Conformational changes at the tetramerization site of erythroid alpha-spectrin upon binding beta-spectrin: a spin label EPR study

We used cysteine scanning, isothermal titration calorimetry (ITC) and spin label EPR methods to study the two regions that flank the partial domain Helix C' of the N-terminal end of alpha-spectrin (residues 14-20 and residues 44-54) in the absence and presence of a model protein of the beta-spectrin C-terminal end. In the absence of beta-spectrin, residues 14-20 and 46-52 were known to be unstructured. The EPR spectral values of the inverse line width (Delta H (-1)) and of the width between the low field peak and the central peak ( aZ) of residues in part of the first unstructured region (residues 17-20) and of most residues in the second unstructured junction region (residues 46-52) changed dramatically upon association with beta-spectrin, suggesting that the two regions undergo a conformational change, becoming more rigid and likely becoming helical. ITC results showed that three of the seven residues in the junction region (residues 46-52) were very important in its association with beta-spectrin, in the following order: L49 > G46 > K48. In general, our results suggest that any mutations that affect the propensity of helical formation in the region spanning residues 17-52 in alpha-spectrin, or that affect hydrophobic clustering and/or salt-bridge stabilization of the bundled helices, would affect spectrin tetramer formation, and may lead to blood disorders.     

Conformational change of erythroid alpha-spectrin at the tetramerization site upon binding beta-spectrin

We previously determined the solution structures of the first 156 residues of human erythroid alpha-spectrin (SpalphaI-1-156, or simply Spalpha). Spalpha consists of the tetramerization site of alpha-spectrin and associates with a model beta-spectrin protein (Spbeta) with an affinity similar to that of native alpha- and beta-spectrin. Upon alphabeta-complex formation, our previous results indicate that there is an increase in helicity in the complex, suggesting conformational change in either Spalpha or Spbeta or in both. We have now used isothermal titration calorimetry, circular dichroism, static and dynamic light scattering, and solution NMR methods to investigate properties of the complex as well as the conformation of Spalpha in the complex. The results reveal a highly asymmetric complex, with a Perrin shape parameter of 1.23, which could correspond to a prolate ellipsoid with a major axis of about five and a minor axis of about one. We identified 12 residues, five prior to and seven following the partial domain helix in Spalpha that moved freely relative to the structural domain in the absence of Spbeta but when in the complex moved with a mobility similar to that of the structural domain. Thus, it appears that the association with Spbeta induced an unstructured-to-helical conformational transition in these residues to produce a rigid and asymmetric complex. Our findings may provide insight toward understanding different association affinities of alphabeta-spectrin at the tetramerization site for erythroid and non-erythroid spectrin and a possible mechanism to understand some of the clinical mutations, such as L49F of alpha-spectrin, which occur outside the functional partial domain region.     

YEAST two-hybrid and itc studies of alpha and beta spectrin interaction at the tetramerization site

Yeast two-hybrid (Y2H) and isothermal titration calorimetry (ITC) methods were used to further study the mutational effect of non-erythroid alpha spectrin (αII) at position 22 in tetramer formation with beta spectrin (βII). Four mutants, αII-V22D, V22F, V22M and V22W, were studied. For the Y2H system, we used plasmids pGBKT7, consisting of the cDNA of the first 359 residues at the N-terminal region of αII, and pGADT7, consisting of the cDNA of residues 1697–2145 at the C-terminal region of βII. Strain AH109 yeast cells were used for colony growth assays and strain Y187 was used for β-galactosidase activity assays. Y2H results showed that the C-terminal region of βII interacts with the N-terminal region of αII, either the wild type, or those with V22F, V22M or V22W mutations. The V22D mutant did not interact with βII. For ITC studies, we used recombinant proteins of the αII N-terminal fragment and of the erythroid beta spectrin (βI) C-terminal fragment; results showed that the K_d values for V22F were similar to those for the wild-type (about 7 nM), whereas the K_d values were about 35 nM for V22M and about 90 nM for V22W. We were not able to detect any binding for V22D with ITC methods. This study clearly demonstrates that the single mutation at position 22 of αII, a region critical to the function of nonerythroid α spectrin, may lead to a reduced level of spectrin tetramers and abnormal spectrin-based membrane skeleton. These abnormalities could cause abnormal neural activities in cells.     

