Chemical ligation of the influenza M2 protein for solid‐state NMR characterization of the cytoplasmic domain

Solid‐state NMR‐based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly ¹³C‐labeled. A strategy to meet this challenge is chemical ligation combined with site‐specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water‐soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the influenza M2 protein, which contains a transmembrane (TM) domain and two extra‐membrane domains. The cytoplasmic domain, which contains an amphipathic helix (AH) and a cytoplasmic tail, is important for regulating virus assembly, virus budding, and the proton channel activity. A recent study of uniformly ¹³C‐labeled full‐length M2 by spectral simulation suggested that the cytoplasmic tail is unstructured. To further test this hypothesis, we conducted native chemical ligation of the TM segment and part of the cytoplasmic domain. Solid‐phase peptide synthesis of the two segments allowed several residues to be labeled in each segment. The post‐AH cytoplasmic residues exhibit random‐coil chemical shifts, low bond order parameters, and a surface‐bound location, thus indicating that this domain is a dynamic random coil on the membrane surface. Interestingly, the protein spectra are similar between a model membrane and a virus‐mimetic membrane, indicating that the structure and dynamics of the post‐AH segment is insensitive to the lipid composition. This chemical ligation approach is generally applicable to medium‐sized membrane proteins to provide site‐specific structural constraints, which complement the information obtained from uniformly ¹³C, ¹⁵N‐labeled proteins.     

NMR determination of protein partitioning into membrane domains with different curvatures and application to the influenza M2 peptide

The M2 protein of the influenza A virus acts both as a drug-sensitive proton channel and mediates virus budding through membrane scission. The segment responsible for causing membrane curvature is an amphipathic helix in the cytoplasmic domain of the protein. Here, we use ³¹P and ¹³C solid-state NMR to examine M2-induced membrane curvature. M2(22–46), which includes only the transmembrane (TM) helix, and M2(21–61), which contains an additional amphipathic helix, are studied. ³¹P chemical shift lineshapes indicate that M2(21–61) causes a high-curvature isotropic phase to both cholesterol-rich virus-mimetic membranes and 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers, whereas M2(22–46) has minimal effect. The lamellar and isotropic domains have distinct ³¹P isotropic chemical shifts, indicating perturbation of the lipid headgroup conformation by the amphipathic helix. ³¹P- and ¹³C-detected ¹H T₂ relaxation and two-dimensional peptide-lipid correlation spectra show that M2(21–61) preferentially binds to the high-curvature domain. ³¹P linewidths indicate that the isotropic vesicles induced by M2(21–61) are 10–35 nm in diameter, and the virus-mimetic vesicles are smaller than the 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles. A strong correlation is found between high membrane curvature and weak drug-binding ability of the TM helix. Thus, the M2 amphipathic helix causes membrane curvature, which in turn perturbs the TM helix conformation, abolishing drug binding. These NMR experiments are applicable to other curvature-inducing membrane proteins such as fusion proteins and antimicrobial peptides.     

Clustering of tetrameric influenza M2 peptides in lipid bilayers investigated by 19F solid-state NMR

The influenza M2 protein forms a drug-targeted tetrameric proton channel to mediate virus uncoating, and carries out membrane scission to enable virus release. While the proton channel function of M2 has been extensively studied, the mechanism by which M2 catalyzes membrane scission is still not well understood. Previous fluorescence and electron microscopy studies indicated that M2 tetramers concentrate at the neck of the budding virus in the host plasma membrane. However, molecular evidence for this clustering is scarce. Here, we use ¹⁹F solid-state NMR to investigate M2 clustering in phospholipid bilayers. By mixing equimolar amounts of 4F-Phe47 labeled M2 peptide and CF₃-Phe47 labeled M2 peptide and measuring F-CF₃ cross peaks in 2D ¹⁹F¹⁹F correlation spectra, we show that M2 tetramers form nanometer-scale clusters in lipid bilayers. This clustering is stronger in cholesterol-containing membranes and phosphatidylethanolamine (PE) membranes than in cholesterol-free phosphatidylcholine and phosphatidylglycerol membranes. The observed correlation peaks indicate that Phe47 sidechains from different tetramers are less than ~2 nm apart. ¹H¹⁹F correlation peaks between lipid chain protons and fluorinated Phe47 indicate that Phe47 is more deeply inserted into the lipid bilayer in the presence of cholesterol than in its absence, suggesting that Phe47 preferentially interacts with cholesterol. Static ³¹P NMR spectra indicate that M2 induces negative Gaussian curvature in the PE membrane. These results suggest that M2 tetramers cluster at cholesterol- and PE-rich regions of cell membranes to cause membrane curvature, which in turn can facilitate membrane scission in the last step of virus budding and release.     

