Disulfi de constrained Fabs overcome target size limitation for high-resolution single-particle cryo-EM.

High-resolution structures of proteins are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. Cryo-EM has revolutionized structure determination of large proteins and their complexes, but a vast majority of proteins that underlie human diseases are small (< 50 kDa) and usually beyond its reach due to low signal-to-noise images and difficulties in particle alignment. Current strategies to overcome this problem increase the overall size of small protein targets using scaffold proteins that bind to the target, but are limited by inherent flexibility and not being bound to their targets in a rigid manner, resulting in the target being poorly resolved compared to the scaffolds.

Cryo-EM reveals an unprecedented binding site for Na1.7 inhibitors enabling rational design of potent hybrid inhibitors.

The voltage-gated sodium (Na) channel Na1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available Na channel-blocking drugs are not selective among the nine Na channel subtypes, Na1.

A Flexible and Scalable High-Throughput Platform for Recombinant Membrane Protein Production.

Integral membrane proteins (MP) are implicated in many disease processes and are the primary targets of numerous marketed drugs. Despite recent advances in the areas of MP solubilization, stabilization, and reconstitution, it remains a time-consuming task to identify the combination of constructs and purification conditions that will enable MP structure-function studies outside of the lipid bilayer. In this chapter, we describe a strategy for rapidly identifying and optimizing the solubilization and purification conditions for nearly any recombinant MP, based on the use of a noninvasive fluorescent probe (His-Glow) that specifically binds to the common hexahistidine affinity tag of expressed targets.

A Novel Inhibitor of the LolCDE ABC Transporter Essential for Lipoprotein Trafficking in Gram-Negative Bacteria.

The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral β-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability.

From Constructs to Crystals - Towards Structure Determination of β-barrel Outer Membrane Proteins.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, α-helical and β-barrel. While α-helical membrane proteins can be found in nearly all cellular membranes, β-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria.

Visual arrestin interaction with clathrin adaptor AP-2 regulates photoreceptor survival in the vertebrate retina.

Arrestins bind ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) and terminate the activation of G proteins. Additionally, nonvisual arrestin/GPCR complex can initiate G protein-independent intracellular signals through their ability to act as scaffolds that bring other signaling molecules to the internalized GPCR. Like nonvisual arrestins, vertebrate visual arrestin (ARR1) terminates G protein signaling from light-activated, phosphorylated GPCR, rhodopsin.

What the N-terminal domain of Atg13 looks like and what it does: a HORMA fold required for PtdIns 3-kinase recruitment.

Atg13 is a subunit of the Atg1 complex that is involved in autophagy. The middle and C-terminal regions of Atg13 are intrinsically disordered and rich in regulatory phosphorylation sites. Thus far, there have been no structural data for any part of Atg13, and no function assigned to its N-terminal domain.

A HORMA domain in Atg13 mediates PI 3-kinase recruitment in autophagy.

Autophagy-related 13 (Atg13) is a key early-acting factor in autophagy and the major locus for nutrient-dependent regulation of autophagy by Tor. The 2.3-Å resolution crystal structure of the N-terminal domain of Atg13 reveals a previously unidentified HORMA (Hop1p, Rev1p and Mad2) domain similar to that of the spindle checkpoint protein Mad2.

Membrane binding and self-association of the epsin N-terminal homology domain.

Epsin possesses a conserved epsin N-terminal homology (ENTH) domain that acts as a phosphatidylinositol 4,5-bisphosphate-lipid-targeting and membrane-curvature-generating element. Upon binding phosphatidylinositol 4,5-bisphosphate, the N-terminal helix (H(0)) of the ENTH domain becomes structured and aids in the aggregation of ENTH domains, which results in extensive membrane remodeling. In this article, atomistic and coarse-grained (CG) molecular dynamics (MD) simulations are used to investigate the structure and the stability of ENTH domain aggregates on lipid bilayers.

Membrane curvature sensing by amphipathic helices: a single liposome study using α-synuclein and annexin B12.

Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic and hydrophilic faces of the helix greatly influence binding and sensing.

Computer modeling of nitroxide spin labels on proteins.

Electron paramagnetic resonance using site-directed spin labeling can be used as an approach for determination of protein structures that are difficult to solve by other methods. One important aspect of this approach is the measurement of interlabel distances using the double electron-electron resonance (DEER) method. Interpretation of experimental data could be facilitated by a computational approach to calculation of interlabel distances.

Membrane curvature induction and tubulation are common features of synucleins and apolipoproteins.

Synucleins and apolipoproteins have been implicated in a number of membrane and lipid trafficking events. Lipid interaction for both types of proteins is mediated by 11 amino acid repeats that form amphipathic helices. This similarity suggests that synucleins and apolipoproteins might have comparable effects on lipid membranes, but this has not been shown directly.

A combinatorial NMR and EPR approach for evaluating the structural ensemble of partially folded proteins.

