Thermal Stability Of Actin Research Articles

Investigating the impact of PACAP on thermal stability of G-actin by differential scanning calorimetry

AbstractPituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide known for its diverse effects on the nervous system. While numerous studies have demonstrated the neuroprotective properties of PACAP, its role in tissue regeneration and potential as a therapeutic agent remain to be fully understood. Specifically, the understanding of PACAP’s impact on cytoskeletal dynamics, particularly the organization and disorganization of actin filament networks, is limited due to the scarcity of in vitro studies in this area. Additionally, the interaction between PACAP and actin has been minimally explored, and the influence of PACAP on the thermal stability of actin is completely unknown. To address these gaps, the current study aimed to investigate the impact of different forms and fragments of PACAP on the thermal denaturation and renaturation of Ca2+-G-actin using a differential scanning calorimetry (DSC) approach. Our primary objective was to determine whether PACAP modulates the thermal stability of Ca2+-G-actin and establish a temperature-dependent pattern of any structural alterations that may occur as a result of PACAP interaction. Two PACAP forms exist in vivo: the 38 amino-acid length PACAP38 and the PACAP27, the latter truncated at the C-terminal. In the PACAP38 + Ca2+-G-actin mixture, the DSC scans exhibited a mild decrease in actin denaturation temperature compared to the control, plus an exotherm appeared in the high-temperature range with a significantly increased calorimetric enthalpy. The truncated PACAP27 produced a slight increase in actin denaturation temperature with the same exotherm without significant alteration in enthalpy. In PACAP6-38 mixture (i.e., an artificial fragment of PACAP38 + Ca2+-G-actin), there was no change in the denaturation temperature of actin and no plus exotherm, but significant ΔHcal was observed. With the modified PACAP6-27 (another artificial fragment), the exotherm was absent, but the actin denaturation temperature and enthalpy increased compared to the control. Through this research, we sought to elucidate the underlying mechanisms of PACAP’s effects on actin dynamics and provide valuable insights into the potential therapeutic applications of PACAP in the context of cytoskeletal organization and neuronal regeneration. The findings of this study may contribute to the development of novel strategies targeting actin-related processes for neuroprotection and neural tissue repair.

Exploring the response of PACAP on thermal endurance of F-actin by differential scanning calorimetry

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide known for its diverse effects on the nervous system. While numerous studies have demonstrated the neuroprotective properties of PACAP, its role in tissue regeneration and potential as a therapeutic agent remain to be fully understood. Specifically, the understanding of PACAP’s impact on cytoskeletal dynamics, particularly the organization and disorganization of actin filament networks, is limited due to the scarcity of in vitro studies in this area. Additionally, the interaction between PACAP and actin has been minimally explored, and the influence of PACAP on the thermal stability of actin is completely unknown. To address these gaps, the current study aimed to investigate the impact of different forms and fragments of PACAP on the thermal denaturation and renaturation of Ca2+-F-actin using a differential scanning calorimetry (DSC) approach. Our primary objective was to determine whether PACAP modulates the thermal stability of Ca2+-F-actin and establish a temperature-dependent pattern of any structural alterations that may occur as a result of PACAP interaction. Two PACAP forms exist in vivo: the 38 amino acid length PACAP38 and the PACAP27, the latter truncated at the C-terminal. Both in the PACAP38 + Ca2+-F-actin and in the PACAP6-38 + Ca2+-F-actin mixtures, the DSC scans exhibited a significant decrease of actin denaturation temperature compared to the control; however, the PACAP27 + Ca2+-F-actin and PACAP6-27 + Ca2+-F-actin revealed no remarkable differences compared to the actin control sample. The calorimetric enthalpy of the truncated PACAP27 and PACAP6-27 + actin mixture also followed the tendencies mentioned above. Thus, in PACAP27 and PACAP6-27 mixture, there was no change in the denaturation temperature of actin, and no significant ΔHcal was observed. Through this research, we sought to elucidate the underlying mechanisms of PACAP’s effects on actin dynamics using thermal de- and renaturation cycles.

Influence of stewing conditions on tenderness and protein structure in beef

In this study, the effects of heating temperature (70°C ~ 100°C) and time (0 ~ 180 min) on beef M. longissimus dorsi tenderness, the oxidation characteristics of myofibrillar protein (MP), and the correlation between tenderness with protein oxidation characteristics were studied. The results showed that heating temperature and time significantly influenced beef tenderness and MP oxidation characteristics. SDS-PAGE analysis indicated that the thermal stability of actin was significantly higher than that of myosin heavy chain. Myosin heavy chain bands disappeared completely when heated for 120 min at 90°C or for 30 min at 100°C. The actin bands faded slightly and they were still clearly visible when heated for 180 min at 100°C. Correlation analysis showed that beef shear force value was significant negatively correlated with the dityrosine content (p < .01). This will provide a theoretical basis for the quality control and cooking process optimization of Chinese traditional beef stew dishes. Novelty impact statement This study investigated the changes of beef tenderness and myofibrillar protein structure during stewing and elucidated the correlation between beef tenderness and myofibrillar protein structure. The results will provide a theoretical basis for the quality control and cooking process optimization of Chinese traditional beef stew dishes.

