A specific C-terminal deletion in tropomyosin results in a stronger head-to-tail interaction and increased polymerization.

Tropomyosin is a 284 residue dimeric coiled-coil protein that interacts in a head-to-tail manner to form linear filaments at low ionic strengths. Polymerization is related to tropomyosin's ability to bind actin, and both properties depend on intact N- and C-termini as well as alpha-amino acetylation of the N-terminus of the muscle protein. Nalpha-acetylation can be mimicked by an N-terminal Ala-Ser fusion in recombinant tropomyosin (ASTm) produced in Escherichia coli. Here we show that a recombinant tropomyosin fragment, corresponding to the protein's first 260 residues plus an Ala-Ser fusion [ASTm(1-260)], polymerizes to a much greater extent than the corresponding full-length recombinant protein, despite the absence of the C-terminal 24 amino acids. This polymerization is sensitive to ionic strength and is greatly reduced by the removal of the N-terminal Ala-Ser fusion [nfTm(1-260)]. CD studies show that nonpolymerizable tropomyosin fragments, which terminate at position 260 [Tm(167-260) and Tm(143-260)], as well as Tm(220-284), are able to interact with ASTm(1-142), a nonpolymerizable N-terminal fragment, and that the head-to-tail interactions observed for these fragment pairs are accompanied by a significant degree of folding of the C-terminal tropomyosin fragment. These results suggest that the new C-terminus, created by the deletion, polymerizes in a manner similar to the full-length protein. Head-to-tail binding for fragments terminating at position 260 may be explained by the presence of a greater concentration of negatively charged residues, while, at the same time, maintaining a conserved pattern of charged and hydrophobic residues found in polymerizable tropomyosins from a variety of sources.     

Different effects of trifluoroethanol and glycerol on the stability of tropomyosin helices and the head-to-tail complex

Tropomyosin (Tm) is a dimeric coiled-coil protein, composed of 284 amino acids (410 A), that forms linear homopolymers through head-to-tail interactions at low ionic strength. The head-to-tail complex involves the overlap of approximately nine N-terminal residues of one molecule with nine C-terminal residues of another Tm molecule. In this study, we investigate the influence of 2,2,2-trifluoroethanol (TFE) and glycerol on the stability of recombinant Tm fragments (ASTm1-142, Tm143-284(5OHW269)) and of the dimeric head-to-tail complex formed by the association of these two fragments. The C-terminal fragment (Tm143-284(5OHW269)) contains a 5-hydroxytryptophan (5OHW) probe at position 269 whose fluorescence is sensitive to the head-to-tail interaction and allows us to accompany titrations of Tm143-284(5OHW269) with ASTm1-142 to calculate the dissociation constant (Kd) and the interaction energy at TFE and glycerol concentrations between 0% and 15%. We observe that TFE, but not glycerol, reduces the stability of the head-to-tail complex. Thermal denaturation experiments also showed that the head-to-tail complex increases the overall conformational stability of the Tm fragments. Urea and thermal denaturation assays demonstrated that both TFE and glycerol increase the stability of the isolated N- and C-terminal fragments; however, only TFE caused a significant reduction in the cooperativity of unfolding these fragments. Our results show that these two cosolvents stabilize the structures of individual Tm fragments in different manners and that these differences may be related to their opposing effects on head-to-tail complex formation.     

Equilibrium unfolding and conformational plasticity of troponin I and T.

The structures and stabilities of recombinant chicken muscle troponin I (TnI) and T (TnT) were investigated by a combination of bis-ANS binding and equilibrium unfolding studies. Unlike most folded proteins, isolated TnI and TnT bind the hydrophobic fluorescent probe bis-ANS, indicating the existence of solvent-exposed hydrophobic domains in their structures. Bis-ANS binding to binary or ternary mixtures of TnI, TnT and troponin C (TnC) in solution is significantly lower than binding to the isolated subunits, which can be explained by burial of previously exposed hydrophobic domains upon association of the subunits to form the native troponin complex. Equilibrium unfolding studies of TnT and TnI by guanidine hydrochloride and urea monitored by changes in far-UV CD and bis-ANS fluorescence revealed noncooperative folding transitions for both proteins and the existence of partially folded intermediate states. Taken together, these results indicate that isolated TnI and TnT are partially unstructured proteins, and suggest that conformational plasticity of the isolated subunits may play an important role in macromolecular recognition for the assembly of the troponin complex.     