Global fold of human cannabinoid type 2 receptor probed by solid‐state 13C‐, 15N‐MAS NMR and molecular dynamics simulations

The global fold of human cannabinoid type 2 (CB₂) receptor in the agonist‐bound active state in lipid bilayers was investigated by solid‐state ¹³C‐ and ¹⁵N magic‐angle spinning (MAS) NMR, in combination with chemical‐shift prediction from a structural model of the receptor obtained by microsecond‐long molecular dynamics (MD) simulations. Uniformly ¹³C‐ and ¹⁵N‐labeled CB₂ receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. ¹³C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of C_α, C_β, and C?O bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the ¹³C chemical shift distribution of C_α resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB₂. Thus the shape of the C_α band can be used as an indicator of CB₂ global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥1.5 ppm for carbons and ≥5.0 ppm for nitrogens). Simulated two‐dimensional ¹³C_α(i)? ¹³C?O(i) and ¹³C?O(i)? ¹⁵NH(i + 1) dipolar‐interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB₂ by solid‐state MAS NMR. Proteins 2014; 82:452–465. © 2013 Wiley Periodicals, Inc.     

X‐ray crystal structure of the N‐terminal region of Moloney murine leukemia virus integrase and its implications for viral DNA recognition

The retroviral integrase (IN) carries out the integration of a dsDNA copy of the viral genome into the host DNA, an essential step for viral replication. All IN proteins have three general domains, the N‐terminal domain (NTD), the catalytic core domain, and the C‐terminal domain. The NTD includes an HHCC zinc finger‐like motif, which is conserved in all retroviral IN proteins. Two crystal structures of Moloney murine leukemia virus (M‐MuLV) IN N‐terminal region (NTR) constructs that both include an N‐terminal extension domain (NED, residues 1–44) and an HHCC zinc‐finger NTD (residues 45–105), in two crystal forms are reported. The structures of IN NTR constructs encoding residues 1–105 (NTR_1–105) and 8–105 (NTR_8–105) were determined at 2.7 and 2.15 Å resolution, respectively and belong to different space groups. While both crystal forms have similar protomer structures, NTR_1–105 packs as a dimer and NTR_8–105 packs as a tetramer in the asymmetric unit. The structure of the NED consists of three anti‐parallel β‐strands and an α‐helix, similar to the NED of prototype foamy virus (PFV) IN. These three β‐strands form an extended β‐sheet with another β‐strand in the HHCC Zn²⁺ binding domain, which is a unique structural feature for the M‐MuLV IN. The HHCC Zn²⁺ binding domain structure is similar to that in HIV and PFV INs, with variations within the loop regions. Differences between the PFV and MLV IN NEDs localize at regions identified to interact with the PFV LTR and are compared with established biochemical and virological data for M‐MuLV. Proteins 2017; 85:647–656. © 2016 Wiley Periodicals, Inc.     

C‐terminal juxtamembrane region of full‐length M2 protein forms a membrane surface associated amphipathic helix

The influenza A M2 protein is a 97‐residue integral membrane protein involved in viral budding and proton conductance. Although crystal and NMR structures exist of truncated constructs of the protein, there is disagreement between models and only limited structural data are available for the full‐length protein. Here, the structure of the C‐terminal juxtamembrane region (sites 50–60) is investigated in the full‐length M2 protein using site‐directed spin‐labeling electron paramagnetic resonance (EPR) spectroscopy in lipid bilayers. Sites 50–60 were chosen for study because this region has been shown to be critical to the role the M2 protein plays in viral budding. Continuous wave EPR spectra and power saturation data in the presence of paramagnetic membrane soluble oxygen are consistent with a membrane surface associated amphipathic helix. Comparison between data from the C‐terminal juxtamembrane region in full‐length M2 protein with data from a truncated M2 construct demonstrates that the line shapes and oxygen accessibilities are remarkably similar between the full‐length and truncated form of the protein.     