Partially folded proteins, characterized as exhibiting secondary structure elements with loose or absent tertiary contacts, represent important intermediates in both physiological protein folding and pathological protein misfolding. To aid in the characterization of the structural state(s) of such proteins, a novel structure calculation scheme is presented that combines structural restraints derived from pulsed EPR and NMR spectroscopy. The methodology is established for the protein alpha-synuclein (alphaS), which exhibits characteristics of a partially folded protein when bound to a micelle of the detergent sodium lauroyl sarcosinate (SLAS).

Multiple modes of endophilin-mediated conversion of lipid vesicles into coated tubes: implications for synaptic endocytosis.

Endophilin A1 is a BAR (Bin/amphiphysin/Rvs) protein abundant in neural synapses that senses and induces membrane curvature, contributing to neck formation in presynaptic endocytic vesicles. To investigate its role in membrane remodeling, we used cryoelectron microscopy to characterize structural changes induced in lipid vesicles by exposure to endophilin. The vesicles convert rapidly to coated tubules whose morphology reflects the local concentration of endophilin.

Roles of amphipathic helices and the bin/amphiphysin/rvs (BAR) domain of endophilin in membrane curvature generation.

Control of membrane curvature is required in many important cellular processes, including endocytosis and vesicular trafficking. Endophilin is a bin/amphiphysin/rvs (BAR) domain protein that induces vesicle formation by promotion of membrane curvature through membrane binding as a dimer. Using site-directed spin labeling and EPR spectroscopy, we show that the overall BAR domain structure of the rat endophilin A1 dimer determined crystallographically is maintained under predominantly vesiculating conditions.

Structure of membrane-bound alpha-synuclein from site-directed spin labeling and computational refinement.

alpha-Synuclein is known to play a causative role in Parkinson disease. Although its physiological functions are not fully understood, alpha-synuclein has been shown to interact with synaptic vesicles and modulate neurotransmitter release. However, the structure of its physiologically relevant membrane-bound state remains unknown.

Mechanism of endophilin N-BAR domain-mediated membrane curvature.

Endophilin-A1 is a BAR domain-containing protein enriched at synapses and is implicated in synaptic vesicle endocytosis. It binds to dynamin and synaptojanin via a C-terminal SH3 domain. We examine the mechanism by which the BAR domain and an N-terminal amphipathic helix, which folds upon membrane binding, work as a functional unit (the N-BAR domain) to promote dimerisation and membrane curvature generation.

Calcium- and membrane-induced changes in the structure and dynamics of three helical hairpins in annexin B12.

Annexins are a family of soluble proteins that can undergo reversible Ca(2+)-dependent interaction with the interfacial region of phospholipid membranes. The helical hairpins on the convex face of the crystal structure of soluble annexins are proposed to mediate binding to membranes, but the mechanism is not defined. For this study, we used a site-directed spin labeling (SDSL) experimental approach to investigate Ca(2+) and membrane-induced structural and dynamic changes that occurred in the helical hairpins encompassing three of the four D and E helices of annexin B12.

The conserved core domains of annexins A1, A2, A5, and B12 can be divided into two groups with different Ca2+-dependent membrane-binding properties.

The hallmark of the annexin super family of proteins is Ca(2+)-dependent binding to phospholipid bilayers, a property that resides in the conserved core domain of these proteins. Despite the structural similarity between the core domains, studies reported herein showed that annexins A1, A2, A5, and B12 could be divided into two groups with distinctively different Ca(2+)-dependent membrane-binding properties. The division correlates with the ability of the annexins to form Ca(2+)-dependent membrane-bound trimers.

Structure of membrane-bound alpha-synuclein studied by site-directed spin labeling.

Many of the proposed physiological functions of alpha-synuclein, a protein involved in the pathogenesis of Parkinson's disease, are related to its ability to interact with phospholipids. To better understand the conformational changes that occur upon membrane binding of monomeric alpha-synuclein, we performed EPR analysis of 47 singly labeled alpha-synuclein derivatives. We show that membrane interaction is mediated by major conformational changes within seven N-terminal 11-aa repeats, which reorganize from a highly dynamic structure into an elongated helical structure devoid of significant tertiary packing.

Structural organization of alpha-synuclein fibrils studied by site-directed spin labeling.

Despite its importance in Parkinson's disease, a detailed understanding of the structure and mechanism of alpha-synuclein fibril formation remains elusive. In this study, we used site-directed spin labeling and electron paramagnetic resonance spectroscopy to study the structural features of monomeric and fibrillar alpha-synuclein. Our results indicate that monomeric alpha-synuclein, in solution, has a highly dynamic structure, in agreement with the notion that alpha-synuclein is a natively unfolded protein.

Cys9, Cys104 and Cys207 of simian virus 40 Vp1 are essential for inter-pentamer disulfide-linkage and stabilization in cell-free lysates.

Previous studies have implicated disulfide bonds between Vp1 molecules in the stabilization of the simian virus 40 (SV40) capsid. To identify the cysteine residues involved in intermolecular disulfide interactions, systematic oligo-directed mutagenesis of cysteine codons to serine codons was initiated. Wild-type and mutant Vp1 proteins were produced in rabbit reticulocyte lysates and were allowed to interact post-translationally.