Deciphering the role of the electrostatic interactions in the alpha-tropomyosin head-to-tail complex

Skeletal alpha-tropomyosin (Tm) is a dimeric coiled-coil protein that forms linear assemblies under low ionic strength conditions in vitro through head-to-tail interactions. A previously published NMR structure of the Tm head-to-tail complex revealed that it is formed by the insertion of the N-terminal coiled-coil of one molecule into a cleft formed by the separation of the helices at the C-terminus of a second molecule. To evaluate the contribution of charged residues to complex stability, we employed single and double-mutant Tm fragments in which specific charged residues were changed to alanine in head-to-tail binding assays, and the effects of the mutations were analyzed by thermodynamic double-mutant cycles and protein-protein docking. The results show that residues K5, K7, and D280 are essential to the stability of the complex. Though D2, K6, D275, and H276 are exposed to the solvent and do not participate in intermolecular contacts in the NMR structure, they may contribute to head-to-tail complex stability by modulating the stability of the helices at the Tm termini.     

Structural analysis of smooth muscle tropomyosin α and β isoforms

A large number of tropomyosin (Tm) isoforms function as gatekeepers of the actin filament, controlling the spatiotemporal access of actin-binding proteins to specialized actin networks. Residues ∼40–80 vary significantly among Tm isoforms, but the impact of sequence variation on Tm structure and interactions with actin is poorly understood, because structural studies have focused on skeletal muscle Tmα. We describe structures of N-terminal fragments of smooth muscle Tmα and Tmβ (sm-Tmα and sm-Tmβ). The 2.0-Å structure of sm-Tmα81 (81-aa) resembles that of skeletal Tmα, displaying a similar super-helical twist matching the contours of the actin filament. The 1.8-Å structure of sm-Tmα98 (98-aa) unexpectedly reveals an antiparallel coiled coil, with the two chains staggered by only 4 amino acids and displaying hydrophobic core interactions similar to those of the parallel dimer. In contrast, the 2.5-Å structure of sm-Tmβ98, containing Gly-Ala-Ser at the N terminus to mimic acetylation, reveals a parallel coiled coil. None of the structures contains coiled-coil stabilizing elements, favoring the formation of head-to-tail overlap complexes in four of five crystallographically independent parallel dimers. These complexes show similarly arranged 4-helix bundles stabilized by hydrophobic interactions, but the extent of the overlap varies between sm-Tmβ98 and sm-Tmα81 from 2 to 3 helical turns. The formation of overlap complexes thus appears to be an intrinsic property of the Tm coiled coil, with the specific nature of hydrophobic contacts determining the extent of the overlap. Overall, the results suggest that sequence variation among Tm isoforms has a limited effect on actin binding but could determine its gatekeeper function.     

Local heterogeneity in the pressure denaturation of the coiled-coil tropomyosin because of subdomain folding units

Coiled-coil domains mediate the oligomerization of many proteins. The assembly of long coiled coils, such as tropomyosin, presupposes the existence of intermediates. These intermediates are not well-known for tropomyosin. Hydrostatic pressure affects the equilibrium between denatured and native forms in the direction of the form that occupies a smaller volume. The hydrophobic core is the region more sensitive to pressure, which leads in most cases to the population of intermediates. Here, we used N-(1-pyrenyl)iodoacetamide covalently bound to cysteine residues of tropomyosin (PIATm) and high hydrostatic pressure to assess the chain interaction and the inherent instability of the coiled-coil molecule. The native and denatured states of tropomyosin were determined from the pyrene excimer fluorescence. The combination of low temperature and high pressure permitted the attainment of the full denaturation of tropomyosin without the separation of the subunits. High-temperature denaturation of Tm leads to a great exchange between labeled and unlabeled Tm subunits, indicating subunit dissociation linked to unfolding. In contrast, under high pressure, unlabeled and labeled tropomyosin molecules do not exchange, demonstrating that the denatured species are dimeric. The decrease of the concentration dependence of PIATm corroborates the idea that pressure produces subdomain denaturation and that the polypeptide chains do not separate. Substantial unfolding of tropomyosin was also verified by measurements of tyrosine fluorescence and bis-ANS binding. Our results indicate the presence of independent folding subdomains with different susceptibilities to pressure along the length of the coiled-coil structure of tropomyosin.     