Solution structural studies on human erythrocyte alpha-spectrin tetramerization site

We have determined the solution NMR structure of a recombinant peptide that consists of the first 156 residues of erythroid alpha-spectrin. The first 20 residues preceding the first helix (helix C') are in a disordered conformation. The subsequent three helices (helices A1, B1, and C1) form a triple helical bundle structural domain that is similar, but not identical, to previously published structures for spectrin from Drosophila and chicken brain. Paramagnetic spin label-induced NMR resonance broadening shows that helix C', the partial domain involved in alpha- and beta-spectrin association, exhibits little interaction with the structural domain. Surprisingly, helix C' is connected to helix A1 of the structural domain by a segment of 7 residues (the junction region) that exhibits a flexible disordered conformation, in contrast to the predicted rigid helical structure. We suggest that the flexibility of this particular junction region may play an important role in modulating the association affinity of alpha- and beta-spectrin at the tetramerization site of different isoforms, such as erythroid spectrin and brain spectrin. These findings may provide insight for explaining various physiological and pathological conditions that are a consequence of varying alpha- and beta-subunit self-association affinities in their formation of the various spectrin tetramers.     

Apparent structural differences at the tetramerization region of erythroid and nonerythroid beta spectrin as discriminated by phage displayed scFvs

We have screened a human immunoglobulin single-chain variable fragment (scFv) phage library against the C-terminal tetramerization regions of erythroid and nonerythroid beta spectrin (βI-C1 and βII-C1, respectively) to explore the structural uniqueness of erythroid and nonerythroid β-spectrin isoforms. We have identified interacting scFvs, with clones "G5" and "A2" binding only to βI-C1, and clone "F11" binding only to βII-C1. The K(d) values, estimated by competitive enzyme-linked immunosorbent assay, of these scFvs with their target spectrin proteins were 0.1-0.3 μM. A more quantitative K(d) value from isothermal titration calorimetry experiments with the recombinant G5 and βI-C1 was 0.15 μM. The α-spectrin fragments (model proteins), αI-N1 and αII-N1, competed with the βI-C1, or βII-C1, binding scFvs, with inhibitory concentration (IC(50) ) values of ～50 μM for αI-N1, and ～0.5 μM for αII-N1. Our predicted structures of βI-C1 and βII-C1 suggest that the Helix B' of the C-terminal partial domain of βI differs from that of βII. Consequently, an unstructured region downstream of Helix B' in βI may interact specifically with the unstructured, complementarity determining region H1 of G5 or A2 scFv. The corresponding region in βII was helical, and βII did not bind G5 scFv. Our results suggest that it is possible for cellular proteins to differentially associate with the C-termini of different β-spectrin isoforms to regulate α- and β-spectrin association to form functional spectrin tetramers, and may sort β-spectrin isoforms to their specific cellular localizations.     

Identification of pH-sensitive regions in the mouse prion by the cysteine-scanning spin-labeling ESR technique

We analyzed the pH-induced mobility changes in moPrP(C) alpha-helix and beta-sheets by cysteine-scanning site-directed spin labeling (SDSL) with ESR. Nine amino acid residues of alpha-helix1 (H1, codon 143-151), four amino acid residues of beta-sheet1 (S1, codon 127-130), and four amino acid residues of beta-sheet2 (S2, codon 160-163) were substituted for by cysteine residues. These recombinant mouse PrP(C) (moPrP(C)) mutants were reacted with a methane thiosulfonate sulfhydryl-specific spin labeling reagent (MTSSL). The 1/deltaH of the central (14N hyperfine) component (M(I) = 0) in the ESR spectrum of spin-labeled moPrP(C) was measured as a mobility parameter of nitroxide residues (R1). The mobilities of E145R1 and Y149R1 at pH 7.4, which was identified as a tertiary contact site by a previous NMR study of moPrP, were lower than those of D143R1, R147R1, and R150R1 reported on the helix surface. Thus, the mobility in the H1 region in the neutral solution was observed with the periodicity associated with a helical structure. On the other hand, the values in the S2 region, known to be located in the buried side, were lower than those in the S1 region located in the surface side. These results indicated that the mobility parameter of the nitroxide label was well correlated with the 3D structure of moPrP. Furthermore, the present study clearly demonstrated three pH-sensitive sites in moPrP, i.e., (1) the N-terminal tertiary contact site of H1, (2) the C-terminal end of H1, and (3) the S2 region. In particular, among these pH-sensitive sites, the N-terminal tertiary contact region of H1 was found to be the most pH-sensitive one and was easily converted to a flexible structure by a slight decrease of pH in the solution. These data provided molecular evidence to explain the cellular mechanism for conversion from PrP(C) to PrP(Sc) in acidic organelles such as the endosome.     

