Sequencing by avidity enables high accuracy with low reagent consumption.

We present avidity sequencing, a sequencing chemistry that separately optimizes the processes of stepping along a DNA template and that of identifying each nucleotide within the template. Nucleotide identification uses multivalent nucleotide ligands on dye-labeled cores to form polymerase-polymer-nucleotide complexes bound to clonal copies of DNA targets. These polymer-nucleotide substrates, termed avidites, decrease the required concentration of reporting nucleotides from micromolar to nanomolar and yield negligible dissociation rates. Avidity sequencing achieves high accuracy, with 96.2% and 85.4% of base calls having an average of one error per 1,000 and 10,000 base pairs, respectively. We show that the average error rate of avidity sequencing remained stable following a long homopolymer.

Lysine acetylation regulates the interaction between proteins and membranes.

Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates membrane protein function. Here, we use bioinformatics, biophysical analysis of recombinant proteins, live-cell fluorescent imaging and genetic manipulation of Drosophila to explore lysine acetylation in peripheral membrane proteins. Analysis of 50 peripheral membrane proteins harboring BAR, PX, C2, or EHD membrane-binding domains reveals that lysine acetylation predominates in membrane-interaction regions. Acetylation and acetylation-mimicking mutations in three test proteins, amphiphysin, EHD2, and synaptotagmin1, strongly reduce membrane binding affinity, attenuate membrane remodeling in vitro and alter subcellular localization. This effect is likely due to the loss of positive charge, which weakens interactions with negatively charged membranes. In Drosophila, acetylation-mimicking mutations of amphiphysin cause severe disruption of T-tubule organization and yield a flightless phenotype. Our data provide mechanistic insights into how lysine acetylation regulates membrane protein function, potentially impacting a plethora of membrane-related processes.

O-GlcNAc modification blocks the aggregation and toxicity of the protein α-synuclein associated with Parkinson's disease.

Several aggregation-prone proteins associated with neurodegenerative diseases can be modified by O-linked N-acetyl-glucosamine (O-GlcNAc) in vivo. One of these proteins, α-synuclein, is a toxic aggregating protein associated with synucleinopathies, including Parkinson's disease. However, the effect of O-GlcNAcylation on α-synuclein is not clear. Here, we use synthetic protein chemistry to generate both unmodified α-synuclein and α-synuclein bearing a site-specific O-GlcNAc modification at the physiologically relevant threonine residue 72. We show that this single modification has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, while not affecting the membrane binding or bending properties of α-synuclein. O-GlcNAcylation is also shown to affect the phosphorylation of α-synuclein in vitro and block the toxicity of α-synuclein that was exogenously added to cells in culture. These results suggest that increasing O-GlcNAcylation may slow the progression of synucleinopathies and further support a general function for O-GlcNAc in preventing protein aggregation.

Structural Characterization of Membrane-Curving Proteins: Site-Directed Spin Labeling, EPR, and Computational Refinement.

Endocytosis and other membrane remodeling processes require the coordinated generation of different membrane shapes. Proteins capable of manipulating lipid bilayers mediate these events using mechanisms that are not fully understood. Progress is limited by the small number of structures solved for proteins bound to different membrane shapes and tools capable of resolving such information. However, recent studies have shown site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) to be capable of obtaining high-resolution structural information for proteins bound to different membrane shapes. This technique can be applied to proteins with no known structure or proteins with structures known in solution. By refining the data obtained by EPR with computational modeling, 3D structures or structural models of membrane-bound proteins can be generated. In this chapter, we highlight the basic considerations and steps required to investigate the structures of membrane-bound proteins using SDSL, EPR, and computational refinement.

Tubulation by amphiphysin requires concentration-dependent switching from wedging to scaffolding.

BAR proteins are involved in a variety of membrane remodeling events but how they can mold membranes into different shapes remains poorly understood. Using electron paramagnetic resonance, we find that vesicle binding of the N-BAR protein amphiphysin is predominantly mediated by the shallow insertion of amphipathic N-terminal helices. In contrast, the interaction with tubes involves deeply inserted N-terminal helices together with the concave surface of the BAR domain, which acts as a scaffold. Combined with the observed concentration dependence of tubulation and BAR domain scaffolding, the data indicate that initial membrane deformations and vesicle binding are mediated by insertion of amphipathic helical wedges, while tubulation requires high protein densities at which oligomeric BAR domain scaffolds form. In addition, we identify a pocket of residues on the concave surface of the BAR domain that insert deeply into tube membrane. Interestingly, this pocket harbors a number of disease mutants in the homologous amphiphysin 2.

Endophilin A1 induces different membrane shapes using a conformational switch that is regulated by phosphorylation.

