Structural-stability studies on recombinant human transferrin

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. The release of iron induces opening of transferrin, which results in a negative effect on its stability. Presence of NaCl, arginine, and histidine leads to opening of the transferrin N-lobe 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, timeconsuming, 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 simulation, allows us to understand the behavior of transferrin on a molecular level.

Reinforcing Protein Biochemistry: A Two-Week Experiment Studying Iron(III) Binding by the Transferrin Protein through Stoichiometric Determination, Stability Analysis, and Visualization of the Binding Site

“… 21 − 25 The protein conformation closure increases the protein’s stability. Fe(III) binding to one site results in the stabilization of the other site without bound Fe(III), indicative of lobe cooperativity 26 similar to the subunit cooperativity observed when O 2 binds to the heme groups in hemoglobin. The fully lobe-closed Fe(III) binding is termed the canonical form of Fe(III) coordination by sTf ( Figure 2 A).…”

“…In addition to high Fe­(III) affinity (log K C‑lobe = 22.2 and log K N‑lobe = 21.3), , the closed conformations are supported by the N-lobe dilysine interaction and by the C-lobe triad amino acid interaction. − The protein conformation closure increases the protein’s stability. Fe­(III) binding to one site results in the stabilization of the other site without bound Fe­(III), indicative of lobe cooperativity similar to the subunit cooperativity observed when O 2 binds to the heme groups in hemoglobin. The fully lobe-closed Fe­(III) binding is termed the canonical form of Fe­(III) coordination by sTf (Figure A).…”

X-ray Characterization of Conformational Changes of Human Apo- and Holo-Transferrin

“…The aim of this contribution is to provide insight into the structural complexity of native human serum transferrin and the challenges that go along with it. When crystallizing transferrin, two important factors must be considered: its great conformational flexibility and the fact that its mechanism of iron release is pH-dependent [2,3,6,15] and has an important impact on its conformational states [16]. In this work, we first summarize the efforts to improve crystal quality which yielded little success in solving the structure via crystallographic methods at a very high resolution.…”