Small angle X-ray scattering and molecular dynamic simulations provide molecular insight for stability of recombinant human transferrin

Alina Kulakova,Sowmya Indrakumar,Pernille Sønderby,Lorenzo Gentiluomo,Werner Streicher,Dierk Roessner,Wolfgang Frieß,Günther H.J Peters,Pernille Harris

doi:10.1016/j.yjsbx.2019.100017

Abstract

Transferrin is an attractive candidate for drug delivery due to its ability to cross the blood brain barrier. However, in order to be able to use it for therapeutic purposes, it is important to investigate how its stability depends on different formulation conditions. Combining high-throughput thermal and chemical denaturation studies with small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, it was possible to connect the stability of transferrin with its conformational changes. Lowering pH induces opening of the transferrin N-lobe, which results in a negative effect on the stability. Presence of NaCl or arginine at low pH enhances the opening and has a negative impact on the overall protein stability. Statement of SignificanceProtein-based therapeutics have become an essential part of medical treatment. They are highly specific, have high affinity and fewer off-target effects. However, stabilization of proteins is critical, time-consuming, and expensive, and it is not yet possible to predict the behavior of proteins under different conditions. The current work is focused on a molecular understanding of the stability of human serum transferrin; a protein which is abundant in blood serum, may pass the blood brain barrier and therefore with high potential in drug delivery. Combination of high throughput unfolding techniques and structural studies, using small angle X-ray scattering and molecular dynamic simulations, allows us to understand the behavior of transferrin on a molecular level.

Highlights

Over the last decades, the number of approved protein-based therapeutics has increased significantly and these drugs have become essential for the treatment of various diseases, such as diabetes, hemophilia, hepatitis C, and cancer (Wishart et al, 2006)
Combining high-throughput thermal and chemical denaturation studies with small angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations, it was possible to connect the stability of transferrin with its conformational changes
The current work is focused on a molecular understanding of the stability of human serum transferrin; a protein which is abundant in blood serum, may pass the blood brain barrier and with high potential in drug delivery

Summary

Introduction

The number of approved protein-based therapeutics has increased significantly and these drugs have become essential for the treatment of various diseases, such as diabetes, hemophilia, hepatitis C, and cancer (Wishart et al, 2006). This is because, compared to small molecules, protein-based therapeutics show higher specificity and generally, have less side effects. Due to their high complexity, protein-drugs are less stable and require special conditions (formulations) that will preserve their stability during production and storage. It is important to obain a detailed molecular understanding of the rationale behind protein stability and conformational changes

Methods

Results

Conclusion