Solid‐state NMR and simulation studies of equinatoxin II N‐terminus interaction with lipid bilayers

The interaction with model membranes of a peptide, EqtII_1–32, corresponding to the N‐terminal region of the pore‐forming toxin equinatoxin II (EqtII) has been studied using solid‐state NMR and molecular dynamics (MD) simulations. The distances between specifically labeled nuclei in [¹⁹F‐para]Phe16‐[1‐¹³C]Leu19 and [¹⁹F‐para]Phe16‐[¹⁵N]Leu23 analogs of EqtII_1–32 measured by REDOR in lyophilized peptide were in agreement with published crystal and solution structures. However, in both DMPC and mixed DMPC:SM membrane environments, significant changes in the distances between the labeled amino acid pairs were observed, suggesting changes in helical content around the experimentally studied region, 16–23, in the presence of bilayers. ¹⁹F‐³¹P REDOR experiments indicated that the aromatic ring of Phe16 is in contact with lipid headgroups in both membrane environments. For the DMPC:SM mixed bilayers, a closer interaction between Phe16 side chains and lipid headgroups was observed, but an increase in distances was observed for both labeled amino acid pairs compared with those measured for EqtII_1–32 in pure DMPC bilayers. The observed differences between DMPC and DMPC:SM bilayers may be due to the greater affinity of EqtII for the latter. MD simulations of EqtII_1–32 in water, on a pure DMPC bilayer, and on a mixed DMPC:SM bilayer indicate significant peptide secondary structural differences in the different environments, with the DMPC‐bound peptide adopting helical formations at residues 16–24, whereas the DMPC:SM‐bound peptide exhibits a longer helical stretch, which may contribute to its enhanced activity against PC:SM compared with pure PC bilayers. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Expression and initial structural insights from solid-state NMR of the M2 proton channel from influenza A virus

The M2 protein from influenza A virus has been expressed, purified, and reconstituted into DMPC/DMPG liposomes. SDS-PAGE analysis of reconstituted M2 protein in DMPC/DMPG liposomes demonstrates a stable tetrameric preparation. Circular dichroism spectra of the intact M2 protein in detergent indicate 67% alpha-helix. The uniformly (15)N-labeled M2 protein and both (15)N-Val- and (15)N-Leu-labeled M2 protein have been expressed from defined M9 media. The (1)H-(15)N HSQC (heteronuclear single quantum correlation) solution NMR experiments have been performed on the amino acid specific labeled protein in 300 mM SDS-d(25) micelles, and the data indicate a homogeneous preparation. The reconstituted M2/DMPC/DMPG proteoliposomes were used for preparing uniformly aligned solid-state NMR samples for (15)N-(1)H dipolar/(15)N chemical shift correlation experiments. The spectra support a transmembrane helix in M2 protein having a tilt angle of approximate 25 degrees, quantitatively similar to results obtained on the isolated M2 transmembrane peptide reconstituted in DMPC bilayers (38 degrees ). In addition, the spectra suggest that the tetrameric protein forms a symmetric or at least pseudosymmetric bundle consistent with data obtained by other research groups based on electrophysiological measurements and substituted cysteine scanning mutagenesis experiments that characterize a tetrameric structure.     

Structural origins of pH‐dependent chemical shifts in the B1 domain of protein G

We report chemical shifts for H^N, N, and C′ nuclei in the His‐tagged B1 domain of protein G (GB1) over a range of pH values from pH 2.0 to 9.0, which fit well to standard pH‐dependent equations. We also report a 1.2 Å resolution crystal structure of GB1 at pH 3.0. Comparison of this crystal structure with published crystal structures at higher pHs provides details of the structural changes in GB1 associated with protonation of the carboxylate groups, in particular a conformational change in the C‐terminus of the protein at low pH. An additional change described recently is not seen in the crystal structure because of crystal contacts. We show that the pH‐dependent changes in chemical shifts can be almost entirely understood based on structural changes, thereby providing insight into the relationship between structure and chemical shift. In particular, we describe through‐bond effects extending up to five bonds, affecting N and C′ but not H^N; through‐space effects of carboxylates, which fit well to a simple electric field model; and effects due to conformational change, which have a similar magnitude to many of the direct effects. Finally, we discuss cooperative effects, demonstrating a lack of cooperative unfolding in the helix, and the existence of a β‐sheet “iceberg” extending over three of the four strands. This study therefore extends the application of chemical shifts to understanding protein structure. Proteins 2010; © 2010 Wiley‐Liss, Inc.     

