Olga P. Nikolaeva

The effect of mutations in alpha tropomyosin (E40K and E54K), that cause familial dilated cardiomyopathy, on the regulatory mechanism of cardiac muscle thin filaments

E40K and E54K mutations in α-tropomyosin cause inherited dilated cardiomyopathy. Previously we showed, using Ala-Ser α-tropomyosin (AS-α-Tm) expressed in Escherichia coli, that both mutations decrease Ca2+ sensitivity. E40K also reduces Vmax of actin-Tm-activated S-1 ATPase by 18%. We investigated cooperative allosteric regulation by native Tm, AS-α-Tm, and the two dilated cardiomyopathy-causing mutants. AS-α-Tm has a lower cooperative unit size (6.5) than native α-tropomyosin (10.0). The E40K mutation reduced the size of the cooperative unit to 3.7, whereas E54K increased it to 8.0. For the equilibrium between On and Off states, the KT value was the same for all actin-Tm species; however, the KT value of actin-Tm-troponin at pCa 5 was 50% less for AS-α-Tm E40K than for AS-α-Tm and AS-α-Tm E54K. Kb, the “closed” to “blocked” equilibrium constant, was the same for all tropomyosin species. The E40K mutation reduced the affinity of tropomyosin for actin by 1.74-fold, but only when in the On state (in the presence of S-1). In contrast the E54K mutation reduced affinity by 3.5-fold only in the Off state. Differential scanning calorimetry measurements of AS-α-Tm showed that domain 3, assigned to the N terminus of tropomyosin, was strongly destabilized by both mutations. Additionally with AS-α-Tm E54K, we observed a unique new domain at 55 °C accounting for 25% of enthalpy indicating stabilization of part of the tropomyosin. The disease-causing mechanism of the E40K mutation may be accounted for by destabilization of the On state of the thin filaments; however, the E54K mutation has a more complex effect on tropomyosin structure and function.

Thermal unfolding and aggregation of actin.

Actin is one of the most abundant proteins in nature. It is found in all eukaryotes and plays a fundamental role in many diverse and dynamic cellular processes. Also, actin is one of the most ubiquitous proteins because actin‐like proteins have recently been identified in bacteria. Actin filament (F‐actin) is a highly dynamic structure that can exist in different conformational states, and transitions between these states may be important in cytoskeletal dynamics and cell motility. These transitions can be modulated by various factors causing the stabilization or destabilization of actin filaments. In this review, we look at actin stabilization and destabilization as expressed by changes in the thermal stability of actin; specifically, we summarize and analyze the existing data on the thermal unfolding of actin as measured by differential scanning calorimetry. We also analyze in vitro data on the heat‐induced aggregation of actin, the process that normally accompanies actin thermal denaturation. In this respect, we focus on the effects of small heat shock proteins, which can prevent the aggregation of thermally denatured actin with no effect on actin thermal unfolding. As a result, we have proposed a mechanism describing the thermal denaturation and aggregation of F‐actin. This mechanism explains some of the special features of the thermal unfolding of actin filaments, including the effects of their stabilization and destabilization; it can also explain how small heat shock proteins protect the actin cytoskeleton from damage caused by the accumulation of large insoluble aggregates under heat shock conditions.

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

We used differential scanning calorimetry (DSC) and circular dichroism (CD) to investigate thermal unfolding of recombinant fibroblast isoforms of α‐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 α‐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 α‐Tm thermal stability. Thus, our data show for the first time a significant difference in the thermal unfolding between muscle and nonmuscle α‐Tm isoforms, and indicate that replacement of alternatively spliced exons alters the stability of the entire Tm molecule.

