Backbone dynamics of an alamethicin in methanol and aqueous detergent solution determined by heteronuclear 1H−15N NMR spectroscopy

Summary The ¹⁵N relaxation rates of the -aminoisobutyric acid (Aib)-rich peptide alamethicin dissolved in methanol at 27°C and 5°C, and dissolved in aqueous sodium dodecylsulfate (SDS) at 27°C, were measured using inverse-detected one-and two-dimensional ¹H–¹⁵N NMR spectroscopy. Measurements of ¹⁵N longitudinal (R_N(N_z)) and transverse (R_N(N_x,y)) relaxation rates and the {¹H} ¹⁵N nuclear Overhauser enhancement (NOE) at 11.7 Tesla were used to calculate (quasi-) spectral density values at 0, 50, and 450 MHz for the peptide in methanol and in SDS. Spectral density mapping at 0, 50, 450, 500, and 550 MHz was done using additional measurements of the ¹H–¹⁵N lingitudinal two-spin order, R_NH(2H _infZ ^supN N_Z), two-spin antiphase coherence, R_NH(2H _infN ^supZ N_x,y), and the proton longitudinal relaxation rate, R_H(H _infN ^supZ ), for the peptide dissolved in methanol only. The spectral density of motions was also modeled using the three-parameter Lipari-Szabo function. The overall rotational correlation times were determined to be 1.1, 2.5, and 5.7 ns for alamethicin in methanol at 27°C and 5°C, and in SDS at 27°C, respectively. From the rotational correlation time determined in SDS the number of detergent molecules associated with the peptide was estimated to be about 40. The average order parameter was about 0.7 and the internal correlation times were about 70 ps for the majority of backbone amide ¹⁵N sites of alamethicin in methanol and in SDS. The relaxation data, spectral densities, and order parameters suggest that the peptide N-H vectors of alamethicin are not as highly constrained as the core regions of folded globular proteins. However, the peptide backbone is clearly not as mobile as the most unconstrained regions of folded proteins, such as those found in the frayed C-and N-termini of some proteins, or in randomcoil peptides. The data also suggest significant mobility at both ends of the peptide dissolved in methanol. In SDS the mobility in the middle and at the ends of the peptide is reduced. The implications of the results with respect to the sterically hindered Aib residues and the biological activities of the peptide are discussed.To whom correspondence should be addressed.     

α-Helical transmembrane peptides: A “Divide and Conquer” approach to membrane proteins

α-Helical membrane proteins fulfill many vital roles in all living cells and constitute the majority of drug targets. However, their relevance is in no way paralleled by our current understanding of their structures and functions. This is because membrane proteins present a number of experimental obstacles that are difficult to surmount by classical methods developed for water-soluble proteins. Moreover, membrane proteins are not only challenging on their very own but, when embedded in a biological membrane, also reside in an outstandingly complex milieu. These difficulties have fostered a “divide and conquer” approach, in which a membrane protein is dissected into shorter and easier-to-handle transmembrane (TM) peptides. Under suitable conditions, such peptides fold independently and retain many of the properties displayed in the context of the full-length parent protein.This contribution reviews some of the most notable insights into α-helical membrane proteins gleaned from experiments on protein-derived TM peptides. We recapitulate some peculiar properties of lipid bilayers that render them such a complex and unique environment and discuss generic features pertaining to hydrophobic peptides derived from α-helical membrane proteins. The main part of the review is devoted to a critical discussion of particularly interesting examples of TM peptides studied in membrane-mimetic systems of increasing complexity: isotropic solvents, detergent micelles, lipid bilayers, and biological membranes. The unifying theme is to explore to what extent TM peptides in combination with different membrane-mimetic systems can aid in advancing our knowledge and comprehension of α-helical membrane proteins as well as in developing new pharmacological tools.     