Studies of the erythrocyte spectrin tetramerization region

Human erythrocyte spectrin dimers associate at the N-terminal region of alpha spectrin (alpha N) and the C-terminal region of beta-spectrin (beta C) to form tetramers. We have prepared model peptides to study the tetramerization region. Based on phasing information obtained from enzyme digests, we prepared spectrin fragments consisting of the first 156 amino-acid residues and the first 368 amino-acid residues of alpha-spectrin (Sp alpha 1-156 and Sp alpha 1-368, respectively), and found that both peptides associate with a beta-spectrin model peptide, with an affinity similar to that found in alpha beta dimer tetramerization. Spin label EPR studies show that the region consisting of residues 21-46 in alpha-spectrin is helical even in the absence of its beta-partner. Multi-dimensional nuclear magnetic resonance studies of samples with and without a spin label attached to residue 154 show that Sp alpha 1-156 consists of four helices, with the first helix unassociated with the remaining three helices, which bundle to form a triple helical coiled coil bundle. A comparison of the structures of erythrocyte spectrin with other published structures of Drosophila and chicken brain spectrin is discussed. Circular dichroism studies show that the lone helix in Sp alpha-156 associates with helices in the beta peptide to form a coiled coil bundle. Based on NMR and CD results, we suggest that the helices in Sp alpha 1-156 exhibit a looser (frayed) conformation, and that the helices convert to a tighter conformation upon association with its beta-partner. This suggestion does not rule out possible conversion of a non-structured conformation to a structured conformation in various parts of the molecule upon association. Spectrin mutations at residues 28 and 45 of alpha-spectrin have been found in patients with hereditary elliptocytosis. NMR studies were also carried out on Sp alpha 1-156R28S, Sp alpha 1-156R45S and Sp alpha 1-156R45T. A comparison of the structures of Sp alpha 1-156 and Sp alpha 1-156R28S, Sp alpha 1-156R45S and Sp alpha 1-156R45T is discussed.     

Conformational stabilities of the structural repeats of erythroid spectrin and their functional implications

The two polypeptide chains of the erythroid spectrin heterodimer contain between them 36 structural repeating modules, which can function as independently folding units. We have expressed all 36 and determined their thermal stabilities. These vary widely, with unfolding transition mid-points (T(m)) ranging from 21 to 72 degrees C. Eight of the isolated repeats are largely unfolded at physiological temperature. Constructs comprising two or more adjacent repeats show inter-repeat coupling with coupling free energies of several kcal mol(-1). Constructs comprising five successive repeats from the beta-chain displayed cooperativity and strong temperature dependence in forced unfolding by atomic force microscopy. Analysis of aligned sequences and molecular modeling suggests that high stability is conferred by large hydrophobic side chains at position e of the heptad hydrophobic repeats in the first helix of the three-helix bundle that makes up each repeat. This inference was borne out by the properties of mutants in which the critical residues have been replaced. The marginal stability of the tertiary structure at several points in the spectrin chains is moderated by energetic coupling with adjoining structural elements but may be expected to permit adaptation of the membrane to the large distortions that the red cell experiences in the circulation.     

Brain proteins interacting with the tetramerization region of non-erythroid alpha spectrin

The N-terminal region of non-erythroid alpha spectrin (SpαII) is responsible for interacting with its binding partner, beta spectrin, to form functional spectrin tetramers. We used a yeast-two-hybrid system, with an N-terminal segment of alpha spectrin representing the functional tetramerization site, as a bait to screen human brain c-DNA library for proteins that interact with the alpha spectrin segment. In addition to several beta spectrin isoforms, we identified 14 proteins that interact with SpαII. Seven of the 14 were matched to 6 known proteins: Duo protein, Lysyl-tRNA synthetase, TBP associated factor 1, two isoforms (b and c) of a protein kinase A interacting protein and Zinc finger protein 333 (2 different segments). Four of the 6 proteins are located primarily in the nucleus, suggesting that spectrin plays important roles in nuclear functions. The remaining 7 proteins were unknown to the protein data base. Structural predictions show that many of the 14 proteins consist of a large portion of unstructured regions, suggesting that many of these proteins fold into a rather flexible conformation. It is interesting to note that all but 3 of the 14 proteins are predicted to consist of one to four coiled coils (amphiphilic helices). A mutation in SpαII, V22D, which interferes with the coiled coil bundling of SpαII with beta spectrin, also affects SpαII interaction with Duo protein, TBP associated factor 1 and Lysyl-tRNA synthetase, suggesting that they may compete with beta spectrin for interaction with SpαII. Future structural and functional studies of these proteins to provide interaction mechanisms will no doubt lead to a better understanding of brain physiology and pathophysiology.     

