Highlights from Faraday discussion 232: Peptide-membrane interactions, 8th-10th September 2021 (online).

The Faraday discussions meeting on peptide-membrane interactions was designed with the goal of repositioning peptides as powerful model systems that are indispensable in contemporary membrane biophysics and biology research. The meeting, originally scheduled for September 2020, was finally held in a virtual format during September 8th-10th, 2021 due to the COVID-19 pandemic. The meeting saw enthusiastic participation by ∼120 scientists from 23 countries. There were 23 talks delivered during the four sessions and 25 posters presented in two poster sessions (the published volume can be accessed online). The meeting drew attention to the multitude of open questions that persist in our understanding of the behavior of membrane peptides and proteins in spite of nearly half-a-century of intensive interdisciplinary research, and was beyond successful in throwing into sharp relief the enduring relevance of exploring membrane biophysics and biology through the looking glass of peptide-membrane interactions.

Comparative Analysis of Tryptophan Dynamics in Spectrin and Its Constituent Domains: Insights from Fluorescence.

Spectrin is a cytoskeletal protein ubiquitous in metazoan cells that acts as a liaison between the plasma membrane and the cellular interior and imparts mechanical stability to the plasma membrane. Spectrin is known to be highly dynamic, with an appreciable degree of torsional and segmental mobility. In this context, we have earlier utilized the red edge excitation shift (REES) approach to report the retention of restricted solvation dynamics and local structure in the vicinity of spectrin tryptophans on urea denaturation and loss of spectrin secondary structure. As a natural progression of our earlier work, in this work, we carried out a biophysical dissection of tryptophan solvation and rotational dynamics in spectrin and its constituent domains, in order to trace the origin of local structure retention observed in denatured spectrin. Our results show that the ankyrin binding domain (and, to a lesser extent, the ß-tetramerization domain) is capable of retention of local structure, similar to that observed for intact spectrin. However, all α-chain domains studied exhibit negligible retention of local structure on urea denaturation. Such a stark chain-specific retention of local structure could originate from the fact that the ß-chain domains possess specialized functions, where conservation of local (structural) integrity may be a prerequisite for optimum cellular function. To the best of our knowledge, these observations represent one of the first systematic biophysical dissections of spectrin dynamics in terms of its constituent domains and add to emerging literature on comprehensive domain-based analysis of spectrin organization, dynamics, and function.

Hydration Dynamics in Biological Membranes: Emerging Applications of Terahertz Spectroscopy.

Water drives the spontaneous self-assembly of lipids and proteins into quasi two-dimensional biological membranes that act as catalytic scaffolds for numerous processes central to life. However, the functional relevance of hydration in membrane biology is only beginning to be addressed, predominantly because of challenges associated with direct measurements of hydration microstructure and dynamics in a biological milieu. Our recent work on the novel interplay of membrane electrostatics and crowding in shaping membrane hydration dynamics utilizing terahertz (THz) spectroscopy represents an important step in this context. In this Perspective, we provide a glimpse into the ever-broadening functional landscape of hydration dynamics in biological membranes in the backdrop of the unique physical chemistry of water molecules. We further highlight the immense (and largely untapped) potential of the THz toolbox in addressing contemporary problems in membrane biology, while emphasizing the adaptability of the analytical framework reported recently by us to such studies.

Membrane electrostatics sensed by tryptophan anchors in hydrophobic model peptides depends on non-aromatic interfacial amino acids: implications in hydrophobic mismatch.

WALPs are synthetic α-helical membrane-spanning peptides that constitute a well-studied system for exploring hydrophobic mismatch. These peptides represent a simplified consensus motif for transmembrane domains of intrinsic membrane proteins due to their hydrophobic core of alternating leucine and alanine flanked by membrane-anchoring aromatic tryptophan residues. Although the modulation of mismatch responses in WALPs by tryptophan anchors has been reported earlier, there have been limited attempts to utilize the intrinsic tryptophan fluorescence of this class of peptides in mismatch sensors. We have previously shown, utilizing the red edge excitation shift (REES) approach, that interfacial WALP tryptophan residues in fluid phase bilayers experience a dynamically constrained membrane microenvironment. Interestingly, emerging reports suggest the involvement of non-aromatic interfacially localized residues in modulating local structure and dynamics in WALP analogs. In this backdrop, we have explored the effect of interfacial amino acids, such as lysine (in KWALPs) and glycine (in GWALPs), on the tryptophan microenvironment of WALP analogs in zwitterionic and negatively charged membranes. We show that interfacial tryptophans in KWALP and GWALP experience a more restricted microenvironment, as reflected in the substantial increase in magnitude of REES and apparent rotational correlation time, relative to those in WALP in zwitterionic membranes. Interestingly, in contrast to WALP, the tryptophan anchors in KWALP and GWALP appear insensitive to the presence of negatively charged lipids in the membrane. These results reveal a subtle interplay between non-aromatic flanking residues in transmembrane helices and negatively charged lipids at the membrane interface, which could modulate the membrane microenvironment experienced by interfacially localized tryptophan residues. Since interfacial tryptophans are known to influence mismatch responses in WALPs, our results highlight the possibility of utilizing the fluorescence signatures of tryptophans in membrane proteins or model peptides such as WALP as markers for assessing protein responses to hydrophobic mismatch. More importantly, these results constitute one of the first reports on the influence of lipid headgroup charge in fine-tuning hydrophobic mismatch in membrane bilayers, thereby enriching the existing framework of hydrophobic mismatch.