Structure determination of α-helical membrane proteins by solution-state NMR: Emphasis on retinal proteins

The biochemical processes of living cells involve a numerous series of reactions that work with exceptional specificity and efficiency. The tight control of this intricate reaction network stems from the architecture of the proteins that drive the chemical reactions and mediate protein–protein interactions. Indeed, the structure of these proteins will determine both their function and interaction partners. A detailed understanding of the proximity and orientation of pivotal functional groups can reveal the molecular mechanistic basis for the activity of a protein. Together with X-ray crystallography and electron microscopy, NMR spectroscopy plays an important role in solving three-dimensional structures of proteins at atomic resolution. In the challenging field of membrane proteins, retinal-binding proteins are often employed as model systems and prototypes to develop biophysical techniques for the study of structural and functional mechanistic aspects. The recent determination of two 3D structures of seven-helical trans-membrane retinal proteins by solution-state NMR spectroscopy highlights the potential of solution NMR techniques in contributing to our understanding of membrane proteins. This review summarizes the multiple strategies available for expression of isotopically labeled membrane proteins. Different environments for mimicking lipid bilayers will be presented, along with the most important NMR methods and labeling schemes used to generate high-quality NMR spectra. The article concludes with an overview of types of conformational restraints used for generation of high-resolution structures of membrane proteins. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

Immunolocalization of the acid-sensing ion channel 2a in the rat cerebellum

The acid-sensing ion channels (ASICs) are members of the DEG/ENaC superfamily of Na+ channels. Acid-gated cation currents have been detected in neurons from multiple regions of the brain including the cerebellum, but little is known about their molecular identity and function. Recently, one of ASICs (ASIC1a) was implicated in synaptic plasticity. In this study we examined the subcellular distribution of ASIC2a in rat cerebellum by immunostaining and confocal microscopy. Monoclonal antibodies for labeling of defined brain structures, for example, astroglia, Purkinje cell dendrites, nuclei, and presynaptic terminals were used for colocalization analyses. In the gray matter, the anti-ASIC2a antibody intensively stained dendrite branches of Purkinje cells evenly distributed throughout the entire molecular layer (ML). In the granule cell layer (GL), anti-ASIC2a antibody stained synaptic glomeruli. Neuronal localization of ASIC2a was confirmed by lack of co-staining with glial fibrillary acidic protein. Anti-ASIC2a staining in the ML colocalized with metabotropic glutamate receptor 1alpha (mGluR1alpha) in Purkinje cell dendrites and dendritic spines. Both proteins, mGluR1alpha and ASIC2a, were enriched in a crude synaptic membrane fraction prepared from cerebellum, suggesting synaptic expression of these proteins. Dual staining with anti-syntaxin 1A and anti-ASIC2a antibodies demonstrates characteristic complementary distribution of two proteins in both ML and GL. Because syntaxin 1A localized in presynaptic membranes and synaptic vesicles, complementary distribution with ASIC2a suggests postsynaptic localization of ASIC2a in these structures. This study shows specific localization of ASIC2a in both Purkinje and granule cell dendrites of the cerebellum and enrichment of ASIC2a in a crude cerebellar synaptic membrane fraction. The study is the first report of synaptic localization of ASIC2a in the CNS. The synaptic localization of ASIC2a in the cerebellum makes this channel a candidate for a role in motor coordination and learning.     

Application of DFT methods to the study of the coordination environment of the VO2+ ion in V?proteins

