Peter J. Judge

Detergent-free formation and physicochemical characterization of nanosized lipid-polymer complexes: Lipodisq.

Lipodisq particles are polymer-lipid complexes formed by detergent-free methods. Lipodisq particles containing dimyristoylphosphatidylcholine (DMPC) are characterized by increased lipid ordering co ...

Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers

Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.

Biophysical characterization of Vpu from HIV‐1 suggests a channel‐pore dualism

Vpu from HIV‐1 is an 81 amino acid type I integral membrane protein which consists of a cytoplasmic and a transmembrane (TM) domain. The TM domain is known to alter membrane permeability for ions and substrates when inserted into artificial membranes. Peptides corresponding to the TM domain of Vpu (Vpu1‐32) and mutant peptides (Vpu1‐32‐W23L, Vpu1‐32‐R31V, Vpu1‐32‐S24L) have been synthesized and reconstituted into artificial lipid bilayers. All peptides show channel activity with a main conductance level of around 20 pS. Vpu1‐32‐W23L has a considerable flickering pattern in the recordings and longer open times than Vpu1‐32. Whilst recordings for Vpu1‐32‐R31V are almost indistinguishable from those of the WT peptide, recordings for Vpu1‐32‐S24L do not exhibit any noticeable channel activity. Recordings of WT peptide and Vpu1‐32‐W23L indicate Michaelis–Menten behavior when the salt concentration is increased. Both peptide channels follow the Eisenman series I, indicative for a weak ion channel with almost pore like characteristics. Proteins 2008.

Recent contributions from solid-state NMR to the understanding of membrane protein structure and function.

The plasma membrane functions as a semi-permeable barrier, defining the interior (or cytoplasm) of an individual cell. This highly dynamic and complex macromolecular assembly comprises predominantly lipids and proteins held together by entropic forces and provide the interface through which a cell interacts with its immediate environment. The extended sheet-like bilayer structure formed by the phospholipids is a highly adaptable platform whose structure and composition may be tuned to provide specialised functionality. Although a number of biophysical techniques including X-ray crystallography have been used to determine membrane protein structures, these methods are unable to replicate and accommodate the complexity and diversity of natural membranes. Solid state NMR (ssNMR) is a versatile method for structural biology and can be used to provide new insights into the structures of membrane components and their mutual interactions. The extensive variety of sample forms amenable for study by ssNMR, allows data to be collected from proteins in conditions that more faithfully resemble those of native environment, and therefore is much closer to a functional state.

The Conformation of Bacteriorhodopsin Loops in Purple Membranes Resolved by Solid-State MAS NMR Spectroscopy†

Membrane proteins (and rhodopsin-like G-protein coupled receptors (GPCRs), in particular) are of significant biological and medical importance since they represent over 50% (GPCRs 25%) of current drug targets. However, the structure determination of membrane proteins is challenging: currently they account for < 1% of the unique protein structures deposited in the Protein Databank. X-ray crystallography has been used to make major contributions towards the structure determination of membrane proteins, but it suffers from the fact that the proteins are rarely crystallized in their native lipid environment or are unmodified, and exposed loop regions are often either dynamic and not visible, or involved in crystal contacts. NMR spectroscopic studies of membrane proteins in solution are generally also reliant on an artificial detergent environment and are also limited by protein size. Solid-state NMR (ssNMR) spectroscopy, in contrast, has the advantage that membrane proteins can be studied in a lipid environment. Although ssNMR does not suffer from the same intrinsic size limitation as solution NMR spectroscopy, spectral overlap is often severe for large proteins and hampers their study. However, magicangle-spinning (MAS) NMR spectroscopy, in particular, has been used to make substantial methodological advances in recent years, and the first membrane protein structures have now been determined using this technique. Herein we report on how solid-state MAS NMR spectroscopy can be used to complement X-ray crystallographic studies of a large seven transmembrane (7TM) helical protein by validating and redefining the loop structures. The structure of bacteriorhodopsin (bR) has been determined in a range of two(2D) and three-dimensional (3D) crystalline environments with the loops showing the highest degree of structural variation. Solid-state MAS NMR spectra of uniformly [C,N]-labeled bR in its native purple membrane have been used to assign the signals of the loop regions of the protein. Extraction of dihedral angle information from chemical shifts has allowed us to validate several loop conformations in the crystal structure and recalculate the structure where there are differences in conformation. Ab initio assignment of the resonances of the loop regions of bR was carried out using 2D DARR spectra (mixing times of 15 and 50 ms) and 3D NCACX (20 ms), 3D NCOCX (20 ms), 3D CANCO and 3D CAN(CO)CX (45 ms) spectra. Assignment of the loops is made possible by the fact that the loop resonances are generally well separated and amenable to assignment in contrast to many of the helical regions, where leucine and valine resonances, in particular, exhibit intractable degrees of spectral overlap. Figure 1 shows the assignment of the section Met68–Gly72 in the BC loop as an example; further 2D spectra and strip plots are provided in the Supporting Information (Figures S1–S3). In total, we have assigned roughly 55% of loop residues covering all loops, except for the CD loop, as well as several residues located in the helices (Figure 2, Table S2 in the Supporting Information, and BMRB Accession code 17361). Interestingly, residues from all loops (except those in the unassigned CD loop) are visible in our cross-polarization (CP)-based spectra; this is in contrast to the spectra of sensory rhodopsin II from Natronomonas pharaonis (NpSRII) where most loops were visible only in INEPT-based spectra, reflecting a higher degree of loop mobility in NpSRII. Our observations are more similar to those made in a recent study of proteorhodopsin in which only isolated residues were observed in INEPT-based spectra. A C,C INEPT-COSY spectrum of bR contains resonances with random-coil chemical shifts from amino acid types that are consistent with the Nand C-terminal tails (see Figures S4 and S5 in Supporting Information). Some chemical shifts for side chains in non-random-coil conformations are also found for residues Lys, Glu, Ala, and Ser, which may belong to the KAES motif in the EF loop. Sequential [*] Dr. V. A. Higman, Dr. P. J. Judge, Prof. A. Watts Department of Biochemistry, University of Oxford South Parks Road, Oxford, OX1 3QU (UK) E-mail: anthony.watts@bioch.ox.ac.uk Dr. K. Varga Department of Chemistry, University of Wyoming Laramie, WY 82071 (USA)