Lipid Headgroup Charge Controls Melittin Oligomerization in Membranes: Implications in Membrane Lysis.

Melittin, a hemolytic peptide present in bee venom, represents one of the most well-studied amphipathic antimicrobial peptides, particularly in terms of its membrane interaction and activity. Nevertheless, no consensus exists on the oligomeric state of membrane-bound melittin. We previously reported on the differential microenvironments experienced by melittin in zwitterionic and negatively charged phospholipid membranes. In this work, we explore the role of negatively charged lipids in the oligomerization of membrane-bound melittin (labeled with 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)) utilizing a quantitative photobleaching homo-FRET assay. Our results show that the presence of negatively charged lipids decreases melittin oligomeric size to ∼50% of that observed in zwitterionic membranes. This is possibly due to differential energetics of binding of the peptide monomer to membranes of different compositions and could explain the reduced lytic activity yet tighter binding of melittin in negatively charged membranes. These results constitute one of the first experimental observations on the role of phospholipid headgroup charge in the oligomerization of melittin in membranes and is relevant in light of previous apparently contradictory reports on oligomerization of membrane-bound melittin. Our results highlight the synergistic interplay of peptide-membrane binding events and peptide oligomerization in modulating the organization, dynamics, and function of amphipathic α-helical peptides.

Extramembranous Regions in G Protein-Coupled Receptors: Cinderella in Receptor Biology?

G protein-coupled receptors (GPCRs) are the largest class of membrane proteins involved in signal transduction and are characterized by seven transmembrane domain architecture interconnected by extra- and intracellular loops. These loops, along with the N- and C-terminal domains, constitute the extramembranous regions in GPCRs. These regions, accounting for ~ 40% or more amino acid residues across different GPCR classes, are distinct from the conserved transmembrane domains in terms of nonconservation of sequence, diversity in length, and conformational heterogeneity. Due to technical challenges in exploring the molecular basis underlying the relation between structure, dynamics, and function in these regions, their contribution to GPCR organization and signaling remain underappreciated. Despite existing literature on the involvement of GPCR loops in numerous aspects of GPCR biology, the functional relevance of GPCR loops in the context of their inherent conformational heterogeneity and probable membrane interaction are not well understood. This review focuses on highlighting these aspects of GPCR extramembranous regions in the overall context of GPCR organization, dynamics, and biology. We envision that a judicious combination of insights obtained from structured transmembrane domains and disordered extramembranous regions in GPCRs would be crucial in arriving at a comprehensive understanding of GPCR structure, function, and dynamics, thereby leading to efficient drug discovery.

Effect of Local Anesthetics on the Organization and Dynamics of Hippocampal Membranes: A Fluorescence Approach.

Understanding the mechanism of action of local anesthetics has been challenging. We previously showed that the local anesthetic phenylethanol (PEtOH) inhibits the function of serotonin1A receptor, which is a member of the G protein-coupled receptor family and a neurotransmitter receptor. With the objective of gaining insight into the molecular mechanism underlying the anesthetic (PEtOH) action, we monitored the organization and dynamics of hippocampal membranes using multiple fluorescent reporters, which include a molecular rotor (BODIPY-C12) and a voltage-sensitive probe (4-(2-(6-(dioctylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-pyridinium inner salt) (di-8-ANEPPS), besides pyrene. These interfacial membrane probes were chosen because membrane partitioning of PEtOH would be reflected in the membrane interfacial environment. Taken together, we report a reduction in dipole potential and microviscosity of hippocampal membranes, with a concomitant increase in lateral diffusion in the presence of PEtOH. The reduction in membrane dipole potential induced by PEtOH constitutes one of the first experimental demonstrations on the modulation of membrane dipole potential by local anesthetics. Our results assume significance in view of previous reports that correlate membrane-perturbing effects of local anesthetics to their anesthetic action. We envision that insights into the interaction of local anesthetics with membranes could serve as a crucial link in developing a comprehensive understanding of the molecular mechanisms involved in anesthesia.

Wavelength-Selective Fluorescence of a Model Transmembrane Peptide: Constrained Dynamics of Interfacial Tryptophan Anchors.