Identification of a potential hydrophobic peptide binding site in the C-terminal arm of trigger factor

Trigger factor (TF) is the first chaperone to interact with nascent chains and facilitate their folding in bacteria. Escherichia coli TF is 432 residues in length and contains three domains with distinct structural and functional properties. The N-terminal domain of TF is important for ribosome binding, and the M-domain carries the PPIase activity. However, the function of the C-terminal domain remains unclear, and the residues or regions directly involved in substrate binding have not yet been identified. Here, a hydrophobic probe, bis-ANS, was used to characterize potential substrate-binding regions. Results showed that bis-ANS binds TF with a 1:1 stoichiometry and a K(d) of 16 microM, and it can be covalently incorporated into TF by UV-light irradiation. A single bis-ANS-labeled peptide was obtained by tryptic digestion and identified by MALDI-TOF mass spectrometry as Asn391-Lys392. In silico docking analysis identified a single potential binding site for bis-ANS on the TF molecule, which is adjacent to this dipeptide and lies in the pocket formed by the C-terminal arms. The bis-ANS-labeled TF completely lost the ability to assist GAPDH or lysozyme refolding and showed increased protection toward cleavage by alpha-chymotrypsin, suggesting blocking of hydrophobic residues. The C-terminal truncation mutant TF389 also showed no chaperone activity and could not bind bis-ANS. These results suggest that bis-ANS binding may mimic binding of a substrate peptide and that the C-terminal region of TF plays an important role in hydrophobic binding and chaperone function.     

Folding intermediates of the prion protein stabilized by hydrostatic pressure and low temperature

Prion diseases are associated with conformational conversion of the cellular prion protein, PrPC, into a misfolded form, PrPSc. We have investigated the equilibrium unfolding of the structured domain of recombinant murine prion protein, comprising residues 121-231 (mPrP-(121-231)). The equilibrium unfolding of mPrP-(121-231) by urea monitored by intrinsic fluorescence and circular dichroism (CD) spectroscopies indicated a two-state transition, without detectable folding intermediates. The fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5-disulfonic acid (bis-ANS) binds to native mPrP-(121-231), indicating exposure of hydrophobic domains on the protein surface. Increasing concentrations of urea (up to 4 M) caused the release of bound bis-ANS, whereas changes in intrinsic fluorescence and CD of mPrP took place only above 4 M urea. This indicates the existence of a partially unfolded conformation of mPrP, characterized by loss of bis-ANS binding and preservation of the overall structure of the protein, stabilized at low concentrations of urea. Hydrostatic pressure and low temperatures were also used to stabilize partially folded intermediates that are not detectable in the presence of chemical denaturants. Compression of mPrP to 3.5 kbar at 25 degrees C and pH 7 caused a slight decrease in intrinsic fluorescence emission and an 8-fold increase in bis-ANS fluorescence. Lowering the temperature to -9 degrees C under pressure reversed the decrease in intrinsic fluorescence and caused a marked (approximately 40-fold) increase in bis-ANS fluorescence. The increase in bis-ANS fluorescence at low temperatures was similar to that observed for mPrP at 1 atm at pH 4. These results suggest that pressure-assisted cold denaturation of mPrP stabilizes a partially folded intermediate that is qualitatively similar to the state obtained at acidic pH. Compression of mPrP in the presence of a subdenaturing concentration of urea stabilized another partially folded intermediate, and cold denaturation under these conditions led to complete unfolding of the protein. Possible implications of the existence of such partially folded intermediates in the folding of the prion protein and in the conversion to the PrPSc conformer are discussed.     

