 subunits on the calcium and voltage sensitivity of the channel, analyzing the data in the context of an allosteric model for BK channel activation by calcium and voltage (Horrigan and Aldrich, 2002). In this study, we used a β2 subunit without its N-type inactivation domain (β2IR). The results indicate that the β2IR subunit, like the β1 subunit, has a small effect on the calcium binding affinity of the channel. Unlike the β1 subunit, the β2IR subunit also has no effect on the voltage sensitivity of the channel. The limiting voltage dependence for steady-state channel activation, unrelated to voltage sensor movements, is unaffected by any of the studied β subunits. The same is observed for the limiting voltage dependence of the deactivation time constant. Thus, the β1 subunit must affect the voltage sensitivity by altering the function of the voltage sensors of the channel. Both β subunits reduce the intrinsic equilibrium constant for channel opening (L 0). In the allosteric activation model, the reduction of the voltage dependence for the activation of the voltage sensors accounts for most of the macroscopic steady-state effects of the β1 subunit, including the increase of the apparent calcium sensitivity of the BK channel. All allosteric coupling factors need to be increased in order to explain the observed effects when the α subunit is coexpressed with the β2IR subunit.

Biochemical properties of V91G calmodulin: A calmodulin point mutation that deregulates muscle contraction in Drosophila

A mutation (Cam7) to the single endogenous calmodulin gene of Drosophila generates a mutant protein with valine 91 changed to glycine (V91G D-CaM). This mutation produces a unique pupal lethal phenotype distinct from that of a null mutation. Genetic studies indicate that the phenotype reflects deregulation of calcium fluxes within the larval muscles, leading to hypercontraction followed by muscle failure. We investigated the biochemical properties of V91G D-CaM. The effects of the mutation on free CaM are minor: Calcium binding, and overall secondary and tertiary structure are indistinguishable from those of wild type. A slight destabilization of the C-terminal domain is detectable in the calcium-free (apo-) form, and the calcium-bound (holo-) form has a somewhat lower surface hydrophobicity. These findings reinforce the indications from the in vivo work that interaction with a specific CaM target(s) underlies the mutant defects. In particular, defective regulation of ryanodine receptor (RyR) channels was indicated by genetic interaction analysis. Studies described here establish that the putative CaM binding region of the Drosophila RyR (D-RyR) binds wild-type D-CaM comparably to the equivalent CaM-RyR interactions seen for the mammalian skeletal muscle RyR channel isoform (RYR1). The V91G mutation weakens the interaction of both apo- and holo-D-CaM with this binding region, and decreases the enhancement of the calcium-binding affinity of CaM that is detectable in the presence of the RyR target peptide. The predicted functional consequences of these changes are consonant with the in vivo phenotype, and indicate that D-RyR is one, if not the major, target affected by the V91G mutation in CaM.

The adaptive intracellular control of cardiac muscle function.

This study explores the mechanisms dominating the regulation of the biochemical energy consumption and the mechanical output of the actin-myosin motor units, the crossbridges (Xbs), in the cardiac sarcomere. Our analytical model, which couples Xbs cycling dynamics with the kinetics of the free Ca(2+) binding to troponin-C (Tn-C), includes two feedback mechanisms: (1) a cooperativity mechanism, whereby the amount of force generating Xbs determines the affinity of calcium binding to the regulatory protein and the force-length relationship (FLR); and (2) a mechanical (negative) feedback, whereby the filament shortening velocity affects the rate of Xb turnover from the force- to the nonforce-generating state, allows the analytical solution for the muscle force-velocity relationship (FVR), and the linear relation between energy consumption and the generated mechanical energy. Our experimental and analytical studies of the force response to large-amplitude sarcomere length (SL) oscillations at various frequencies and constant [Ca(2+)] in the isolated tetanized rat trabeculae reveal that the generated force depends on the history of contraction and establishes the validity of these two feedbacks. The cooperativity mechanism generates counterclockwise (CCW) hystereses, where the muscle generates external work; while at higher frequencies the mechanical feedback produces clockwise (CW) hystereses, where the muscle behaves as a damper. The cooperativity provides the adaptive control of the cardiac response to short-term changes in the load by modulating Xb recruitment. The cardiac efficiency, defined as the ratio of the generated mechanical energy (i.e., external work and pseudo-potential energy) to the sarcomere energy consumption, is determined by the mechanical feedback, reflecting an inherent property of the single Xb. The efficiency is thus independent of the number of strong Xbs and is constant and load independent.

Controlling the mechanism of trypsin inhibition by the numbers of α-cyclodextrins and carboxyl groups in carboxyethylester-polyrotaxanes

Carboxyethylester-polyrotaxanes (CEE-polyrotaxanes) with the various number of CEE-modified α-cyclodextrins (CEE-α-CDs) were synthesized, and the effects of the number of CEE-α-CDs on calcium binding and trypsin inhibition were investigated. Calcium binding affinity was dependent on the density of the CEE groups accompanied with the number of α-CD threading in the CEE-polyrotaxanes. The high number of CEE-α-CDs leads to greater inhibition of trypsin activity than poly(acrylic acid), which is mainly due to the good calcium binding affinity. The CEE-polyrotaxane with the smallest number of CEE-α-CDs temporally interacted with trypsin, which was well correlated with the inhibition and recovery of trypsin activity. Therefore, the number of CEE-α-CDs in the CEE-polyrotaxanes can control the inhibition mechanism of trypsin activity.