Conserved Noncanonical Residue Gly-126 Confers Instability to the Middle Part of the Tropomyosin Molecule

Tropomyosin (Tm) is a two-stranded α-helical coiled-coil protein with a well established role in regulation of actin cytoskeleton and muscle contraction. It is believed that many Tm functions are enabled by its flexibility whose nature has not been completely understood. We hypothesized that the well conserved non-canonical residue Gly-126 causes local destabilization of Tm. To test this, we substituted Gly-126 in skeletal muscle α-Tm either with an Ala residue, which should stabilize the Tm α-helix, or with an Arg residue, which is expected to stabilize both α-helix and coiled-coil structure of Tm. We have shown that both mutations dramatically reduce the rate of Tm proteolysis by trypsin at Asp-133. Differential scanning calorimetry was used for detailed investigation of thermal unfolding of the Tm mutants, both free in solution and bound to F-actin. It was shown that a significant part of wild type Tm unfolds in a non-cooperative manner at low temperature, and both mutations confer cooperativity to this part of the Tm molecule. The size of the flexible middle part of Tm is estimated to be 60–70 amino acid residues, about a quarter of the Tm molecule. Thus, our results show that flexibility is unevenly distributed in the Tm molecule and achieves the highest extent in its middle part. We conclude that the highly conserved Gly-126, acting in concert with the previously identified non-canonical Asp-137, destabilizes the middle part of Tm, resulting in a more flexible region that is important for Tm function.

Differential scanning calorimetric study of the complexes of modified myosin subfragment 1 with ADP and vanadate or beryllium fluoride

SummaryThe effects of various modifications of rabbit skeletal myosin subfragment 1 on thermal denaturation of subfragment 1 in ternary complexes with Mg-ADP and orthovanadate (Vi) or beryllium fluoride (BeFx) have been studied by differential scanning calorimetry. It has been shown that specific modifications of SH1 group of Cys-707 by different sulfhydryl reagents, trinitrophenylation of Lys-83, and reductive methylation of lysine residues promote the decomposition of the S1·ADP·Vi complex and change the character of structural transitions of the subfragment 1 molecule induced by the formation of this complex, but they have much less or no influence on subfragment 1 thermal stability in the S1·ADP·BeFx complex. Thus, the differential scanning calorimetric studies on modified subfragment 1 preparations reveal a significant difference between S1·ADP·Vi and S1·ADP·BeFx complexes. It is suggested that S1·ADP·Vi and S1·ADP·BeFx complexes represent structural analogues of different transition states of the ATPase cycle, namely the intermediate states S1**·ADP·Pi and S1*·ATP, respectively. It is also proposed that during formation of the S1·ADP·Vi complex the region containing both Cys-707 and Lys-83 plays an important role in the spread of conformational changes from the active site of subfragment 1 ATPase throughout the structure of the entire subfragment 1 molecule. In such a case, the effects of reductive methylation of lysine residues on the subfragment 1 structure in the S1·ADP·Vi complex are related to the modification of Lys-83.

Interaction of myosin subfragment 1 with F‐actin studied by differential scanning calorimetry

The thermal unfolding of the myosin subfragment 1 (S1) and of filamentous actin (F‐actin) in their strong complex obtained in the presence of ADP was studied by differential scanning calorimetry (DSC). It is shown that in the acto‐S1 complexes S1 and F‐actin melt separately, and thermal transitions of each protein can be easily followed. Interaction of S1 with F‐actin significantly increases S1 thermal stability and also affects the thermal stability of F‐actin. Although S1 unfolds at much lower temperature than F‐actin, the molecules of S1 remain bound to F‐actin even after full denaturation. Under these conditions S1 may induce cross‐linking between actin filaments. It is concluded that DSC studies on the acto‐S1 complexes offer a new and promising approach to investigate the structural changes which occur in the myosin head and in F‐actin due to their interaction.