Specific sequences determine the stability and cooperativity of folding of the C-terminal half of tropomyosin

Tropomyosin is a flexible 410 A coiled-coil protein in which the relative stabilities of specific regions may be important for its proper function in the control of muscle contraction. In addition, tropomyosin can be used as a simple model of natural occurrence to understand the inter- and intramolecular interactions that govern the stability of coiled-coils. We have produced eight recombinant tropomyosin fragments (Tm(143-284(5OHW),) Tm(189-284(5OHW)), Tm(189-284), Tm(220-284(5OHW)), Tm(220-284), Tm(143-235), Tm(167-260), and Tm(143-260)) and one synthetic peptide (Ac-Tm(215-235)) to investigate the relative conformational stability of different regions derived from the C-terminal region of the protein, which is known to interact with the troponin complex. Analytical ultracentrifugation experiments show that the fragments that include the last 24 residues of the molecule (Tm(143-284(5OHW)), Tm(189-284(5OHW)), Tm(220-284(5OHW)), Tm(220-284)) are completely dimerized at 10 microm dimer (50 mm phosphate, 100 mm NaCl, 1.0 mm dithiothreitol, and 0.5 mm EDTA, 10 degrees C), whereas fragments that lack the native C terminus (Tm(143-235),Tm(167-260), and Tm(143-260)) are in a monomer-dimer equilibrium under these conditions. The presence of trifluoroethanol resulted in a reduction in the [theta](222)/[theta](208) circular dichroism ratio in all of the fragments and induced stable trimer formation only in those containing residues 261-284. Urea denaturation monitored by circular dichroism and fluorescence revealed that residues 261-284 of tropomyosin are very important for the stability of the C-terminal half of the molecule as a whole. Furthermore, the absence of this region greatly increases the cooperativity of urea-induced unfolding. Temperature and urea denaturation experiments show that Tm(143-235) is less stable than other fragments of the same size. We have identified a number of factors that may contribute to this particular instability, including an interhelix repulsion between g and e' positions of the heptad repeat, a charged residue at the hydrophobic coiled-coil interface, and a greater fraction of beta-branched residues located at d positions.     

Altering the stability of the Cdc8 overlap region modulates the ability of this tropomyosin to bind co-operatively to actin and regulate myosin

Tm (tropomyosin) is an evolutionarily conserved α-helical coiled-coil protein, dimers of which form end-to-end polymers capable of associating with and stabilizing actin filaments, and regulating myosin function. The fission yeast Schizosaccharomyces pombe possesses a single essential Tm, Cdc8, which can be acetylated on its N-terminal methionine residue to increase its affinity for actin and enhance its ability to regulate myosin function. We have designed and generated a number of novel Cdc8 mutant proteins with N-terminal substitutions to explore how stability of the Cdc8 overlap region affects the regulatory function of this Tm. By correlating the stability of each protein, its propensity to form stable polymers, its ability to associate with actin and to regulate myosin, we have shown that the stability of the N-terminal of the Cdc8 α-helix is crucial for Tm function. In addition we have identified a novel Cdc8 mutant with increased N-terminal stability, dimers of which are capable of forming Tm polymers significantly longer than the wild-type protein. This protein had a reduced affinity for actin with respect to wild-type, and was unable to regulate actomyosin interactions. The results of the present paper are consistent with acetylation providing a mechanism for modulating the formation and stability of Cdc8 polymers within the fission yeast cell. The data also provide evidence for a mechanism in which Tm dimers form end-to-end polymers on the actin filament, consistent with a co-operative model for Tm binding to actin.     

Structural rearrangements and the unfolding mechanism of a Trigger Factor mutant studied by multiple structural probes

Trigger Factor (TF) is a three-domain chaperone which catalyzes nascent peptide folding and harbors peptidyl–prolyl cis–trans isomerase activity. The multi-domain structure of TF makes it an interesting and challenging candidate for studies of the structural properties and functional behavior of individual domains or combined domain constructs. Here we constructed a TF mutant, NC, combining the N- and C-domains that are responsible for TF's chaperone function, and compared structural changes and unfolding characteristics of NC with wild-type TF by monitoring fluorescence spectra, far-UV CD, chemical crosslinking, DSC and binding with hydrophobic probes (ANS or bis-ANS). The results showed that the NC construct, like intact TF, could bind to hydrophobic probes, form dimers in solution, and showed a similar 3-state guanidine-induced unfolding profile. However, the NC fragment showed reduced stability towards both guanidine unfolding and thermal denaturation, suggesting that the presence of the M-domain of TF contributes to the stability of the intact TF structure.     