Kinetics of heat induced muscle protein denaturation of brown shrimp (Crangon crangon)

To optimize the quality of cooked brown shrimp (Crangon crangon), quantitative data on the influence of all relevant process parameters (treatment time, temperature, NaCl concentration) on several quality attributes are required. Surprisingly, kinetic data and models on heat induced quality changes of brown shrimp are scarce. In this study, the denaturation of muscle proteins was studied by differential scanning calorimetry. The thermal denaturation kinetics of actin, the most heat resistant muscle protein, were determined and showed a first order decay with k72 °C = 0.038 ± 0.001 min−1 and Ea = 388 ± 7.3 kJ/mol. Spiking experiments showed that NaCl (concentrations in the range from 1 to 5% NaCl) had a negligible influence on the actin thermal stability. The kinetic data obtained can be used to quantitatively evaluate the doneness of cooked shrimp.

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.

Lipid and protein deterioration during the chilled storage of minced sea salmon (Pseudopercis semifasciata)

AbstractSea salmon is a very appreciated seafood. The aim of this work was to analyze changes in lipid and protein fractions of minced muscle during chilled storage (1 ± 1 °C). Lipid oxidation was important during the first 6 days of storage according to 2‐thiobarbituric acid (TBA) values determined, decreasing mainly ω3 22:6 fatty acid content. Lipid hydrolysis was evident after 9 days of storage. Interaction compounds between oxidation products and other cellular components were analyzed by fluorescence measurements. The results obtained showed evidence of the formation of interaction products involved, mainly polar components such as proteins. Decreases in myosin and actin thermal stability and myosin denaturation were recorded by differential scanning calorimetry. Solubility of total and myofibrillar proteins decreased after 6 days of storage. The electrophoretic profile of soluble fractions showed an increase in the intensity of bands corresponding to low‐molecular‐weight polypeptides and a decrease in high‐molecular‐weight species. Available lysine content did not change during chilled storage. Copyright © 2007 Society of Chemical Industry

Structural stability of actin filaments as studied by DSC and EPR

Thermal stability of actin, isolated from skeletal muscle, was studied in monomeric and polymerized form using DSC and EPR spectroscopy. The analysis of the DSC profiles in both forms enabled the assignment of the components: two endothermic components in the G-form (47.3±1.4°C and 53.4±1.8°C with enthalpies of 184.4±10.9 and 120.0±7.5 kJ/mol), and three endothermic components in the F-form ( T m1=59.7±1.4°C, H 1=171.3±1.7 kJ/mol; T m2=60.6±1.6°C, H 2=231.2±9.6 kJ/mol; T m3=61.3±1.4°C, H 3=148.3±4.6 kJ/mol). Actin was labeled with the paramagnetic derivative of maleimide at the reactive thiol site Cys-374 in the C-terminal subdomain-1. The rotational correlation time for MSL-F-actin is 100 μs, as obtained from the peak ratio L″/ L=0.745±0.05 ( n=6), which indicates that there is significant internal flexibility in F-actin. The unfolding of actin induced by heating resulted in a single transition in both forms of actin. The comparison of G- and F-actins showed remarkable differences in transition temperature and enthalpy, suggesting intramolecular interactions between subdomains and monomers.

Thermal denaturation of proteins in Post rigor muscle tissue as studied by differential scanning calorimetry

AbstractPost rigor bovine M. semimembranosus was analysed by differential scanning calorimetry (d.s.c.). After extractive removal of sarcoplasmic proteins, subsequent pH adjustment and manual connective tissue removal, d.s.c. yielded reproducible thermograms which permitted investigation of the individual major myofibrillar proteins in various pH and salt environments without prior isolation. The positions of two major peaks, interpreted as myosin transitions, proved to be strongly pH dependent. At pH 5.4, the peak maxima occurred at 58 and 65°C, respectively, at a heating rate of 10°C min‐1. Above pH 6.5 their order of denaturation was reversed. In the pH range 5.4–6.5 the peak ascribed to actin had its maximum near 80°C in intact muscle. Above this pH range it was displaced to lower temperature. The thermal stability of actin was studied after treatment of the muscle tissue with different salt solutions. At equal ionic strengths (μ = 0.15) at pH 5.5, calcium chloride and sodium chloride caused 6.5°C and 4°C displacement to lower temperature, respectively. The thermograms of bovine semimembranosus muscle were compared to those of two red and two white muscle types (bovine cardiac and rabbit soleus muscles, chicken breast and rabbit semimembranosus muscles, respectively) at two pH levels. Greater myosin differences were found between red and white muscles than between muscles from different animal species. All muscles gave similar actin transitions, with exception of the heart muscle where the actin peak appeared at 3 °C lower temperature. The necessity of a strict pH control in order to obtain reproducible muscle thermograms is demonstrated.