Pro-islet amyloid polypeptide in micelles contains a helical prohormone segment.

Pro-islet amyloid polypeptide (proIAPP) is the prohormone precursor molecule to IAPP, also known as amylin. IAPP is a calcitonin family peptide hormone that is cosecreted with insulin, and largely responsible for hunger satiation and metabolic homeostasis. Amyloid plaques containing mixtures of mature IAPP and misprocessed proIAPP deposit on, and destroy pancreatic ß-cell membranes, and they are recognized as a clinical hallmark of type 2 diabetes mellitus. In order to better understand the interaction with cellular membranes, we solved the solution NMR structure of proIAPP bound to dodecylphosphocholine micelles at pH 4.5. We show that proIAPP is a dynamic molecule with four α-helices. The first two helices are contained within the mature IAPP sequence, while the second two helices are part of the C-terminal prohormone segment (Cpro). We mapped the membrane topology of the amphipathic helices by paramagnetic relaxation enhancement, and we used CD and diffusion-ordered spectroscopy to identify environmental factors that impact proIAPP membrane affinity. We discuss how our structural results relate to prohormone processing based on the varied pH environments and lipid compositions of organelle membranes within the regulated secretory pathway, and the likelihood of Cpro survival for cosecretion with IAPP. DATABASE: The assigned resonances have been deposited in the Biological Magnetic Resonance Bank (BMRB) with accession numbers 50007 and 50019 for proIAPP and Cpro, respectively. The lowest energy structures have been deposited in the Protein Data Bank (PDB) with access codes 6UCJ and 6UCK.

Partial alignment, residual dipolar couplings and molecular symmetry in solution NMR.

Residual dipolar couplings (RDCs) and residual anisotropic chemical shifts (RACSs) are produced by the partial alignment of solution NMR samples. RDCs and RACSs yield high-resolution structural and dynamic information on the orientation of bonds and chemical groups in molecules. Many molecules form oligomers or have intrinsic symmetries, which may simplify the analysis of their partial alignment datasets. In this report, we explore the theory of partial alignment using an irreducible spherical representation, and we investigate the impact of molecular symmetry on the alignment of molecules. Though previous studies have reported simplified relationships on the partial alignment of molecules bearing different symmetry groups, we show that these simplified relationships may not be universal and only apply to a limited set of systems.

Super resolution NOESY spectra of proteins.

Spectral resolution remains one of the most significant limitations in the NMR study of biomolecules. We present the srNOESY (super resolution nuclear overhauser effect spectroscopy) experiment, which enhances the resolution of NOESY cross-peaks at the expense of the diagonal peak line-width. We studied two proteins, ubiquitin and the influenza hemagglutinin fusion peptide in bicelles, and we achieved average resolution enhancements of 21-47% and individual peak enhancements as large as ca. 450%. New peaks were observed over the conventional NOESY experiment in both proteins as a result of these improvements, and the final structures generated from the calculated restraints matched published models. We discuss the impact of the experimental parameters, spin diffusion and the information content of the srNOESY lineshape.

Mollib: a molecular and NMR data analysis software.

Mollib is a software framework for the analysis of molecular structures, properties and data with an emphasis on data collected by NMR. It uses an open source model and a plugin framework to promote community-driven development of new and enhanced features. Mollib includes tools for the automatic retrieval and caching of protein databank (PDB) structures, the hydrogenation of biomolecules, the analysis of backbone dihedral angles and hydrogen bonds, and the fitting of residual dipolar coupling (RDC) and residual anisotropic chemical shift (RACS) data. In this article, we release version 1.0 of mollib and demonstrate its application to common molecular and NMR data analyses.

Structure and Dynamics of Membrane Proteins and Membrane Associated Proteins with Native Bicelles from Eukaryotic Tissues.

