Naslov
Design of short catalytic sequences inspired by enzymatic active sites

Autori
Janković, Patrizia ; Todorovski, Toni ; Kalafatovic, Daniela

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Skup
Women in Supramolecular Chemistry (WISC) Workshop

Mjesto i datum
Cagliari, Italija, 06.09.2021. - 08.09.2021

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
self-assembly, catalytic, peptides, minimalistic, co-assembly

Sažetak
Short catalytic peptides offer simple, tunable, low-cost biomolecules able to catalyze chemical reactions such as ester hydrolysis1. The main drawback of this approach is their low catalytic efficiency attributed to the lack of well-defined tridimensional structures characteristic for enzymes. Peptide self-assembly offers the possibility to obtain nanostructures with a higher degree of order and improved catalytic efficiency. However, better understanding of the principles that govern the catalytic activity of short peptides at the sequence level is needed. Our aim is to design short peptides, inspired by active sites found in hydrolases containing the catalytic triad. For this purpose, the amino acids sequences of 23 hydrolases acting on ester bonds (EC 3.1.) were shortened to peptides composed of the catalytic triad and the residues forming the oxyanion hole. We present the design of three sequences that will be evaluated experimentally: SGNYDYLHGD, CTLGLGSHCGG, GGESTGHTGAGNDK obtained from the active sites of 1-alkyl-2- acetylglycerophosphocholine esterase and protein- glutamate methylesterases (CheD and CheB), respectively. The sequences were designed to include the core fragment derived from the active site containing important residues found in proximity at the sequence level. In the case of 1- alkyl-2-acetylglycerophosphocholine esterase, the core sequence is based on the Tyr191-His195 segment. Other important residues that are far away from the selected core, are added at the N- or C-terminus, respecting their original sequence order (e.g. Ser47, Gly74 and Asn104 were added at the N-terminus of the YDYLH core sequence). Additionally, residues that allow the cyclization were included at the C-terminus, where Gly acts as spacer and Asp is used for the head-to-side-chain cyclization with the N-terminal amino acid. The peptides were synthesized by solid-phase peptide synthesis (SPPS) as linear and cyclic analogues. The cyclization was achieved by three different methods (head-to-side-chain, cysteine mediated and coupling of side chains) depending on the sequence composition. The peptides were purified using semipreparative-HPLC and analyzed using LC-MS and analytical HPLC. Linear and cyclic analogues of the proposed sequences will be tested to assess whether the enhanced rigidity obtained by cyclization will improve their catalytic activity. The peptides will be further analyzed using FTIR, CD, fluorescence spectroscopy (CAC) to investigate their ability to self-assemble and to determine how the cyclization affects nanostructure formation. The catalytic efficiency of ester hydrolysis for both linear and cyclic analogues will be evaluated and compared using the standard colorimetric test with 4-nitrophenyl acetate (p- NPA). A successful outcome of this project could result in general rules applicable to the design of short catalytic peptides with esterase activity.

Izvorni jezik
Engleski

Znanstvena područja
Interdisciplinarne prirodne znanosti, Biotehnologija, Interdisciplinarne biotehničke znanosti, Biotehnologija u biomedicini (prirodno područje, biomedicina i zdravstvo, biotehničko područje)

POVEZANOST RADA