Single-molecule force spectroscopy of mycobacterial adhesin-adhesin interactions

The heparin-binding hemagglutinin (HBHA) is one of the few virulence factors identified for Mycobacterium tuberculosis. It is a surface-associated adhesin that expresses a number of different activities, including mycobacterial adhesion to nonphagocytic cells and microbial aggregation. Previous evidence indicated that HBHA is likely to form homodimers or homopolymers via a predicted coiled-coil region located within the N-terminal portion of the molecule. Here, we used single-molecule atomic-force microscopy to measure individual homophilic HBHA-HBHA interaction forces. Force curves recorded between tips and supports derivatized with HBHA proteins exposing their N-terminal domains showed a bimodal distribution of binding forces reflecting the formation of dimers or multimers. Moreover, the binding peaks showed elongation forces that were consistent with the unfolding of α-helical coiled-coil structures. By contrast, force curves obtained for proteins exposing their lysine-rich C-terminal domains showed a broader distribution of binding events, suggesting that they originate primarily from intermolecular electrostatic bridges between cationic and anionic residues rather than from specific coiled-coil interactions. Notably, similar homophilic HBHA-HBHA interactions were demonstrated on live mycobacteria producing HBHA, while they were not observed on an HBHA-deficient mutant. Together with the fact that HBHA mediates bacterial aggregation, these observations suggest that the single homophilic HBHA interactions measured here reflect the formation of multimers that may promote mycobacterial aggregation.     

Functional homodimers and heterodimers of recombinant smooth muscle tropomyosin

Skeletal and smooth muscle tropomyosin (Tm) require acetylation of their N-termini to bind strongly to actin. Tm containing an N-terminal alanine-serine (AS) extension to mimic acetylation has been widely used to increase binding. The current study investigates the ability of an N-terminal AS extension to mimic native acetylation for both alpha alpha and beta beta smooth Tm homodimers. We show that (1) AS alpha-Tm binds actin 100-fold tighter than alpha-Tm and 2-fold tighter than native smooth alphabeta-Tm, (2) beta-Tm requires an AS extension to bind actin, and (3) AS beta-Tm binds actin 10-fold weaker than AS alpha-Tm. Tm is present in smooth muscle tissues as >95% heterodimer; therefore, we studied the binding of recombinant alphabeta heterodimers with different AS extensions. This study shows that recombinant Tm requires an AS extension on both alpha and beta chains to bind like native Tm and that the alpha chain contributes more to actin binding than the beta chain. Once assembled onto an actin filament, all smooth muscle Tm's regulate S1 binding to actin Tm in the same way, irrespective of the presence of an AS extension.     

Characterization of the Head-to-Tail Overlap Complexes Formed by Human Lamin A, B1 and B2 “Half-minilamin” Dimers

Half-minilamins, representing amino- and carboxy-terminal fragments of human lamins A, B1 and B2 with a truncated central rod domain, were investigated for their ability to form distinct head-to-tail-type dimer complexes. This mode of interaction represents an essential step in the longitudinal assembly reaction exhibited by full-length lamin dimers. As determined by analytical ultracentrifugation, the amino-terminal fragments were soluble under low ionic strength conditions sedimenting with distinct profiles and s-values (1.6-1.8 S) indicating the formation of coiled-coil dimers. The smaller carboxy-terminal fragments were, except for lamin B2, largely insoluble under these conditions. However, after equimolar amounts of homotypic amino- and carboxy-terminal lamin fragments had been mixed in 4 M urea, upon subsequent renaturation the carboxy-terminal fragments were completely rescued from precipitation and distinct soluble complexes with higher s-values (2.3-2.7 S) were obtained. From this behavior, we conclude that the amino- and carboxy-terminal coiled-coil dimers interact to form distinct oligomers (i.e. tetramers). Furthermore, a corresponding interaction occurred also between heterotypic pairs of A- and B-type lamin fragments. Hence, A-type lamin dimers may interact with B-type lamin dimers head-to-tail to yield linear polymers. These findings indicate that a lamin dimer principally has the freedom for a “combinatorial” head-to-tail association with all types of lamins, a property that might be of significant importance for the assembly of the nuclear lamina. Furthermore, we suggest that the head-to-tail interaction of the rod end domains represents a principal step in the assembly of cytoplasmic intermediate filament proteins too.     