The flexible C‐terminal arm of the Lassa arenavirus Z‐protein mediates interactions with multiple binding partners

The arenavirus genome encodes for a Z‐protein, which contains a RING domain that coordinates two zinc ions, and has been identified as having several functional roles at various stages of the virus life cycle. Z‐protein binds to multiple host proteins and has been directly implicated in the promotion of viral budding, repression of mRNA translation, and apoptosis of infected cells. Using homology models of the Z‐protein from Lassa strain arenavirus, replica exchange molecular dynamics (MD) was used to refine the structures, which were then subsequently clustered. Population‐weighted ensembles of low‐energy cluster representatives were predicted based upon optimal agreement of the chemical shifts computed with the SPARTA program with the experimental NMR chemical shifts. A member of the refined ensemble was indentified to be a potential binder of budding factor Tsg101 based on its correspondence to the structure of the HIV‐1 Gag late domain when bound to Tsg101. Members of these ensembles were docked against the crystal structure of human eIF4E translation initiation factor. Two plausible binding modes emerged based upon their agreement with experimental observation, favorable interaction energies and stability during MD trajectories. Mutations to Z are proposed that would either inhibit both binding mechanisms or selectively inhibit only one mode. The C‐terminal domain conformation of the most populated member of the representative ensemble shielded protein‐binding recognition motifs for Tsg101 and eIF4E and represents the most populated state free in solution. We propose that C‐terminal flexibility is key for mediating the different functional states of the Z‐protein. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Molecules‐in‐molecules fragment‐based method for the calculation of chiroptical spectra of large molecules: Vibrational circular dichroism and Raman optical activity spectra of alanine polypeptides

The molecules‐in‐molecules (MIM) fragment‐based method has recently been adapted to evaluate the chiroptical (vibrational circular dichroism [VCD] and Raman optical activity [ROA]) spectra of large molecules such as peptides. In the MIM‐VCD and MIM‐ROA methods, the relevant higher energy derivatives of the parent molecule are assembled from the corresponding derivatives of smaller fragment subsystems. In addition, the missing long‐range interfragment interactions are accounted at a computationally less expensive level of theory (MIM2). In this work we employed the MIM‐VCD and MIM‐ROA fragment‐based methods to explore the evolution of the chiroptical spectroscopic characteristics of 3₁₀‐helix, α‐helix, β‐hairpin, γ‐turn, and β‐extended conformers of gas phase polyalanine (chain length n = 6–14). The different conformers of polyalanine show distinctive features in the MIM chiroptical spectra and the associated spectral intensities increase with evolution of system size. For a better understanding the site‐specific effects on the vibrational spectra, isotopic substitutions were also performed employing the MIM method. An increasing redshift with the number of isotopically labeled ¹³C=O functional groups in the peptide molecule was seen. For larger polypeptides, we implemented the two‐step‐MIM model to circumvent the high computational expense associated with the evaluation of chiroptical spectra at a high level of theory using large basis sets. The chiroptical spectra of α‐(alanine)₂₀ polypeptide obtained using the two‐step‐MIM model, including continuum solvation effects, show good agreement with the full calculations and experiment. This benchmark study suggests that the MIM‐fragment approach can assist in predicting and interpreting chiroptical spectra of large polypeptides.     

Helix‐sheet packing in proteins

The three‐dimensional structure of a protein is organized around the packing of its secondary structure elements. Although much is known about the packing geometry observed between α‐helices and between β‐sheets, there has been little progress on characterizing helix–sheet interactions. We present an analysis of the conformation of αβ₂ motifs in proteins, corresponding to all occurrences of helices in contact with two strands that are hydrogen bonded. The geometry of the αβ₂ motif is characterized by the azimuthal angle θ between the helix axis and an average vector representing the two strands, the elevation angle ψ between the helix axis and the plane containing the two strands, and the distance D between the helix and the strands. We observe that the helix tends to align to the two strands, with a preference for an antiparallel orientation if the two strands are parallel; this preference is diminished for other topologies of the β‐sheet. Side‐chain packing at the interface between the helix and the strands is mostly hydrophobic, with a preference for aliphatic amino acids in the strand and aromatic amino acids in the helix. From the knowledge of the geometry and amino acid propensities of αβ₂ motifs in proteins, we have derived different statistical potentials that are shown to be efficient in picking native‐like conformations among a set of non‐native conformations in well‐known decoy datasets. The information on the geometry of αβ₂ motifs as well as the related statistical potentials have applications in the field of protein structure prediction. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Design of high‐affinity S100‐target hybrid proteins

