PhD Scientific Days 2022

Molecular Sciences I. (Poster discussion will take place in the Aula during the Coffee Break)

Effect of PEVK on actin polymerization

Text of the abstract

Actin is the most abundant protein in the human body. It plays an important role in a wide range of biological processes. in the striated muscle sarcomere actin interacts with a number of proteins: myosin binding results in sarcomeric contraction; tropomyosin and troponin binding regulates contractility. It has been shown that the giant filamentous protein titin also binds actin with several of its domains, including the unstructured PEVK domain. The physiological role of titin-actin interaction are yet to be understood.
The middle, approximately 700-residue-long segment (named PEVKII) of the full-length PEVK domain was cloned and expressed for actin-polymerization experiments. We chose this sequence because, due to the conformational dynamics of the unstructured PEVK domain, this segment appears to be the most accessible for binding to actin within the sarcomeric environment. The PEVKII DNA was cloned into pet-28a vector, expressed in E.coli BL21(Rosetta2), then purified on a Ni2+-NTA column under native conditions. F-actin was labelled with N-(1-pyrene) iodoacetamide after cycles of ultracentrifugation and dialysis. For the polymerization assay we used G-actin samples pyrene-labeled with an efficiency of 10%. To initiate polymerization, we first mixed G-actin (4 µM) with G-buffer (4mM Tris-HCI, 0.2 mM ATP, 0.2 mM CaCI2, 0.005% NaN3, pH 8.0) and Ca-Mg ion exchange buffer ( 50 µM MgCI2, 0.2 mM EGTA, pH 8.0) at room temperature, then, after 5 minutes of incubation, we added polymerization buffer (100 mM KCI, 2 mM MgCI2, 2 mM Tris-HCI, 0,005% NaN3, 1 mM EGTA, pH 8.0). Actin polymerization was monitored by measuring fluorescence intensity (at λex 365 nm and λem 407 nm) for 2000 seconds.
We found that the addition of PEVKII accelerated actin polymerization in a concentration-dependent manner (2 nM - 40 nM). While the initial polymerization rate increased 3-fold across the investigated PEVKII concentration range, the final fluorescence intensity remained unaffected, suggesting that the total F-actin amount is independent of the presence of PEVKII. Our results indicate that titin's PEVK domain is an actin-binding protein that facilitates actin's polymerization. Conceivably, the actin-PEVK interaction might play a role in modulating actin turnover in the sarcomere.

Funding: EFOP-3.6.3-VEKOP-16-2017-00009