Alteration around the active site of rhodanese during urea-induced denaturation and its implications for folding

The enzyme rhodanese contains two globular domains connected by a tether region and associated by strong hydrophobic interactions. The protein has proven to be very difficult to refold without assistance to prevent oxidation and aggregation. For this study, the active site cysteine 247, near the interdomain region, was modified with the environmentally sensitive fluorescent probe, 2-(4'-(iodoacetamido)anilino)naphthalene-6-sulfonic acid (IAANS), to yield a derivative that reversibly unfolds. Structural transitions during urea unfolding/refolding were complex and multiphasic. Increasing urea concentrations increased the IAANS fluorescence intensity and polarization. Both values reached maxima at approximately 4 m urea, where there is a concomitant large exposure of hydrophobic sites as reported by both IAANS and the noncovalent fluorescent probe, bis-ANS. The exposure of the hydrophobic sites arises from the decrease in strong interaction between the domain interfaces, which lead to their partial separation. This correlates with the loss of activity of the unlabeled enzyme. Above 4.5 m urea, there is progressive loss of rigid, hydrophobic surfaces, and both fluorescence and polarization of IAANS decrease, with accompanying loss of secondary structure. These results are consistent with a folding model in which there is an initial, rapid hydrophobic collapse of the denatured form to an intermediate with native like secondary structure, with exposed interdomain, hydrophobic surfaces. This step is followed by adjustment of the domain-domain interactions and the proper positioning of reduced cysteine 247 at the active site.     

The binding of bis-ANS to the isolated GroEL apical domain fragment induces the formation of a folding intermediate with increased hydrophobic surface not observed in tetradecameric GroEL

The extent of hydrophobic exposure upon bis-ANS binding to the functional apical domain fragment of GroEL, or minichaperone (residues 191-345), was investigated and compared with that of the GroEL tetradecamer. Although a total of seven molecules of bis-ANS bind cooperatively to this minichaperone, most of the hydrophobic sites were induced following initial binding of one to two molecules of probe. From the equilibrium and kinetics studies at low bis-ANS concentrations, it is evident that the native apical domain is converted to an intermediate conformation with increased hydrophobic surfaces. This intermediate binds additional bis-ANS molecules. Tyrosine fluorescence detected denaturation demonstrated that bis-ANS can destabilize the apical domain. The results from (i) bis-ANS titrations, (ii) urea denaturation studies in the presence and absence of bis-ANS, and (iii) intrinsic tyrosine fluorescence studies of the apical domain are consistent with a model in which bis-ANS binds tightly to the intermediate state, relatively weakly to the native state, and little to the denatured state. The results suggest that the conformational changes seen in apical domain fragments are not seen in the intact GroEL oligomer due to restrictions imposed by connections of the apical domain to the intermediate domain and suppression of movement due to quaternary structure.     

Effect of signal peptide on the stability and folding kinetics of maltose binding protein

While the role of the signal sequence in targeting proteins to specific subcellular compartments is well characterized, there are fewer studies that characterize its effects on the stability and folding kinetics of the protein. We report a detailed characterization of the folding kinetics and thermodynamic stabilities of maltose binding protein (MBP) and its precursor form, preMBP. Isothermal GdmCl and urea denaturation as a function of temperature and thermal denaturation studies have been carried out to compare stabilities of the two proteins. preMBP was found to be destabilized by about 2-6 kcal/mol (20-40%) with respect to MBP. Rapid cleavage of the signal peptide by various proteases shows that the signal peptide is accessible in the native form of preMBP. The observed rate constant of the major slow phase in folding was decreased 5-fold in preMBP relative to MBP. The rate constants of unfolding were similar at 25 degrees C, but preMBP also exhibited a large burst phase change in unfolding that was absent in MBP. At 10 degrees C, preMBP exhibited a higher unfolding rate than MBP as well as a large burst phase. The appreciable destabilization of MBP by signal peptide is functionally relevant, because it enhances the likelihood of finding the protein in an unfolded translocation-competent form and may influence the interactions of the protein with the translocation machinery. Destabilization is likely to result from favorable interactions between the hydrophobic signal peptide and other hydrophobic regions that are exposed in the unfolded state.     

