NMR Reveals Functionally Relevant Thermally Induced Structural Changes within the Native Ensemble of G-CSF

Kellerman, Mark-Adam W, Almeida, Teresa, Rudd, Timothy R ORCID: 0000-0003-4434-0333, Matejtschuk, Paul and Dalby, Paul A (2022) NMR Reveals Functionally Relevant Thermally Induced Structural Changes within the Native Ensemble of G-CSF. MOLECULAR PHARMACEUTICS, 19 (9). pp. 3242-3255.

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Abstract

Structure-function relationships in proteins refer to a trade-off between stability and bioactivity, molded by evolution of the molecule. Identifying which protein amino acid residues jeopardize global or local stability for the benefit of bioactivity would reveal residues pivotal to this structure-function trade-off. Here, we use ¹⁵N-¹H heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectroscopy to probe the microenvironment and dynamics of residues in granulocyte colony-stimulating factor (G-CSF) through thermal perturbation. From this analysis, we identified four residues (G4, A6, T133, and Q134) that we classed as significant to global stability, given that they all experienced large environmental and dynamic changes and were closely correlated to each other in their NMR characteristics. Additionally, we observe that roughly four structural clusters are subject to localized conformational changes or partial unfolding prior to global unfolding at higher temperature. Combining NMR observables with structure relaxation methods reveals that these structural clusters concentrate around loop AB (binding site III inclusive). This loop has been previously implicated in conformational changes that result in an aggregation prone state of G-CSF. Residues H43, V48, and S63 appear to be pivotal to an opening motion of loop AB, a change that is possibly also important for function. Hence, we present here an approach to profiling residues in order to highlight their potential roles in the two vital characteristics of proteins: stability and bioactivity.

Item Type:	Article
Uncontrolled Keywords:	denaturation, remodeling, structure-function, ''switch'', aggregation
Divisions:	Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User:	Symplectic Admin
Date Deposited:	17 Oct 2022 14:36
Last Modified:	18 Jan 2023 20:36
DOI:	10.1021/acs.molpharmaceut.2c00398
Open Access URL:	https://doi.org/10.1021/acs.molpharmaceut.2c00398
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3165550