Conformational specificity of the lac repressor coiled-coil tetramerization domain

Predictive understanding of how the folded, functional shape of a native protein is encoded in the linear sequence of its amino acid residues remains an unsolved challenge in modern structural biology. Antiparallel four-stranded coiled coils are relatively simple protein structures that embody a heptad sequence repeat and rich diversity for tertiary packing of alpha-helices. To explore specific sequence determinants of the lac repressor coiled-coil tetramerization domain, we have engineered a set of buried nonpolar side chains at the a-, d-, and e-positions into the hydrophobic interior of the dimeric GCN4 leucine zipper. Circular dichroism and equilibrium ultracentrifugation studies show that this core variant (GCN4-pAeLV) forms a stable tetrameric structure with a reversible and highly cooperative thermal unfolding transition. The X-ray crystal structure at 1.9 A reveals that GCN4-pAeLV is an antiparallel four-stranded coiled coil of the lac repressor type in which the a, d, and e side chains associate by means of combined knobs-against-knobs and knobs-into-holes packing with a characteristic interhelical offset of 0.25 heptad. Comparison of the side chain shape and packing in the antiparallel tetramers shows that the burial of alanine residues at the e positions between the neighboring helices of GCN4-pAeLV dictates both the antiparallel orientation and helix offset. This study fills in a gap in our knowledge of the determinants of structural specificity in antiparallel coiled coils and improves our understanding of how specific side chain packing forms the teritiary structure of a functional protein.     

Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC(1)]-Gm; [TOAC(1), Ser(2,6,11,15)]-Gm; [Trp(7)]-Gm; [Ser(2,6,11,15), Trp(7)]-Gm; [Trp(9)]-Gm; and [Ser(2,6,11,15), Trp(9)]-Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a beta-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC(1)]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.     

Conformational and functional studies of gomesin analogues by CD, EPR and fluorescence spectroscopies

The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC¹]-Gm; [TOAC¹, Ser^2,6,11,15]-Gm; [Trp⁷]-Gm; [Ser^2,6,11,15, Trp⁷]-Gm; [Trp⁹]-Gm; and [Ser^2,6,11,15, Trp⁹]-Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a β-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC¹]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.     

Identification of the main ubiquitination site in human erythroid alpha-spectrin

Erythroid spectrin is the main component of the red cell membrane skeleton, which is very important in determining the shape, resistance to mechanical stresses and deformability of red cells. Previously we demonstrated that human erythroid alpha-spectrin is ubiquitinated in vitro and in vivo, and using recombinant peptides we identified on repeat 17 the main ubiquitination site of alpha-spectrin. In order to identify the lysine(s) involved in the ubiquitination process, in the present study we mutated the lysines by site-directed mutagenesis. We found that ubiquitination was dramatically inhibited in peptides carrying the mutation of lysine 27 on repeat 17 (mutants K25,27R and K27R). We also demonstrated that the correct folding of this protein is fundamental for its recognition by the ubiquitin conjugating system. Furthermore, the region flanking lysine 27 showed a 75% similarity with the leucine zipper pattern present in many regulatory proteins. Thus, a new potential ubiquitin recognition motif was identified in alpha-spectrin and may be present in several other proteins.     

Physical properties of the lipid bilayer membrane made of cortical and nuclear bovine lens lipids: EPR spin-labeling studies

The physical properties of membranes derived from the total lipids extracted from the lens cortex and nucleus of a 2-year-old cow were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves show that spin labels detect a single homogenous environment in membranes made from cortical lipids. Properties of these membranes are very similar to those reported by us for membranes made of the total lipid extract of 6-month-old calf lenses (J. Widomska, M. Raguz, J. Dillon, E. R. Gaillard, W. K. Subczynski, Biochim. Biophys. Acta 1768 (2007) 1454-1465). However, in membranes made from nuclear lipids, two domains were detected by the EPR discrimination by oxygen transport method using the cholesterol analogue spin label and were assigned to the bulk phospholipid-cholesterol domain (PCD) and the immiscible cholesterol crystalline domain (CCD), respectively. Profiles of the order parameter, hydrophobicity, and the oxygen transport parameter are practically identical in the bulk PCD when measured for either the cortical or nuclear lipid membranes. In both membranes, lipids in the bulk PCD are strongly immobilized at all depths. Hydrophobicity and oxygen transport parameter profiles have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The permeability coefficient for oxygen, estimated at 35 °C, across the bulk PCD in both membranes is slightly lower than across the water layer of the same thickness. However, the evaluated upper limit of the permeability coefficient for oxygen across the CCD (34.4 cm/s) is significantly lower than across the water layer of the same thickness (85.9 cm/s), indicating that the CCD can significantly reduce oxygen transport in the lens nucleus.     