Effects of Troponin on Thermal Unfolding of Actin-Bound Tropomyosin

Differential scanning calorimetry (DSC) was used to study the effect of troponin (Tn) and its isolated components on the thermal unfolding of skeletal muscle tropomyosin (Tm) bound to F-actin. It is shown that in the absence of actin the thermal unfolding of Tm is expressed in two well-distinguished thermal transitions with maxima at 42.8 and 53.8°C. Interaction with F-actin affects the character of thermal unfolding of Tm leading to appearance of a new Tm transition with maximum at about 48°C, but it has no influence on the thermal denaturation of F-actin stabilized by aluminum fluoride, which occurs within the temperature region above 70°C. Addition of troponin leads to significant increase in the cooperativity and enthalpy of the thermal transition of the actin-bound Tm. The most pronounced effect of Tn was observed in the absence of calcium. To elucidate how troponin complex affects the properties of Tm, we studied the influence of its isolated components, troponin I (TnI) and troponin T (TnT), on the thermal unfolding of actin-bound Tm. Isolated TnT and TnI do not demonstrate cooperative thermal transitions on heating up to 100°C. However, addition of TnI, and especially of TnT, to the F-actin–Tm complex significantly increased the cooperativity of the thermal unfolding of actin-bound tropomyosin.

Interaction of F‐actin with phosphate analogues studied by differential scanning calorimetry

The thermal unfolding of F‐actin and the changes induced in it by the binding of phosphate analogues were studied by differential scanning calorimetry. It is shown that the conformation of actin is drastically altered by interaction with beryllium fluoride or aluminium fluoride, while the effects of vanadate and phosphate are negligible. The effect of beryllium fluoride on the F‐actin structure, as reflected in a significant increase of the actin thermal stability, is much more pronounced in the presence of Mg2+ than in the case of F‐actin polymerized by KCl or LiCl in the absence of Mg2+. It is concluded that differential scanning calorimetry is a very convenient method for probing the conformational changes in F‐actin caused by the interaction with phosphate analogues.

Thermal denaturation and aggregation of myosin subfragment 1 isoforms with different essential light chains.

We compared thermally induced denaturation and aggregation of two isoforms of the isolated myosin head (myosin subfragment 1, S1) containing different “essential” (or “alkali”) light chains, A1 or A2. We applied differential scanning calorimetry (DSC) to investigate the domain structure of these two S1 isoforms. For this purpose, a special calorimetric approach was developed to analyze the DSC profiles of irreversibly denaturing multidomain proteins. Using this approach, we revealed two calorimetric domains in the S1 molecule, the more thermostable domain denaturing in two steps. Comparing the DSC data with temperature dependences of intrinsic fluorescence parameters and S1 ATPase inactivation, we have identified these two calorimetric domains as motor domain and regulatory domain of the myosin head, the motor domain being more thermostable. Some difference between the two S1 isoforms was only revealed by DSC in thermal denaturation of the regulatory domain. We also applied dynamic light scattering (DLS) to analyze the aggregation of S1 isoforms induced by their thermal denaturation. We have found no appreciable difference between these S1 isoforms in their aggregation properties under ionic strength conditions close to those in the muscle fiber (in the presence of 100 mM KCl). Under these conditions kinetics of this process was independent of protein concentration, and the aggregation rate was limited by irreversible denaturation of the S1 motor domain.

Changes in the thermal unfolding of p‐phenylenedimaleimide‐modified myosin subfragment 1 induced by its ‘weak’ binding to F‐actin

Differential scanning calorimetry (DSC) was used to analyze the thermal unfolding of myosin subfragment 1 (S1) with the SH1 (Cys‐707) and SH2 (Cys‐697) groups cross‐linked by N,N′‐p‐phenylenedimaleimide (pPDM‐S1). It has been shown that F‐actin affects the thermal unfolding of pPDM‐S1 only at very low ionic strength, when some part of pPDM‐S1 binds weakly to F‐actin, but not at higher ionic strength (200 mM KCl). The weak binding of pPDM‐S1 to F‐actin shifted the thermal transition of pPDM‐S1 by about 5°C to a higher temperature. This actin‐induced increase in thermal stability of pPDM‐S1 was similar to that observed with ‘strong’ binding of unmodified S1 to F‐actin. Our results show that actin‐induced structural changes revealed by DSC in the myosin head occur not only upon strong binding but also on weak binding of the head to F‐actin, thus suggesting that these changes may occur before the power‐stroke and play an important role in the motor function of the head.