Elsevier

Journal of Magnetic Resonance

Volume 253, April 2015, Pages 138-142
Journal of Magnetic Resonance

Progression of NMR studies of membrane-active peptides from lipid bilayers to live cells

https://doi.org/10.1016/j.jmr.2014.11.016Get rights and content

Highlights

  • A perspective on solid-state NMR studies of membrane peptides is given.

  • The structure and dynamics of both the peptide and lipid membranes can be studied.

  • Heteronuclear distances between peptide and membrane atoms can be measured.

  • Model membranes give structure–activity insights but in-cell studies are required.

Abstract

Understanding the structure of membrane-active peptides faces many challenges associated with the development of appropriate model membrane systems as the peptide structure depends strongly on the lipid environment. This perspective provides a brief overview of the approach taken to study antimicrobial and amyloid peptides in phospholipid bilayers using oriented bilayers and magic angle spinning techniques. In particular, Boltzmann statistics REDOR and maximum entropy analysis of spinning side bands are used to analyse systems where multiple states of peptide or lipid molecules may co-exist. We propose that in future, rather than model membranes, structural studies in whole cells are feasible.

Introduction

As a tool of structural biology, NMR spectroscopy is employed primarily to determine the structure of proteins in the solution-state. However, solid-state NMR spectroscopy (ss-NMR) is being used increasingly to determine the structure of biomolecules despite limitations due to line broadening and sensitivity. Developments in ss-NMR have resulted in structural information being gained from membrane peptides, and even proteins, in complex lipid systems, that is difficult to obtain by other techniques. Starting from 13C and 15N studies of the antibiotic peptide, gramicidin A, in phospholipid bilayers, ss-NMR structural studies have been extended to membrane receptors [1]. Progressively more complex model membranes are being used and we present some novel ss-NMR experiments (BS-REDOR, MeMAS) of these mixed lipid systems. Although structures in model membranes are seen as having biological relevance, the Holy Grail is to determine the structure at atomic resolution of these important biomolecules in their native environment. Solution NMR has been used to reveal the structure of over expressed proteins in living cells [2], but the structure of membrane-active peptides in cells has not been reported. Using current ss-NMR technology and isotopic labelling, high-resolution structures of peptides in membranes of live cells are within reach.

Section snippets

Orientational dependence in NMR interactions

In typical solution NMR experiments, biological molecules reorient with a correlation time well within the fast motion regime w.r.t. NMR time scale thereby averaging all angle-dependent interactions between the nuclear spins and the magnetic field, B0, aligned with the z-axis of the laboratory frame. With multidimensional approaches, NMR structures of soluble proteins have been resolved, up to 1000 kDa with TROSY-based pulse sequences [3], [4]. The presence of a lipid membrane environment for

Gramicidin A in aligned lipid bilayers

By aligning phospholipid bilayers between thin glass plates well resolved spectra can be obtained. Aligned multilayers can support up to 50% w/w water, which is sufficient to fully hydrate the phospholipids with about 2 nm of water between each bilayer [18]. These aligned systems result in two dimensional liquid crystals with respect to the magnetic field as the phospholipid long axis is oriented perpendicular to the glass surface. Using Eq. (1) the orientation of incorporated membrane proteins

Membrane-active peptides in model systems

The structure and orientation of the lytic peptide, melittin, in aligned lipid bilayers was also determined using 13C NMR [23]. Melittin formed a transmembrane α-helix with a kink at Pro-14. The structure of the peptide around Pro-14 was better defined using MAS techniques and double labels [17]. Interestingly, the peptide structure depended on the state of lipid bilayer above and below the lipid gel–fluid phase transition temperature. Similarly, antimicrobial peptides from Australian tree

BS-REDOR

REDOR is a useful MAS technique for determination of heteronuclear distances and is used to gain details of peptide structures and lipid–peptide interactions. However, care is required in setting up the experiment and in analysing the data since the signal intensity is prone to artefacts and complex spin–spin interactions. Thus, improvements to the original REDOR pulse sequence have been made, including: compensating for imperfect pulse angles (especially in low-sensitivity samples where pulse

Maximum entropy analysis of spinning side bands for accurate CSA determination

Since lipid composition plays an important role in the regulation of membrane-active peptides and proteins, more complex lipid membranes are required which poses difficulty in retaining high resolution spectra. For instance, each lipid has its own chemical environment and thermotropism and thus a phospholipid mixture at a specific temperature displays a distribution in 31P δiso, Δδ and η. To discern specific interactions between a peptide and a lipid in a mixture of lipids can become very

Towards live cell studies

One of the biggest challenges in structural biology is how to deal with heterogeneous systems such as cell membranes. Ultimately, one would like to perform ss-NMR experiments in live cells under physiological conditions. However, several drawbacks need to be faced, such as:

  • (1)

    Natural membranes are extremely heterogeneous, which precludes control of specific interactions with lipid species or domains.

  • (2)

    The limited lifetime of cells undergoing MAS conditions prevents long multidimensional experiments.

  • (3)

Conclusion

With the progress in pulse sequences, protein expression methods that enable specific in-situ labelling and a renewed appreciation of functionality of the lipid membrane, solving the structure–function relationship of membrane-active peptides or proteins and lipid composition is entering a new era. While great focus has been on solution NMR over the past three decades, ss-NMR now has the attention with the hope that it will reach similar success in determining the structure of biopolymers in

Acknowledgment

FS is grateful to the Australian Research Council Grant DP140102127 for financial support.

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