Density functional theory (DFT) methods were used to simulate the environment of vanadium in several V proteins, such as vanadyl-substituted carboxypeptidase (sites A and B), vanadyl-substituted chloroplast F(1)-ATPase (CF(1); site 3), the reduced inactive form of vanadium bromoperoxidase (VBrPO; low- and high-pH sites), and vanadyl-substituted imidazole glycerol phosphate dehydratase (IGPD; sites α, β, and γ). Structural, electron paramagnetic resonance, and electron spin echo envelope modulation parameters were calculated and compared with the experimental values. All the simulations were performed in water within the framework of the polarizable continuum model. The angular dependence of [Formula: see text] and [Formula: see text] on the dihedral angle θ between the V=O and N-C bonds and on the angle φ between the V=O and V-N bonds, where N is the coordinated aromatic nitrogen atom, was also found. From the results it emerges that it is possible to model the active site of a vanadium protein through DFT methods and determine its structure through the comparison between the calculated and experimental spectroscopic parameters. The calculations confirm that the donor sets of sites B and A of vanadyl-substituted carboxypeptidase are [[Formula: see text], H(2)O, H(2)O, H(2)O] and [N(His)(||), N(His)(⊥), [Formula: see text], H(2)O], and that the donor set of site 3 of CF(1)-ATPase is [[Formula: see text], OH(Thr), H(2)O, H(2)O, [Formula: see text]]. For VBrPO, the coordination modes [N(His)(||), N(His)(∠), OH(Ser), H(2)O, H(2)O(ax)] for the low-pH site and [N(His)(||), N(His)(∠), OH(Ser), OH(-), H(2)O(ax)] or [N(His)(||), N(His)(∠), [Formula: see text], H(2)O] for the high-pH site, with an imidazole ring of histidine strongly displaced from the equatorial plane, can be proposed. Finally, for sites α, β, and γ of IGPD, the subsequent deprotonation of one, two, and three imidazole rings of histidine and the participation of a carboxylate group of a glutamate residue ([N(His)(||), [Formula: see text], H(2)O, H(2)O], [N(His)(||), N(His)(||), [Formula: see text], H(2)O], and [N(His)(||), N(His)(||), [Formula: see text], OH(-), [Formula: see text]], respectively) seems to be the most plausible hypothesis.     

Residue-specific membrane location of peptides and proteins using specifically and extensively deuterated lipids and 13C–2H rotational-echo double-resonance solid-state NMR

Residue-specific location of peptides in the hydrophobic core of membranes was examined using ¹³C–²H REDOR and samples in which the lipids were selectively deuterated. The transmembrane topology of the KALP peptide was validated with this approach with substantial dephasing observed for deuteration in the bilayer center and reduced or no dephasing for deuteration closer to the headgroups. Insertion of β sheet HIV and helical and β sheet influenza virus fusion peptides into the hydrophobic core of the membrane was validated in samples with extensively deuterated lipids.     

Identification and removal of nitroxide spin label contaminant: impact on PRE studies of α-helical membrane proteins in detergent

NMR paramagnetic relaxation enhancement (PRE) provides long‐range distance constraints (～15–25 Å) that can be critical to determining overall protein topology, especially where long‐range NOE information is unavailable such as in the case of larger proteins that require deuteration. However, several challenges currently limit the use of NMR PRE for α‐helical membrane proteins. One challenge is the nonspecific association of the nitroxide spin label to the protein‐detergent complex that can result in spurious PRE derived distance restraints. The effect of the nitroxide spin label contaminant is evaluated and quantified and a robust method for the removal of the contaminant is provided to advance the application of PRE restraints to membrane protein NMR structure determination.     

Misfolding of membrane proteins in health and disease: the lady or the tiger?

Protein misfolding is increasingly recognized as a factor in many diseases, including cystic fibrosis, Parkinson's, Alzheimer's and atherosclerosis. Many proteins involved in misfolding-based pathologies are membrane-associated, such that the bilayer may play roles in normal and aberrant folding. It can be argued that the in vivo partitioning of eukaryotic membrane proteins between folding and misfolding pathways is under kinetic control. Moreover, the balance between these pathways can be surprisingly delicate.     

NMR relaxation parameters of methyl groups as a tool to map the interfaces of helix–helix interactions in membrane proteins

A number of recent advances in the field of magic-angle-spinning (MAS) solid-state NMR have enabled its application to a range of biological systems of ever increasing complexity. To retain biological relevance, these samples are increasingly studied in a hydrated state. At the same time, experimental feasibility requires the sample preparation process to attain a high sample concentration within the final MAS rotor. We discuss these considerations, and how they have led to a number of different approaches to MAS NMR sample preparation. We describe our experience of how custom-made (or commercially available) ultracentrifugal devices can facilitate a simple, fast and reliable sample preparation process. A number of groups have since adopted such tools, in some cases to prepare samples for sedimentation-style MAS NMR experiments. Here we argue for a more widespread adoption of their use for routine MAS NMR sample preparation.     

