Effect of Lysine Acetylation on Protein Secondary Structure Stability, RNA Recognition, and Cell Penetration
Date Issued
2015
Date
2015
Author(s)
Chen, Chin-Yih
Abstract
Lysine acetylation is a very common and highly regulated post-translational modification (PTM) and is crucial for the protein structures and many biological functions in living organisms. The main effect of lysine acetylation is the neutralization of the lysine positive charge and consequently the regulation of the biochemical properties of lysine-containing proteins. The roles of lysine acetylation in cellular functions have been studied extensively. However, the effect of acetylation on protein secondary structures remains unclear. To study the effect of lysine acetylation on protein secondary structures, α-helical monomeric peptides and β-hairpin peptides were designed as basic models and acetyl-Lysine (AcK) was introduced into each guest position. Hydrogen-bonds have significant impact on protein stability, therefore the effect of replacing a methyl group with an amine group in acetylated Lys residues on secondary structure stability was also studied. The helical content of the α-helical peptides were determined by circular dichorism spectroscopy (CD), and the β-hairpin peptides were analyzed by NMR spectroscopy (TOCSY , ROESY , NOESY , DQF-COSY) to determine the sheet propensity. The result showed that compared to Lys, Lys acetylation increased both α-helix and β-sheet stability. In an α-helix, AcK increased the helix propensity, but lowered the C-Cap parameters. In a β-hairpin, the folding percentage of HPTAcKAla was 1.2 fold higher than that of HPTLysAla. Post-translational modification of RNA binding proteins (RBPs) plays an important role in regulating many cellular functions. The Tat protein, one of the RBPs, is essential for the life cycle of HIV-1 and is known to undergo acetylation on specific lysines to modulate various Tat functions. Tat, like most of the RBPs, contain a region (Tat49-57) rich in positively charged amino acids such as Arg and Lys to mediate the initial recognition of the negatively charged phosphate backbone RNA and the efficiency of cellular uptake. To investigate the effect of Lys side chain charge on RNA recognition and on cellular uptake, each positively charged Lys in Tat49-57 was replaced with one AcK at a time. The dissociation constant for the binding affinity of TAR RNA-Tat derived peptide was studied by gel shift assays, and the cellular uptake efficiency for peptide-treated Jurkat cells was assessed by flow cytometry. The result showed that removing the Lys positive charge by lysine acetylation affected RNA recognition and cell penetration of HIV-1 Tat. Furthermore, the effects showed position dependence. Acetylation of Lys 50 and 51 decreased the binding affinity between Tat49-57 and TAR RNA and could not be recovered by introducing hydrogen bond donors. Acetylation of Lys 50 and 51 on Tat49-57 decreased the cell penetration efficiency. However, adding hydrogen bond donors could compensate this decrease.
Subjects
Lysine
Acetylation
Tat-derived peptides
RNA recognition
Cellular uptake
Type
thesis
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