The nuclear Overhauser Effect (NOE) as a tool to study macromolecular confinement: Elucidation and disentangling of crowding and encapsulation effects.

Philipp Honegger,Othmar Steinhauser

doi:10.1063/1.5135816

Abstract

We propose a methodology to capture short-lived but biophysically important contacts of biomacromolecules using the biomolecule-water nuclear Overhauser effect as an indirect microscope. Thus, instead of probing the direct correlation with the foreign biomolecule, we detect its presence by the disturbance it causes in the surrounding water. In addition, this information obtained is spatially resolved and can thus be attributed to specific sites. We extend this approach to the influence of more than one change in chemical environment and show a methodological way of resolution. This is achieved by taking double differences of corresponding σNOE/σROE ratios of the systems studied and separating specific, unspecific, and intermediate influence. While applied to crowding and encapsulation in this study, this method is generally suitable for any combination of changes in chemical environment.

Highlights

In order to test our hypothesis whether indirect nuclear Overhauser Effect (NOE) measurements can capture macromolecular crowding, we repurposed molecular dynamics (MD) simulations featured in previous articles:
This study focuses on changes in NOE observables upon introducing a new chemical influence
The physicochemical properties of biomolecules in living matter are strongly impacted by both encapsulation and the concentrated presence of organic matter, e.g., other biomacromolecules

Summary

Introduction

Protein-protein interactions are profoundly important in molecular recognition.[9] For instance, the protein ubiquitin (UBQ) controls many protein degradation pathways[10] by forming specific complexes with partner molecules.[11–14] Beyond stable protein-protein complexes, more recent studies emphasize the importance of less specific interactions between proteins encountered in cytosol, which may contain up to 400 g/l of organic matter.[15]. Havenith et al showed that using biochemically plausible cosolutes stabilizes ubiquitin (UBQ) structure by direct interaction, which is of enthalpic nature. They found that polyethylene glycol (PEG), a popular voluminous cosolute in crowding studies lacking specific interactions with the protein, leads to destabilization instead due to the excluded volume effects of entropic nature.[16]. Candotti et al discouraged the usage of unspecific voluminous crowders such as PEG altogether in favor of realistic bio-organic matter.[17]

Methods

Results

Conclusion