Organization and dynamics of tryptophan residues in erythroid spectrin: novel structural features of denatured spectrin revealed by the wavelength-selective fluorescence approach

We have investigated the organization and dynamics of the functionally important tryptophan residues of erythroid spectrin in native and denatured conditions utilizing the wavelength-selective fluorescence approach. We observed a red edge excitation shift (REES) of 4 nm for the tryptophans in the case of spectrin in its native state. This indicates that tryptophans in spectrin are localized in a microenvironment of restricted mobility, and that the regions surrounding the spectrin tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited state tryptophans. Interestingly, spectrin exhibits a REES of 3 nm even when denatured in 8 M urea. This represents the first report of a denatured protein displaying REES. Observation of REES in the denatured state implies that some of the structural and dynamic features of this microenvironment around the spectrin tryptophans are retained even when the protein is denatured. Fluorescence quenching data of denatured spectrin support this conclusion. In addition, we have deduced the organization and dynamics of the hydrophobic binding site of the polarity-sensitive fluorescent probe PRODAN that binds erythroid spectrin with high affinity. When bound to spectrin, PRODAN exhibits a REES of 9 nm. Because PRODAN binds to a hydrophobic site in spectrin, such a result would directly imply that this region of spectrin offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state fluorophore. The results of our study could provide vital insight into the role of tryptophans in the stability and folding of spectrin.     

Binding of polarity-sensitive hydrophobic ligands to erythroid and nonerythroid spectrin: fluorescence and molecular modeling studies

We have used three polarity-sensitive fluorescence probes, 6-propionyl 2-(N,N-dimethyl-amino) naphthalene (Prodan), pyrene and 8-anilino 1-naphthalene sulphonic acid, to study their binding with erythroid and nonerythroid spectrin, using fluorescence spectroscopy. We have found that both bind to prodan and pyrene with high affinities with apparent dissociation constants (K_d) of .50 and .17?μM, for prodan, and .04 and .02?μM, for pyrene, respectively. The most striking aspect of these bindings have been that the binding stoichiometry have been equal to 1 in erythroid spectrin, both in dimeric and tetrameric form, and in tetrameric nonerythroid spectrin. From an estimate of apparent dielectric constants, the polarity of the binding site in both erythroid and nonerythroid forms have been found to be extremely hydrophobic. Thermodynamic parameters associated with such binding revealed that the binding is favored by positive change in entropy. Molecular docking studies alone indicate that both prodan and pyrene bind to the four major structural domains, following the order in the strength of binding to the Ankyrin binding domain?>?SH3 domain?>?Self-association domain?>?N-terminal domain of α-spectrin of both forms of spectrin. The binding experiments, particularly with the tetrameric nonerythroid spectrin, however, indicate more toward the self association domain in offering the unique binding site, since the binding stoichiometry have been 1 in all forms of dimeric and tetrameric spectrin, so far studied by us. Further studies are needed to characterize the hydrophobic binding sites in both forms of spectrin.     

Conformational studies of human islet amyloid peptide using molecular dynamics and simulated annealing methods

Molecular dynamics simulations and simulated annealing in vacuum, model aqueous solution, and simulated membrane were used to analyze the conformational preferences of a segment spanning 20–29 residues of human islet amyloid polypeptide, [referred to as IAPP^H(20–29)]. Molecular dynamics simulations were conducted at 300 K on IAPP^H(20–29). The minimum energy conformers obtained in model aqueous solution and vacuum exhibited similar structures. Even in the absence of any constraints on peptide bonds, trans conformation was preferred consistently by all the peptide bonds. Analysis of the minimum energy conformers indicated that IAPP^H(20–29) showed a strong preference for turn structures in all the environments. These turn structures were stabilized by the formation of hydrogen bonds between the backbone amide and carbonyl groups. A good agreement was found between the results obtained from the molecular dynamics simulation and solid-state nmr experimental studies. © 1998 John Wiley & Sons, Inc. Biopoly 45: 9–20, 1998     

Calcium-dependent movement of troponin I between troponin C and actin as revealed by spin-labeling EPR

We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca(2+)-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca(2+) state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time tau=1.9ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca(2+) state showed that Cys133 existed on a stable segment (tau=4.8ns) held by TnC. Spectra of reconstituted thin filaments (-Ca(2+) state) revealed that slow mobility (tau=45ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca(2+) concentrations.