A distance geometry program for determining the structures of small proteins and other macromolecules from nuclear magnetic resonance measurements of intramolecular1H−1H proximities in solution

DISGEO is a new implementation of a distance geometry algorithm which has been specialized for the calculation of macromolecular conformation from distance measurements obtained by two-dimensional nuclear Overhauser enhancement spectroscopy. The improvements include (1) a decomposition of the complete embedding process into two successive, more tractable calculations by the use of “substructures”, (2) a compact data structure for storing incomplete distance information on a structure, (3) a more efficient shortest-path algorithm for computing the triangle inequality limits on all distances from this information, (4) a new algorithm for selecting random metric spaces from within these limits, (5) the use of chirality constraints to obtain good covalent geometry without the use ofad hoc weights or excessive optimization. The utility of the resultant program with nuclear magnetic resonance data is demonstrated by embedding complete spatial structures for the protein basic pancreatic trypsin inhibitor vs all 508 intramolecular, interresidue proton-proton contacts shorter than 4.0 Å that were present in its crystal structure. The crystal structure could be reproduced from this data set to within 1.3 Å minimum root mean square coordinate difference between the backbone atoms. We conclude that the information potentially available from nuclear magnetic resonance experiments in solution is sufficient to define the spatial structure of small proteins.     

Developmental changes in the cochlear hair cell mechanotransducer channel and their regulation by transmembrane channel–like proteins

Vibration of the stereociliary bundles activates calcium-permeable mechanotransducer (MT) channels to initiate sound detection in cochlear hair cells. Different regions of the cochlea respond preferentially to different acoustic frequencies, with variation in the unitary conductance of the MT channels contributing to this tonotopic organization. Although the molecular identity of the MT channel remains uncertain, two members of the transmembrane channel–like family, Tmc1 and Tmc2, are crucial to hair cell mechanotransduction. We measured MT channel current amplitude and Ca²⁺ permeability along the cochlea’s longitudinal (tonotopic) axis during postnatal development of wild-type mice and mice lacking Tmc1 (Tmc1−/−) or Tmc2 (Tmc2−/−). In wild-type mice older than postnatal day (P) 4, MT current amplitude increased ∼1.5-fold from cochlear apex to base in outer hair cells (OHCs) but showed little change in inner hair cells (IHCs), a pattern apparent in mutant mice during the first postnatal week. After P7, the OHC MT current in Tmc1−/− (dn) mice declined to zero, consistent with their deafness phenotype. In wild-type mice before P6, the relative Ca²⁺ permeability, P_Ca, of the OHC MT channel decreased from cochlear apex to base. This gradient in P_Ca was not apparent in IHCs and disappeared after P7 in OHCs. In Tmc1−/− mice, P_Ca in basal OHCs was larger than that in wild-type mice (to equal that of apical OHCs), whereas in Tmc2−/−, P_Ca in apical and basal OHCs and IHCs was decreased compared with that in wild-type mice. We postulate that differences in Ca²⁺ permeability reflect different subunit compositions of the MT channel determined by expression of Tmc1 and Tmc2, with the latter conferring higher P_Ca in IHCs and immature apical OHCs. Changes in P_Ca with maturation are consistent with a developmental decrease in abundance of Tmc2 in OHCs but not in IHCs.     

Inhibition of 5′-nucleotidase by concanavalin A: Evidence for localization on the outer surface of the plasma membrane

Summary 5-Nucleotidase activity of rat liver plasma membrane is markedly inhibited by concanavalin A. Taken together with a unilateral pattern of labelling of concanavalin A binding sites with hemocyanin, the results indicate that an allosteric site of the enzyme is at the outer surface of the membrane.Work supported in part by NIH CA 13145 to D.J. Morré and by U.S. NASA W-12792(05) to J. Shen-Miller. Argonne National Laboratory is operated under the auspices of the U.S. Energy Research and Development Administration. We thank Keri Safranski, Dorothy Werderitish, and G.T. Chubb for technical assistance     

Investigating the membrane orientation and transversal distribution of 17β-estradiol in lipid membranes by solid-state NMR

