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Probing the binding mechanism of a solid-binding peptide

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posted on 28.03.2022, 17:21 authored by Rachit Bansal
The interactions between biomolecules and solid surfaces play an important role in designing new materials and applications which mimic nature. Recently, solid-binding peptides (SBPs) have emerged as potential molecular building blocks in nanobiotechnology. SBPs are short amino acid sequences (7-12 amino acids) that have the distinctive ability to recognize and bind to the surfaces of specific solid materials, such as metals and metal oxides, semiconductors, carbon-based materials, and polymers. These peptides act as 'molecular linkers' that mediate the simple and controlled attachment of biomolecules onto solid surfaces to confer biological functionality. SBP-solid interactions rely primarily on the peptide adopting structural conformations that maximize the contact between the reactive side chains of its amino acid residues and the solid surface. However, due to the high level of complexity of the SBP-solid interface within the surrounding solution, the exact mechanism that determines the SBP recognition, selectivity, and strong binding affinity remain ill-defined. We have engineered a bifunctional fusion protein composed of a solid-binding peptide (referred to as the 'linker'), which is a tetra-repeat of 21 amino acid sequence with unique binding affinity to silica-based materials, and a Streptococcus protein G, which binds antibodies. Linker protein G (LPG) acts as an anchor for the rapid and oriented immobilization of antibodies onto silica surfaces without using any complex conjugation chemistry. Although we have used this linker technology in biotechnology and biomedicine applications, there is still a lack of knowledge and understanding about the interaction mechanism which facilitates the binding of this SBP to the surface of silica. In this work, different biophysical characterization techniques, namely quartz crystal microbalance for dissipation monitoring (QCM-D), surface plasmon resonance (SPR), circular dichroism spectrometry (CD) and fluorescence spectrometry, were used to study the binding of LPG to silica surfaces and compared to protein G (PG) without the linker. LPG displayed high binding affinity to silica surface (KD=34.77±11.8 nM) with a standing-up orientation in comparison to parent PG, which exhibited no measurable binding affinity. Incorporation of the linker in the fusion protein LPG had no effect on the antibody binding function of PG, which retained its secondary structure and displayed no alteration of its chemical stability. We also engineered several truncated derivatives (1xLPG, 2xLPG, 3xLPG) from LPG to determine the effect of SBP multimerization on the silica binding function of LPG. The quantitative binding analysis for the different truncated derivatives were compared to that of LPG and PG (without linker) using various biophysical characterization techniques. Out of these truncated derivatives, 1xLPG (single linker sequence) displayed no binding to silica surface while the 2xLPG (two linker sequences) displayed minimal binding. Although the three-repeat derivative (3xLPG) binds to silica with a binding affinity (KD) of 53.23 ± 4.5 nM, it was 1.5 times lower than that of the four-repeat sequence (LPG). Spectroscopic techniques like circular dichroism (CD) spectroscopy and fluorescence spectroscopy studies indicated that the SBP degree of multimerization has no effect on the secondary structure and chemical stability of the parent protein G. However, the data from quartz crystal microbalance with dissipation monitoring (QCM-D) showed that multimerization was an important parameter for a strong and stable silica binding. The effect of peptide length on silica binding was evaluated by replacing the 3 sequence repeats by a (GGGGS)12 glycine-rich spacer. The results indicated that the overall length rather than the SBP multimerization mediated the effective binding to silica. A preliminary investigation was performed to assess the linker-protein G (LPG) as a suitable system for potential use in nanomedicine applications. We took advantage of the ability of the LPG bifunctional fusion protein to bind in an end-on-end orientation on the silica surface. The sensitive, selective and efficient detection of the HER2 biomarker was also investigated in the presence of biological fluids. This preliminary work showed that LPG mediates the functionalization of silica-coated nanoparticles with the anti-HER2 antibody trastuzumab easily and under mild conditions. The system was able to detect the HER2 biomarker in the presence of biological fluids demonstrating its potential suitability in nanomedicine applications e.g., nanoparticle-based drug delivery systems and efficient detection of disease biomarkers. This work provides the first insights into the binding mechanism of the aforementioned silica binding SBP. It also highlights the advantage of the LPG as a milder, facile and faster affinity immobilization technique of biomolecules to inorganic surfaces compared to traditional chemical coupling techniques --abstract.

History

Table of Contents

Chapter 1. Experimental and theoretical tools to elucidate the binding mechanisms of solid-binding peptides -- Chapter 2. Elucidating the binding mechanism of a novel silica-binding peptide -- Chapter 3. Effect of solid-binding peptide multimerization on its binding to silica-based materials -- Chapter 4. Solid-binding peptide-based biosensor for efficient detection of HER2 -- Chapter 5. Summary and future perspectives -- Appendix

Notes

Includes bibliographic records Theoretical thesis.

Awarding Institution

Macquarie University

Degree Type

Thesis PhD

Degree

PhD, Macquarie University, Faculty of Science and Engineering, Department of Molecular Sciences

Department, Centre or School

Department of Molecular Sciences

Year of Award

2019

Principal Supervisor

Anwar Sunna

Rights

Copyright Rachit Bansal 2019 Copyright disclaimer: http://mq.edu.au/library/copyright

Language

English

Extent

1 online resource (xi, 110 pages)

Former Identifiers

mq:72096 http://hdl.handle.net/1959.14/1281342