Structural and functional studies of muscle proteins by using differential scanning calorimetry

Levitsky, Dmitrii I.

doi:10.1007/1-4020-2219-0_6

Cited by 7 publications

(5 citation statements)

References 80 publications

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“…2), also observed by us [41,52] and other authors [53], is very useful for DSC studies on F‐actin complexes with other proteins, and we often use phalloidin‐stabilized F‐actin to obtain a better separation of the thermal transitions of F‐actin and actin‐bound proteins (e.g. myosin head, tropomyosin) on DSC curves [54].…”

Section: Thermal Unfolding Of F‐actin Filamentsmentioning

confidence: 88%

Thermal unfolding and aggregation of actin

et al. 2008

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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.

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Section: Thermal Unfolding Of F‐actin Filamentsmentioning

confidence: 88%

Thermal unfolding and aggregation of actin

et al. 2008

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“…This suggests that the stabilizing of Tm on actin results in almost all of the unfolding occurring as a single cooperative process simultaneous with dissociation from actin. It was concluded from our previous studies that F‐actin prevents the actin‐bound Tm from thermal denaturation, which only occurs upon dissociation of Tm from F‐actin [14,45]. As a result, Tm unfolds at higher temperature and with much higher cooperativity.…”

Section: Discussionmentioning

confidence: 99%

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

Kremneva¹,

Nikolaeva²,

Maytum³

et al. 2006

The FEBS Journal

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Tropomyosins (Tm) are a family of actin-binding, a-helical coiled-coil proteins found in most eukaryotic cells [1]. They bind to actin cooperatively along the length of actin filaments and confer cooperativity upon the interaction of actin with myosin heads [2]. The Tm molecules are parallel homo-or heterodimers (encoded from the same or different genes) of two a-helical chains of identical length, although the length can vary according to isoform type. In mammalian cells, alternative splicing produces a variety of muscle and nonmuscle isoforms from four different genes [1]. Muscle cells express two major isoforms of Tm (a and b), each containing 284 residues. Smooth and skeletal muscles express different isoforms resulting from alternative splicing of the a and b genes.There are two major classes of a-Tm: long (or high relative molecular mass) Tm (284 residues) are expressed in muscle and nonmuscle cells whereas short (or low relative molecular mass) Tm (247 residues) are We used differential scanning calorimetry (DSC) and circular dichroism (CD) to investigate thermal unfolding of recombinant fibroblast isoforms of a-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 a-helix content of Tm or on the end-toend 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 a-Tm thermal stability. Thus, our data show for the first time a significant difference in the thermal unfolding between muscle and nonmuscle a-Tm isoforms, and indicate that replacement of alternatively spliced exons alters the stability of the entire Tm molecule.Abbreviations CD, circular dichroism; DSC, differential scanning calorimetry; Tm, tropomyosin; smTm, recombinant smooth muscle Tm; Tm5a and Tm5b, recombinant fibroblast Tm isoforms with alternatively spliced exons 6b and 6a, respectively.

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“…Differential scanning calorimetry (DSC) experiments were performed using a DASM-4 differential scanning microcalorimeter (“Biopribor”, Pushchino, Russia) as described earlier. − Protein samples (0.65–0.7 mg/mL) were heated at a constant rate of 1 K/min and a constant pressure of 2.4 atm. The reversibility of the thermal transitions was assessed by reheating the sample immediately after the cooling step from the previous scan.…”

Section: Methodsmentioning

confidence: 99%

New Insight in Protein–Ligand Interactions. 2. Stability and Properties of Two Mutant Forms of thed-Galactose/d-Glucose-Binding Protein fromE. coli

et al. 2011

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The galactose/glucose-binding protein from E. coli (GGBP) is a 32 kDa protein possessing the typical two-domains structure of the ligand-binding proteins family. GGBP is characterized by low dissociation constant values with respect to glucose binding, displaying an affinity constant for glucose in micromolar range. This feature makes GGBP unsuitable as a sensitive probe for continuous glucose monitoring in blood of diabetic patients. In this work we designed, produced, and characterized two mutant forms of GGBP carrying the following amino acid substitutions in the active center of the protein: W183A or F16A. The two mutant GGBP forms retained a globular structure similar to that of the wild-type GGBP and displayed an affinity for glucose lower than the wild-type GGBP. A deep inspection of the entire set of the obtained results pointed out that the N- and C-terminal domains of GGBP-W183A in the absence of glucose have a stability lower than that of the wild-type protein. In the presence of glucose, the two domains of GGBP-W183A were tightly bound, making the protein structure more stable to the action of denaturing agents. On the contrary, the mutant form GGBP-F16A possesses a very restricted structural stability both in the absence and in the presence of glucose. In this work the role of Phe 16 and W 183 are discussed with regard to the structural and functional features of GGBP. In addition, some general guidelines are reported for the design of a novel glucose biosensor based on the use of GGBP.

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Structural and functional studies of muscle proteins by using differential scanning calorimetry

Cited by 7 publications

References 80 publications

Thermal unfolding and aggregation of actin

Thermal unfolding and aggregation of actin

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

New Insight in Protein–Ligand Interactions. 2. Stability and Properties of Two Mutant Forms of thed-Galactose/d-Glucose-Binding Protein fromE. coli

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