Membrane remodeling is controlled by proteins that can promote the formation of highly curved spherical or cylindrical membranes. How a protein induces these different types of membrane curvature and how cells regulate this process is still unclear. Endophilin A1 is a protein involved in generating endocytotic necks and vesicles during synaptic endocytosis and can transform large vesicles into lipid tubes or small and highly curved vesicles in vitro. By using EM and electron paramagnetic resonance of endophilin A1, we find that tubes are formed by a close interaction with endophilin A1's BIN/amphiphysin/Rvs (BAR) domain and deep insertion of its amphipathic helices. In contrast, vesicles are predominantly stabilized by the shallow insertion of the amphipathic helical wedges with the BAR domain removed from the membrane. By showing that the mechanism of membrane curvature induction is different for vesiculation and tubulation, these data also explain why previous studies arrived at different conclusions with respect to the importance of scaffolding and wedging in the membrane curvature generation of BAR proteins. The Parkinson disease-associated kinase LRRK2 phosphorylates S75 of endophilin A1, a position located in the acyl chain region on tubes and the aqueous environment on vesicles. We find that the phosphomimetic mutation S75D favors vesicle formation by inhibiting this conformational switch, acting to regulate endophilin A1-mediated curvature. As endophilin A1 is part of a protein superfamily, we expect these mechanisms and their regulation by posttranslational modifications to be a general means for controlling different types of membrane curvature in a wide range of processes in vivo.

Hydration dynamics as an intrinsic ruler for refining protein structure at lipid membrane interfaces.

Knowing the topology and location of protein segments at water-membrane interfaces is critical for rationalizing their functions, but their characterization is challenging under physiological conditions. Here, we debut a unique spectroscopic approach by using the hydration dynamics gradient found across the phospholipid bilayer as an intrinsic ruler for determining the topology, immersion depth, and orientation of protein segments in lipid membranes, particularly at water-membrane interfaces. This is achieved through the site-specific quantification of translational diffusion of hydration water using an emerging tool, (1)H Overhauser dynamic nuclear polarization (ODNP)-enhanced NMR relaxometry. ODNP confirms that the membrane-bound region of α-synuclein (αS), an amyloid protein known to insert an amphipathic α-helix into negatively charged phospholipid membranes, forms an extended α-helix parallel to the membrane surface. We extend the current knowledge by showing that residues 90-96 of bound αS, which is a transition segment that links the α-helix and the C terminus, adopt a larger loop than an idealized α-helix. The unstructured C terminus gradually threads through the surface hydration layers of lipid membranes, with the beginning portion residing within 5-15 Å above the phosphate level, and only the very end of C terminus surveying bulk water. Remarkably, the intrinsic hydration dynamics gradient along the bilayer normal extends to 20-30 Å above the phosphate level, as demonstrated with a peripheral membrane protein, annexin B12. ODNP offers the opportunity to reveal previously unresolvable structure and location of protein segments well above the lipid phosphate, whose structure and dynamics critically contribute to the understanding of functional versatility of membrane proteins.

The S100A10 subunit of the annexin A2 heterotetramer facilitates L2-mediated human papillomavirus infection.

Mucosotropic, high-risk human papillomaviruses (HPV) are sexually transmitted viruses that are causally associated with the development of cervical cancer. The most common high-risk genotype, HPV16, is an obligatory intracellular virus that must gain entry into host epithelial cells and deliver its double stranded DNA to the nucleus. HPV capsid proteins play a vital role in these steps. Despite the critical nature of these capsid protein-host cell interactions, the precise cellular components necessary for HPV16 infection of epithelial cells remains unknown. Several neutralizing epitopes have been identified for the HPV16 L2 minor capsid protein that can inhibit infection after initial attachment of the virus to the cell surface, which suggests an L2-specific secondary receptor or cofactor is required for infection, but so far no specific L2-receptor has been identified. Here, we demonstrate that the annexin A2 heterotetramer (A2t) contributes to HPV16 infection and co-immunoprecipitates with HPV16 particles on the surface of epithelial cells in an L2-dependent manner. Inhibiting A2t with an endogenous annexin A2 ligand, secretory leukocyte protease inhibitor (SLPI), or with an annexin A2 antibody significantly reduces HPV16 infection. With electron paramagnetic resonance, we demonstrate that a previously identified neutralizing epitope of L2 (aa 108-120) specifically interacts with the S100A10 subunit of A2t. Additionally, mutation of this L2 region significantly reduces binding to A2t and HPV16 pseudovirus infection. Furthermore, downregulation of A2t with shRNA significantly decreases capsid internalization and infection by HPV16. Taken together, these findings indicate that A2t contributes to HPV16 internalization and infection of epithelial cells and this interaction is dependent on the presence of the L2 minor capsid protein.