WALPs are prototypical, α-helical transmembrane peptides that represent a consensus sequence for transmembrane segments of integral membrane proteins and serve as excellent models for exploring peptide-lipid interactions and hydrophobic mismatch in membranes. Importantly, the WALP peptides are in direct contact with the lipids. They consist of a central stretch of alternating hydrophobic alanine and leucine residues capped at both ends by tryptophans. In this work, we employ wavelength-selective fluorescence approaches to explore the intrinsic fluorescence of tryptophan residues in WALP23 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. Our results show that the four tryptophan residues in WALP23 exhibit an average red edge excitation shift (REES) of 6 nm, implying their localization at the membrane interface, characterized by a restricted microenvironment. This result is supported by fluorescence anisotropy and lifetime measurements as a function of wavelength displayed by WALP23 tryptophans in POPC membranes. These results provide a new approach based on intrinsic fluorescence of interfacial tryptophans to address protein-lipid interaction and hydrophobic mismatch.

Constrained dynamics of the sole tryptophan in the third intracellular loop of the serotonin1A receptor.

G protein-coupled receptors (GPCRs) are major signaling proteins in eukaryotic cells and are important drug targets. In spite of their role in GPCR function, the extramembranous regions of GPCRs are relatively less appreciated. The third intracellular loop (ICL3), which connects transmembrane helices V and VI, is important in this context since its crucial role in signaling has been documented for a number of GPCRs. Unfortunately, the structure of this loop is generally not visualized in x-ray crystallographic studies since this flexible loop is either stabilized using a monoclonal antibody or replaced with lysozyme. In this work, we expressed and purified the ICL3 region of the serotonin1A receptor and monitored its motional restriction and organization utilizing red edge excitation shift (REES) of its sole tryptophan and circular dichroism (CD) spectroscopy. Our results show that the tryptophan in ICL3 exhibits REES of 4 nm, implying that it is localized in a restricted microenvironment. These results are further supported by wavelength-selective changes in fluorescence anisotropy and lifetime. This constrained dynamics was relaxed upon denaturation of the peptide, thereby suggesting the involvement of the peptide secondary structure in the observed motional restriction, as evident from CD spectroscopy and apparent rotational correlation time. To the best of our knowledge, these results constitute one of the first measurements of motional constraint in the ICL3 region of GPCRs. Our results are relevant in the context of the reported intrinsically disordered nature of ICL3 and its role in providing functional diversity to GPCRs due to conformational plasticity.

Effect of Phospholipid Headgroup Charge on the Structure and Dynamics of Water at the Membrane Interface: A Terahertz Spectroscopic Study.

Biological membranes are highly organized supramolecular assemblies of lipids and proteins. The membrane interface separates the outer (bulk) aqueous phase from the hydrophobic membrane interior. In this work, we have explored the microstructure and collective dynamics of the membrane interfacial hydration shell in zwitterionic and negatively charged phospholipid membrane bilayers using terahertz time-domain spectroscopy. We show here that the relaxation time constants of the water hydrogen bond network exhibit a unique "rise and dip" pattern with increasing lipid concentration. More importantly, we observed a dependence of the critical lipid concentration corresponding to the inflection point on the charge of the lipid headgroup, thereby implicating membrane electrostatics as a major factor in the microstructure and dynamics of water at the membrane interface. These results constitute one of the first experimental evidences of the modulation of the dielectric relaxation response of membrane interfacial water by membrane lipid composition in a concentration-dependent manner. Lipid-stringent membrane hydration could be relevant in the broader context of lipid diversity observed in biological membranes and the role of negatively charged lipids in membrane protein structure and function.

Molecular rheology of neuronal membranes explored using a molecular rotor: Implications for receptor function.

The role of membrane cholesterol as a crucial regulator in the structure and function of membrane proteins and receptors is well documented. However, there is a lack of consensus on the mechanism for such regulation. We have previously shown that the function of an important neuronal receptor, the serotonin1A receptor, is modulated by cholesterol in hippocampal membranes. With an overall objective of addressing the role of membrane physical properties in receptor function, we measured the viscosity of hippocampal membranes of varying cholesterol content using a meso-substituted fluorophore (BODIPY-C12) based on the BODIPY probe. BODIPY-C12 acts as a fluorescent molecular rotor and allows measurement of hippocampal membrane viscosity through its characteristic viscosity-sensitive fluorescence depolarization. A striking feature of our results is that specific agonist binding by the serotonin1A receptor exhibits close correlation with hippocampal membrane viscosity, implying the importance of global membrane properties in receptor function. We envision that our results are important in understanding GPCR regulation by the membrane environment, and is relevant in the context of diseases in which GPCR signaling plays a major role and are characterized by altered membrane fluidity.