In vitro studies of protein structure, function, and dynamics typically preclude the complex range of molecular interactions found in living tissues. In vivo studies elucidate these complex relationships, yet they are typically incompatible with the extensive and controlled biophysical experiments available in vitro. We present an alternative approach by extracting membranes from eukaryotic tissues to produce native bicelles to capture the rich and complex molecular environment of in vivo studies while retaining the advantages of in vitro experiments. Native bicelles derived from chicken egg or mouse cerebrum tissues contain a rich composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), lysolipids, cholesterol, ceramides (CM), and sphingomyelin (SM). The bicelles also contain source-specific lipids such as triacylglycerides (TAGs) and sulfatides from egg and brain tissues, respectively. With the influenza hemagglutinin fusion peptide (HAfp) and the C-terminal Src homology domain of lymphocyte-specific protein-tyrosine kinase (lck-cSH2), we show that membrane proteins and membrane associated proteins reconstituted in native bicelles produce high-resolution NMR data and probe native protein-lipid interactions.

Hybrid NMR: A Union of Solution- and Solid-State NMR.

Hybrid NMR (hdNMR) is a powerful new tool that combines the strengths of solution- and solid-state NMR to measure dipolar, chemical shift, and quadrupolar tensors in aqueous solution. We introduce the theory of hdNMR and partially randomly oriented (PRO) crystalline hydrogel samples. PRO samples produce randomly oriented spectra with characteristic Pake patterns from the solid state, yet they maintain the high-resolution dispersion of solution NMR experiments. With new pulse sequences, we show how hdNMR can be used to measure with high precision the 1Hα-13Cα dipolar tensor and carboxylate chemical shift anisotropy tensor of aspartate. These measurements contain detailed information on the distribution of electron density, interatomic distances, and the orientation dependence of molecular motion.

Membrane Fusion and Infection of the Influenza Hemagglutinin.

The influenza virus is a major health concern associated with an estimated 5000 to 30,000 deaths every year (Reed et al. 2015) and a significant economic impact with the development of treatments, vaccinations and research (Molinari et al. 2007). The entirety of the influenza genome is comprised of only eleven coding genes. An enormous degree of variation in non-conserved regions leads to significant challenges in the development of inclusive inhibitors for treatment. The fusion peptide domain of the influenza A hemagglutinin (HA) is a promising candidate for treatment since it is one of the most highly conserved sequences in the influenza genome (Heiny et al. 2007), and it is vital to the viral life cycle. Hemagglutinin is a class I viral fusion protein that catalyzes the membrane fusion process during cellular entry and infection. Impediment of the hemagglutinin's function, either through incomplete post-translational processing (Klenk et al. 1975; Lazarowitz and Choppin 1975) or through mutations (Cross et al. 2001), leads to non-infective virus particles. This review will investigate current research on the role of hemagglutinin in the virus life cycle, its structural biology and mechanism as well as the central role of the hemagglutinin fusion peptide (HAfp) to influenza membrane fusion and infection.

A Positively Charged Liquid Crystalline Medium for Measuring Residual Dipolar Couplings in Membrane Proteins by NMR.

Residual Dipolar Couplings (RDCs) are integral to the refinement of membrane protein structures by NMR since they accurately define the orientation of helices and other structural units. Only a small set of liquid crystals used for RDC measurements are compatible with the detergents needed in membrane protein studies. The available detergent-compatible liquid crystals are negatively charged, thus offering effectively only one of five orthogonal components of the alignment Saupe matrix. In this communication, we present a robust liquid crystalline medium that is positively charged, pinacyanol acetate (PNA), for the determination of orthogonal sets of RDCs in membrane proteins. This new medium promises to enhance the accuracy of membrane protein structures and the measurement of dynamics based on RDCs.

The influenza hemagglutinin fusion domain is an amphipathic helical hairpin that functions by inducing membrane curvature.