17β-Estradiol (E₂) is a potent estrogen, which modulates many important cellular functions by binding to specific estrogen receptors located in the cell nucleus and also on the plasma membrane. We have studied the membrane interaction of E₂ using a combination of solid-state NMR methods. ²H NMR results indicate that E₂ does not cause a condensation effect of the surrounding phospholipids, which is contrary to the effects of cholesterol, and only very modest E₂ induced alterations of the membrane structure were detected. ¹H magic-angle spinning NMR showed well resolved signals from E₂ as well as of POPC in the membrane-lipid layer. Two-dimensional NOESY spectra revealed intense cross-peaks between E₂ and the membrane lipids indicating that E₂ is stably inserted into the membrane. The determination of intermolecular cross-relaxation rates revealed that E₂ is broadly distributed in the membrane with a maximum of the E₂ distribution function in the upper chain region of the membrane. We conclude that E₂ is highly dynamic in lipid membranes and may undergo rotations as it exhibits two polar hydroxyl groups on either side of the molecule.     

Membrane-disruptive abilities of β-hairpin antimicrobial peptides correlate with conformation and activity: A P and H NMR study

The membrane interaction and solution conformation of two mutants of the β-hairpin antimicrobial peptide, protegrin-1 (PG-1), are investigated to understand the structural determinants of antimicrobial potency. One mutant, [A_6,8,13,15] PG-1, does not have the two disulfide bonds in wild-type PG-1, while the other, [Δ_4,18 G₁₀] PG-1, has only half the number of cationic residues. ³¹P solid-state NMR lineshapes of uniaxially aligned membranes indicate that the membrane disorder induced by the three peptides decreases in the order of PG-1>[Δ_4,18 G₁₀] PG-1?[A_6,8,13,15] PG-1. Solution NMR studies of the two mutant peptides indicate that [Δ_4,18 G₁₀] PG-1 preserves the β-hairpin fold of the wild-type peptide while [A_6,8,13,15] PG-1 adopts a random coil conformation. These NMR results correlate well with the known activities of these peptides. Thus, for this class of peptides, the presence of a β-hairpin fold is more essential than the number of cationic charges for antimicrobial activity. This study indicates that ³¹P NMR lineshapes of uniaxially aligned membranes are well correlated with antimicrobial activity, and can be used as a diagnostic tool to understand the peptide-lipid interactions of these antimicrobial peptides.     

PIN-G – A novel reporter for imaging and defining the effects of trafficking signals in membrane proteins

Background  

The identification of protein trafficking signals, and their interacting mechanisms, is a fundamental objective of modern biology. Unfortunately, the analysis of trafficking signals is complicated by their topography, hierarchical nature and regulation. Powerful strategies to test candidate motifs include their ability to direct simpler reporter proteins, to which they are fused, to the appropriate cellular compartment. However, present reporters are limited by their endogenous expression, paucity of cloning sites, and difficult detection in live cells.     

The conformation of peptide thymosin α1 in solution and in a membrane-like environment by circular dichroism and NMR spectroscopy. a possible model for its interaction with the lymphocyte membrane

The 28-residue peptide thymosin α1 was studied by circular dichroism and two-dimensional NMR. Circular dichroism indicates that thymosin α1 in water solution does not assume a preferred conformation, while in the presence of small unilamellar vesicles of dimiristoylphosphatidylcholine and dimiristoylphosphatidic acid (10:1) and in sodium dodecyl sulphate, it assumes a partly structured conformation. Presence of zinc ions produces similar effects. In a more hydrophobic environment like a solution of a mixed solvent water-2,2,2 trifluoroethanol, it adopts a structured conformation. NMR spectra indicated that in this mixture as solvent, thymosin α1 has a structure characterized by two regions. A β-turn is present between residue 5 and residue 8, while the region between residues 17 and 24 shows an α helix conformation. These changes of conformation in different environments may be considered structural requirements in the steps of its interaction with the lymphocyte membrane. In fact, these conformational changes may correspond to the first event of the mechanism of lymphocyte activation in the immune response modulation by thymosin α1.