Structural biology of the cell adhesion protein CD2: from molecular recognition to protein folding and design.

CD2 (cluster of differentiation 2) is a cell adhesion molecule expressed on T cells and is recognized as a target for CD48 (rats) and CD58 (humans). Tremendous progress has been achieved in understanding the function of CD2, the mechanism of molecular recognition and protein folding, thus, leading towards the use of this protein as a scaffold for protein design. CD2 has been shown to set quantitative thresholds in T cell activation both in vivo and in vitro. Further, intracellular CD2 signaling pathways and networks are being discovered by the identification of several cytosolic tail binding proteins. In addition, a new method for directly measuring heterophilic adhesion has been developed. The functional "hot spot" for the adhesion surface of CD2 and CD58 has been dissected. Detailed NMR studies reveal that rat CD2 weakly self-associates to form a homodimeric structure in solution. Dynamic interaction of CD2 with the GYF and SH3 domains has been investigated. CD2 has been shown to form fibrils in the presence of 2,2,2-trifluoroethanol (TFE) and at low pH. Furthermore, kinetic studies have been completed to monitor the effect of surface hydrophobic residues and intramolecular bridges on the folding pathways of CD2. Our lab has de novo designed single calcium-binding sites into domain 1 of rat CD2 (CD2-D1) with strong metal selectivity. In addition, the EF-hand motifs have been grafted into CD2 to understand the site-specific calcium-binding affinity of calmodulin and calcium-dependent cell adhesion.

Obtaining site-specific calcium-binding affinities of calmodulin.

Calmodulin (CaM) is an EF-hand Ca(II)-binding protein involved in the regulation of many important biological processes. To date, there is a wealth of information available concerning studies to obtain site-specific calcium binding affinities of CaM, and further to estimate the cooperativity of calcium binding using mutational studies, peptide models, and proteolytic fragmentation. In this paper, we will discuss the energetics of calcium binding and the strong relationship between calcium binding cooperativity and conformational change. We then explain the difficulty of studying key determinants of calcium binding affinity of CaM due to the large change of calcium binding affinity upon mutation. Subsequently, we will introduce "grafting" as a novel approach to obtain the site-specific metal binding properties of calmodulin.

Rational design of a calcium-binding protein.

Calcium ions play key roles as structural components in biomineralization and as a second messenger in signaling pathways. We have introduced a de novo designed calcium-binding site into the framework of a non-calcium-binding protein, domain 1 of CD2. The resulting protein selectively binds calcium over magnesium with calcium-binding affinity comparable to that of natural extracellular calcium-binding proteins (K(d) of 50 microM). This experiment is the first successful metalloprotein design that has a high coordination number (seven) metal-binding site constructed into a beta-sheet protein. Our results demonstrate the feasibility of designing a single calcium-binding site into a host protein, taking into account only local properties of a calcium-binding site obtained by a survey of natural calcium-binding proteins and chelators. The resulting site exhibits strong metal selectivity, suggesting that it should now be feasible to understand and manipulate signaling processes by designing novel calcium-modulated proteins with specifically desired functions and to affect their stability.

Solution Structure and Dynamics of a Calcium Binding Epidermal Growth Factor-like Domain Pair from the Neonatal Region of Human Fibrillin-1

Fibrillin-1 is a mosaic protein mainly composed of 43 calcium binding epidermal growth factor-like (cbEGF) domains arranged as multiple, tandem repeats. Mutations within the fibrillin-1 gene cause Marfan syndrome (MFS), a heritable disease of connective tissue. More than 60% of MFS-causing mutations identified are localized to cbEGFs, emphasizing that the native properties of these domains are critical for fibrillin-1 function. The cbEGF12-13 domain pair is within the longest run of cbEGFs, and many mutations that cluster in this region are associated with severe, neonatal MFS. The NMR solution structure of Ca(2+)-loaded cbEGF12-13 exhibits a near-linear, rod-like arrangement of domains. This observation supports the hypothesis that all fibrillin-1 (cb)EGF-cbEGF pairs, characterized by a single interdomain linker residue, possess this rod-like structure. The domain arrangement of cbEGF12-13 is stabilized by additional interdomain packing interactions to those observed for cbEGF32-33, which may help to explain the previously reported higher calcium binding affinity of cbEGF13. Based on this structure, a model of cbEGF11-15 that encompasses all known neonatal MFS missense mutations has highlighted a potential binding region. Backbone dynamics data confirm the extended structure of cbEGF12-13 and lend support to the hypothesis that a correlation exists between backbone flexibility and cbEGF domain calcium affinity. These results provide important insight into the potential consequences of MFS-associated mutations for the assembly and biomechanical properties of connective tissue microfibrils.

Metal-binding studies for a de novo designed calcium-binding protein.

To understand the key determinants in calcium-binding affinity, a calcium-binding site with pentagonal bipyramid geometry was designed into a non-calcium-binding protein, domain 1 of CD2. This metal-binding protein has five mutations with a net charge in the coordination sphere of -5 and is termed DEEEE. Fluorescence resonance energy transfer was used to determine the metal-binding affinity of DEEEE to the calcium analog terbium. The addition of protein concentration to Tb(III) solution results in a large enhancement of Tb(III) fluorescence due to energy transfer between terbium ions and aromatic residues in CD2-D1. In addition, both calcium and lanthanum compete with terbium for the same desired metal binding pocket. Our designed protein exhibits a stronger affinity for Tb(III), with a K(d) of 21 microM, than natural calcium-binding proteins with a similar Greek key scaffold.