The highly conserved N-terminal 23 residues of the hemagglutinin glycoprotein, known as the fusion peptide domain (HAfp23), is vital to the membrane fusion and infection mechanism of the influenza virus. HAfp23 has a helical hairpin structure consisting of two tightly packed amphiphilic helices that rest on the membrane surface. We demonstrate that HAfp23 is a new class of amphipathic helix that functions by leveraging the negative curvature induced by two tightly packed helices on membranes. The helical hairpin structure has an inverted wedge shape characteristic of negative curvature lipids, with a bulky hydrophobic region and a relatively small hydrophilic head region. The F3G mutation reduces this inverted wedge shape by reducing the volume of its hydrophobic base. We show that despite maintaining identical backbone structures and dynamics as the wild type HAfp23, the F3G mutant has an attenuated fusion activity that is correlated to its reduced ability to induce negative membrane curvature. The inverted wedge shape of HAfp23 is likely to play a crucial role in the initial stages of membrane fusion by stabilizing negative curvature in the fusion stalk.

Use of isotropically tumbling bicelles to measure curvature induced by membrane components.

Isotropically tumbling discoidal bicelles are a useful biophysical tool for the study of lipids and proteins by NMR, dynamic light scattering, and small-angle X-ray scattering. Isotropically tumbling bicelles present a low-curvature central region, typically enriched with DMPC in the lamellar state, and a highly curved detergent rim, typically composed of DHPC. In this report, we study the impact of the partitioning and induced curvature of a few molecules of a foreign lipid on the bicelle size, structure, and curvature. Previous approaches for studying curvature have focused on macroscopic and bulk properties of membrane curvature. In the approach presented here, we show that the conical shape of the DOPE lipid and the inverted-conical shape of the DPC lipid induce measurable curvature changes in the bicelle size. Bicelles with an average of 1.8 molecules of DOPE have marked increases in the size of bicelles, consistent with negative membrane curvature in the central region of the bicelle. With bicelle curvature models, radii of curvature on the order of -100 Å and below are measured, with a greater degree of curvature observed in the more pliable Lα state above the phase-transition temperature of DMPC. Bicelles with an average of 1.8 molecules of DPC are reduced in size, consistent with positive membrane curvature in the rim, and at higher temperatures, DPC is distributed in the central region to form mixed-micelle structures. We use translational and rotational diffusion measurements by NMR, size-exclusion chromatography, and structural models to quantitate changes in bicelle size, curvature, and lipid dynamics.

Modulating alignment of membrane proteins in liquid-crystalline and oriented gel media by changing the size and charge of phospholipid bicelles.

We demonstrate that alignment of a structured peptide or small protein solubilized in mixed phospholipid:detergent micelles or bicelles, when embedded in a compressed gel or liquid crystalline medium, can be altered by either changing the phospholipid aggregate shape, charge, or both together. For the hemagglutinin fusion peptide solubilized in bicelles, we show that bicelle shape and charge do not change its helical hairpin structure but impact its alignment relative to the alignment medium, both in charged compressed acrylamide gel and in liquid crystalline d(GpG). The method can be used to generate sets of residual dipolar couplings that correspond to orthogonal alignment tensors, and holds promise for high-resolution structural refinement and dynamic mapping of membrane proteins.

The impact of influenza hemagglutinin fusion peptide length and viral subtype on its structure and dynamics.

A peptide comprising no fewer than the first 20 residues of the influenza hemagglutinin HA2 subunit suffices to induce lipid mixing between the membranes of different unilamellar vesicles. This 20-residue peptide was previously reported to adopt an open "boomerang" structure that differs significantly from the closed helical-hairpin structure of a fusion peptide consisting of the first 23 residues of the HA2 sequence. This study investigates the structural and dynamic features of fusion peptides of different length and subtype. Lacking key interactions that stabilize the closed, helical-hairpin structure, the 20-residue peptide is in a dynamic equilibrium between closed and open states, adopting a ca. 11% population of the former when solubilized by DPC micelles. Peptides shorter than 20 residues would have even fewer interactions to stabilize a helical hairpin fold, resulting in a vanishing hairpin population. Considering the conserved nature of hairpin-stabilizing interactions across all serotypes, and the minimum of 20 residues needed for fusion, we postulate that the closed state plays an essential role in the fusion process. However, opening of this hairpin structure may be essential to the formation of a membrane pore at the final stage of the fusion process.

pH-triggered, activated-state conformations of the influenza hemagglutinin fusion peptide revealed by NMR.