Binding of bis-ANS to Bacillus subtilis lipase: A combined computational and experimental investigation

8-Anilino-1-naphthalene sulfonate (ANS) and its covalent dimer bis-ANS are widely used for titrating hydrophobic surfaces of proteins. Interest to understand the nature of interaction of these dyes with proteins was seriously pursued. However as the techniques used in these studies varied, they often provided varied information regarding stoichiometry, binding affinity, actual binding sites etc. In the present study, we used combination of computation methods (docking and MD simulation) and experimental methods (mutations, steady-state and time-resolved fluorescence) to investigate bis-ANS interaction with Bacillus subtilis lipase. We identified seven binding sites for bis-ANS on lipase using computational docking and MD simulation and verified these data using a set of single amino acid substituted mutants. Docking and MD simulation studies indicated that the binding sites were various indentations and grooves on protein surface with hydrophobic characteristics. Both hydrophobic and ionic interactions were involved in each of these binding events. We further examine the fluorescence properties of bis-ANS bound to mutant lipases that either gained or lost a binding site. Our results indicated that neither gain nor loss of single binding site caused any change in fluorescence lifetimes (and their relative amplitudes) of mutant lipase-bound bis-ANS in comparison to that bound to wild type; hence, it suggested that nature of bis-ANS binding to each of the sites in lipase was very similar.     

Effects of cardiomyopathic mutations on the biochemical and biophysical properties of the human alpha-tropomyosin.

Mutations in the protein alpha-tropomyosin (Tm) can cause a disease known as familial hypertrophic cardiomyopathy. In order to understand how such mutations lead to protein dysfunction, three point mutations were introduced into cDNA encoding the human skeletal tropomyosin, and the recombinant Tms were produced at high levels in the yeast Pichia pastoris. Two mutations (A63V and K70T) were located in the N-terminal region of Tm and one (E180G) was located close to the calcium-dependent troponin T binding domain. The functional and structural properties of the mutant Tms were compared to those of the wild type protein. None of the mutations altered the head-to-tail polymerization, although slightly higher actin binding was observed in the mutant Tm K70T, as demonstrated in a cosedimentation assay. The mutations also did not change the cooperativity of the thin filament activation by increasing the concentrations of Ca2+. However, in the absence of troponin, all mutant Tms were less effective than the wild type in regulating the actomyosin subfragment 1 Mg2+ ATPase activity. Circular dichroism spectroscopy revealed no differences in the secondary structure of the Tms. However, the thermally induced unfolding, as monitored by circular dichroism or differential scanning calorimetry, demonstrated that the mutants were less stable than the wild type. These results indicate that the main effect of the mutations is related to the overall stability of Tm as a whole, and that the mutations have only minor effects on the cooperative interactions among proteins that constitute the thin filament.     

Thermal unfolding of smooth muscle and nonmuscle tropomyosin alpha-homodimers with alternatively spliced exons

We used differential scanning calorimetry (DSC) and circular dichroism (CD) to investigate thermal unfolding of recombinant fibroblast isoforms of alpha-tropomyosin (Tm) in comparison with that of smooth muscle Tm. These two nonmuscle Tm isoforms 5a and 5b differ internally only by exons 6b/6a, and they both differ from smooth muscle Tm by the N-terminal exon 1b which replaces the muscle-specific exons 1a and 2a. We show that the presence of exon 1b dramatically decreases the measurable calorimetric enthalpy of the thermal unfolding of Tm observed with DSC, although it has no influence on the alpha-helix content of Tm or on the end-to-end interaction between Tm dimers. The results suggest that a significant part of the molecule of fibroblast Tm (but not smooth muscle Tm) unfolds noncooperatively, with the enthalpy no longer visible in the cooperative thermal transitions measured. On the other hand, both DSC and CD studies show that replacement of muscle exons 1a and 2a by nonmuscle exon 1b not only increases the thermal stability of the N-terminal part of Tm, but also significantly stabilizes Tm by shifting the major thermal transition of Tm to higher temperature. Replacement of exon 6b by exon 6a leads to additional increase in the alpha-Tm thermal stability. Thus, our data show for the first time a significant difference in the thermal unfolding between muscle and nonmuscle alpha-Tm isoforms, and indicate that replacement of alternatively spliced exons alters the stability of the entire Tm molecule.