Investigating the Disordered and Membrane-Active Peptide A-Cage-C Using Conformational Ensembles.

Olena Dobrovolska,Morten L Govasli,Ørjan Sele Handegård,Nils Åge Frøystein,Øyvind Halskau,Åge Aleksander Skjevik,Øyvind Strømland,Knut Teigen,Martin Jakubec

doi:10.3390/molecules26123607

Olena Dobrovolska, Morten L Govasli + Show 7 more

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https://doi.org/10.3390/molecules26123607

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Abstract

The driving forces and conformational pathways leading to amphitropic protein-membrane binding and in some cases also to protein misfolding and aggregation is the subject of intensive research. In this study, a chimeric polypeptide, A-Cage-C, derived from α-Lactalbumin is investigated with the aim of elucidating conformational changes promoting interaction with bilayers. From previous studies, it is known that A-Cage-C causes membrane leakages associated with the sporadic formation of amorphous aggregates on solid-supported bilayers. Here we express and purify double-labelled A-Cage-C and prepare partially deuterated bicelles as a membrane mimicking system. We investigate A-Cage-C in the presence and absence of these bicelles at non-binding (pH 7.0) and binding (pH 4.5) conditions. Using in silico analyses, NMR, conformational clustering, and Molecular Dynamics, we provide tentative insights into the conformations of bound and unbound A-Cage-C. The conformation of each state is dynamic and samples a large amount of overlapping conformational space. We identify one of the clusters as likely representing the binding conformation and conclude tentatively that the unfolding around the central W23 segment and its reorientation may be necessary for full intercalation at binding conditions (pH 4.5). We also see evidence for an overall elongation of A-Cage-C in the presence of model bilayers.

Highlights

We calculated its hydropathy index and secondary structure propensity using the approach of Tanford and Protparam, respectively [37,38]
There are intervals of intermediate to high hydropathy (L14-l24 and P34-V50), suggesting that these segments would intercalate into a hydrophobic lipid environment if present
The previous study using CD and fluorescence support that A-Cage-C is somewhat helical in solution at both pHs

Summary

Introduction

Protein amphitropism is a phenomenon where soluble proteins, usually globular, reversibly interact with the cellular plasma membrane. Amphitropism is involved in numerous enzymatic processes [3,9,10] and membrane-associated cell signalling and transduction [7,9,10,11]. Amphitropic proteins such as tyrosine hydroxylase, monoamine oxidase A, and neutrophil serine proteases are important in biomedical and pharmacological research [12,13,14]. Intrinsic polypeptide disorder and weakly folded domains are common features of amphitropic proteins [4,15]

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