The highly conserved first 23 residues of the influenza hemagglutinin HA2 subunit constitute the fusion domain, which plays a pivotal role in fusing viral and host-cell membranes. At neutral pH, this peptide adopts a tight helical hairpin wedge structure, stabilized by aliphatic hydrogen bonding and charge-dipole interactions. We demonstrate that at low pH, where the fusion process is triggered, the native peptide transiently visits activated states that are very similar to those sampled by a G8A mutant. This mutant retains a small fraction of helical hairpin conformation, in rapid equilibrium with at least two open structures. The exchange rate between the closed and open conformations of the wild-type fusion peptide is ~40 kHz, with a total open-state population of ~20%. Transitions to these activated states are likely to play a crucial role in formation of the fusion pore, an essential structure required in the final stage of membrane fusion.

Whole-body rocking motion of a fusion peptide in lipid bilayers from size-dispersed 15N NMR relaxation.

Biological membranes present a highly fluid environment, and integration of proteins within such membranes is itself highly dynamic: proteins diffuse laterally within the plane of the membrane and rotationally about the normal vector of this plane. We demonstrate that whole-body motions of proteins within a lipid bilayer can be determined from NMR (15)N relaxation rates collected for different-sized bicelles. The importance of membrane integration and interaction is particularly acute for proteins and peptides that function on the membrane itself, as is the case for pore-forming and fusion-inducing proteins. For the influenza hemagglutinin fusion peptide, which lies on the surface of membranes and catalyzes the fusion of membranes and vesicles, we found large-amplitude, rigid-body wobbling motions on the nanosecond time scale relative to the lipid bilayer. This behavior complements prior analyses where data were commonly interpreted in terms of a static oblique angle of insertion for the fusion peptide with respect to the membrane. Quantitative disentanglement of the relative motions of two interacting objects by systematic variation of the size of one is applicable to a wide range of systems beyond protein-membrane interactions.

Helical hairpin structure of influenza hemagglutinin fusion peptide stabilized by charge-dipole interactions between the N-terminal amino group and the second helix.

The fusion domain of the influenza coat protein hemagglutinin HA2, bound to dodecyl phosphocholine micelles, was recently shown to adopt a structure consisting of two antiparallel α-helices, packed in an exceptionally tight hairpin configuration. Four interhelical H(α) to C═O aliphatic H-bonds were identified as factors stabilizing this fold. Here, we report evidence for an additional stabilizing force: a strong charge-dipole interaction between the N-terminal Gly(1) amino group and the dipole moment of helix 2. pH titration of the amino-terminal (15)N resonance, using a methylene-TROSY-based 3D NMR experiment, and observation of Gly(1 13)C' show a strongly elevated pK = 8.8, considerably higher than expected for an N-terminal amino group in a lipophilic environment. Chemical shifts of three C-terminal carbonyl carbons of helix 2 titrate with the protonation state of Gly(1)-N, indicative of a close proximity between the N-terminal amino group and the axis of helix 2, providing an optimal charge-dipole stabilization of the antiparallel hairpin fold. pK values of the side-chain carboxylate groups of Glu(11) and Asp(19) are higher by about 1 and 0.5 unit, respectively, than commonly seen for solvent-exposed side chains in water-soluble proteins, indicative of dielectric constants of ε = ∼30 (Glu(11)) and ∼60 (Asp(19)), placing these groups in the headgroup region of the phospholipid micelle.

The complete influenza hemagglutinin fusion domain adopts a tight helical hairpin arrangement at the lipid:water interface.