Towards a Mechanism of Function of the Viral Ion Channel Vpu from HIV-1

Abstract Vpu, an integral membrane protein encoded in HIV-1, is implicated in the release of new virus particles from infected cells, presumably mediated by ion channel activity of homo- oligomeric Vpu bundles. Reconstitution of both full length Vpu1–81 and a short, the transmembrane (TM) domain comprising peptide Vpu1-32 into bilayers under a constant electric field results in an asymmetric orientation of those channels. For both cases, channel activity with similar kinetics is observed. Channels can open and remain open within a broad series of conductance states even if a small or no electric potential is applied. The mean open time for Vpu peptide channels is voltage-independent. The rate of channel opening shows a biphasic voltage activation, implicating that the gating is influenced by the interaction of the dipole moments of the TM helices with an electric field.

Anti-antimicrobial Peptides FOLDING-MEDIATED HOST DEFENSE ANTAGONISTS

Background: Direct antagonists of native antimicrobial peptide (AMP) sequences are unknown. Results: Complementary antagonistic sequences can co-fold with AMPs into functionally inert assemblies. Conclusion: Antagonists act as anti-AMPs. Significance: The findings offer a molecular rationale for anti-AMP responses with potential implications for antimicrobial resistance. Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance.

Anti-antimicrobial Peptides

Background: Direct antagonists of native antimicrobial peptide (AMP) sequences are unknown. Results: Complementary antagonistic sequences can co-fold with AMPs into functionally inert assemblies. Conclusion: Antagonists act as anti-AMPs. Significance: The findings offer a molecular rationale for anti-AMP responses with potential implications for antimicrobial resistance. Antimicrobial or host defense peptides are innate immune regulators found in all multicellular organisms. Many of them fold into membrane-bound α-helices and function by causing cell wall disruption in microorganisms. Herein we probe the possibility and functional implications of antimicrobial antagonism mediated by complementary coiled-coil interactions between antimicrobial peptides and de novo designed antagonists: anti-antimicrobial peptides. Using sequences from native helical families such as cathelicidins, cecropins, and magainins we demonstrate that designed antagonists can co-fold with antimicrobial peptides into functionally inert helical oligomers. The properties and function of the resulting assemblies were studied in solution, membrane environments, and in bacterial culture by a combination of chiroptical and solid-state NMR spectroscopies, microscopy, bioassays, and molecular dynamics simulations. The findings offer a molecular rationale for anti-antimicrobial responses with potential implications for antimicrobial resistance.

Solid-State Nuclear Magnetic Resonance Spectroscopy for Membrane Protein Structure Determination

Solid-state NMR (ssNMR) is a versatile technique that can provide high-resolution (sub-angstrom) structural data for integral membrane proteins embedded in native and model membrane environments. The methodologies for a priori structure determination have for the most part been developed using samples with crystalline and fibrous morphologies. However, the techniques are now being applied to large, polytopic membrane proteins including receptors, ion channels, and porins. ssNMR data may be used to annotate and refine existing structures in regions of the protein not fully resolved by crystallography (including ligand-binding sites and mobile solvent accessible loop regions). This review describes the spectroscopic experiments and data analysis methods (including assignment) used to generate high-resolution structural data for membrane proteins. We also consider the range of sample morphologies that are appropriate for study by this method.

Mediation mechanism of tyrosine 185 on the retinal isomerization equilibrium and the proton release channel in the seven-transmembrane receptor bacteriorhodopsin.

Electrostatic coupling leading to conformational changes in proteins is challenging to demonstrate directly, it requires that both the local, discrete electronic details and dynamic information relevant to the functional descriptions are probed. Here, as a novel study to address this challenge, the roles of an aromatic residue in influencing the functional conformational changes of a membrane receptor in its natural membrane environment are reported. Previously intractable discrete electronic details have been obtained using 2D solid-state NMR of specifically labelled receptor, reinforced with molecular dynamic simulations, mutational analysis and functional assays, supported by and compared with rigid-atom crystal structural models. Hydrogen bonding and hydrophobic interactions are identified as the mechanistic origin for direct electromechanical coupling to the dynamics of conformational changes within the receptor.