All but five of the N-terminal 23 residues of the HA2 domain of the influenza virus glycoprotein hemagglutinin (HA) are strictly conserved across all 16 serotypes of HA genes. The structure and function of this HA2 fusion peptide (HAfp) continues to be the focus of extensive biophysical, computational, and functional analysis, but most of these analyses are of peptides that do not include the strictly conserved residues Trp(21)-Tyr(22)-Gly(23). The heteronuclear triple resonance NMR study reported here of full length HAfp of sero subtype H1, solubilized in dodecylphosphatidyl choline, reveals a remarkably tight helical hairpin structure, with its N-terminal alpha-helix (Gly(1)-Gly(12)) packed tightly against its second alpha-helix (Trp(14)-Gly(23)), with six of the seven conserved Gly residues at the interhelical interface. The seventh conserved Gly residue in position 13 adopts a positive angle, enabling the hairpin turn that links the two helices. The structure is stabilized by multiple interhelical C(alpha)H to C=O hydrogen bonds, characterized by strong interhelical H(N)-H(alpha) and H(alpha)-H(alpha) NOE contacts. Many of the previously identified mutations that make HA2 nonfusogenic are also incompatible with the tight antiparallel hairpin arrangement of the HAfp helices.(15)N relaxation analysis indicates the structure to be highly ordered on the nanosecond time scale, and NOE analysis indicates HAfp is located at the water-lipid interface, with its hydrophobic surface facing the lipid environment, and the Gly-rich side of the helix-helix interface exposed to solvent.

Conformational dynamics of an intact virus: order parameters for the coat protein of Pf1 bacteriophage.

This study has examined the atomic-level dynamics of the protein in the capsid of filamentous phage Pf1. This capsid consists of approximately 7,300 small subunits of only 46 aa in a helical array around a highly extended, circular single-stranded DNA molecule of 7,349 nt. Measurements were made of site-specific, solid-state NMR order parameters, S, the values which are dimensionless quantities between 0 (mobile) and 1 (static) that characterize the amplitudes of molecular bond angular motions that are faster than microseconds. It was found that the protein subunit backbone is very static, and of particular interest, it appears to be static at residues glycine 15 and glutamine 16 where it had been previously thought to be mobile. In contrast to the backbone, several side chains display large-amplitude angular motions. Side chains on the virion exterior that interact with solvent are highly mobile, but surprisingly, the side chains of residues arginine 44 and lysine 45 near the DNA deep in the interior of the virion are also highly mobile. The large-amplitude dynamic motion of these positively charged side chains in their interactions with the DNA were not previously expected. The results reveal a highly dynamic aspect of a DNA-protein interface within a virus.

Conformational flexibility of a microcrystalline globular protein: order parameters by solid-state NMR spectroscopy.

The majority of protein structures are determined in the crystalline state, yet few methods exist for the characterization of dynamics for crystalline biomolecules. Solid-state NMR can be used to probe detailed dynamic information in crystalline biomolecules. Recent advances in high-resolution solid-state NMR have enabled the site-specific assignment of (13)C and (15)N nuclei in proteins. With the use of multidimensional separated-local-field experiments, we report the backbone and side chain conformational dynamics of ubiquitin, a globular microcrystalline protein. The measurements of molecular conformational order parameters are based on heteronuclear dipolar couplings, and they are correlated to assigned chemical shifts, to obtain a global perspective on the sub-microsecond dynamics in microcrystalline ubiquitin. A total of 38 Calpha, 35 Cbeta and multiple side chain unique order parameters are collected, and they reveal the high mobility of ubiquitin in the microcrystalline state. In general the side chains show elevated motion in comparison with the backbone sites. The data are compared to solution NMR order parameter measurements on ubiquitin. The SSNMR measurements are sensitive to motions on a broader time scale (low microsecond and faster) than solution NMR measurements (low nanosecond and faster), and the SSNMR order parameters are generally lower than the corresponding solution values. Unlike solution NMR relaxation-based order parameters, order parameters for (13)C(1)H(2) spin systems are readily measured from the powder line shape data. These results illustrate the potential for detailed, extensive, and site-specific dynamic studies of biopolymers by solid-state NMR.