S100B and S100A10 are dimeric, EF‐hand proteins. S100B undergoes a calcium‐dependant conformational change allowing it to interact with a short contiguous sequence from the actin‐capping protein CapZ (TRTK12). S100A10 does not bind calcium but is able to recruit the N‐terminus of annexin A2 important for membrane fusion events, and to form larger multiprotein complexes such as that with the cation channel proteins TRPV5/6. In this work, we have designed, expressed, purified, and characterized two S100‐target peptide hybrid proteins comprised of S100A10 and S100B linked in tandem to annexin A2 (residues 1–15) and CapZ (TRTK12), respectively. Different protease cleavage sites (tobacco etch virus, PreScission) were incorporated into the linkers of the hybrid proteins. In situ proteolytic cleavage monitored by ¹H‐¹⁵N HSQC spectra showed the linker did not perturb the structures of the S100A10‐annexin A2 or S100B‐TRTK12 complexes. Furthermore, the analysis of the chemical shift assignments (¹H, ¹⁵N, and ¹³C) showed that residues T102‐S108 of annexin A2 formed a well‐defined α‐helix in the S100A10 hybrid while the TRTK12 region was unstructured at the N‐terminus with a single turn of α‐helix from D108‐K111 in the S100B hybrid protein. The two S100 hybrid proteins provide a simple yet extremely efficient method for obtaining high yields of intact S100 target peptides. Since cleavage of the S100 hybrid protein is not necessary for structural characterization, this approach may be useful as a scaffold for larger S100 complexes.     

Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization‐dependent regulation could occur downstream of the membrane‐bound kinase in RTK‐mediated signaling pathways. Extrapolation to the full‐length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine‐phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated‐tyrosine‐mimic Y492E‐FL‐Grb7 protein (Y492E‐FL‐Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild‐type FL Grb7(WT‐FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11μM. Our studies here measure a FL protein dimerization K_d of WT‐FL‐Grb7 within one order of magnitude at approximately 1μM. The approximate size and shape of the WT‐FL‐Grb7 in comparison the tyrosine‐phosphorylation mimic Y492E‐FL‐Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.     

The presence of non‐native helical structure in the unfolding of a beta‐sheet protein MPT63

MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a β‐sandwich protein containing 11 β strands and a very short stretch of 3₁₀ helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete β‐sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV‐CD and FTIR spectra clearly suggest that the low‐pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete β‐sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non‐native helical propensity of the protein structure.     

Slow formation of aggregation‐resistant β‐sheet folding intermediates

Protein folding has been studied extensively for decades, yet our ability to predict how proteins reach their native state from a mechanistic perspective is still rudimentary at best, limiting our understanding of folding‐related processes in vivo and our ability to manipulate proteins in vitro. Here, we investigate the in vitro refolding mechanism of a large β‐helix protein, pertactin, which has an extended, elongated shape. At 55 kDa, this single domain, all‐β‐sheet protein allows detailed analysis of the formation of β‐sheet structure in larger proteins. Using a combination of fluorescence and far‐UV circular dichroism spectroscopy, we show that the pertactin β‐helix refolds remarkably slowly, with multiexponential kinetics. Surprisingly, despite the slow refolding rates, large size, and β‐sheet‐rich topology, pertactin refolding is reversible and not complicated by off‐pathway aggregation. The slow pertactin refolding rate is not limited by proline isomerization, and 30% of secondary structure formation occurs within the rate‐limiting step. Furthermore, site‐specific labeling experiments indicate that the β‐helix refolds in a multistep but concerted process involving the entire protein, rather than via initial formation of the stable core substructure observed in equilibrium titrations. Hence pertactin provides a valuable system for studying the refolding properties of larger, β‐sheet‐rich proteins, and raises intriguing questions regarding the prevention of aggregation during the prolonged population of partially folded, β‐sheet‐rich refolding intermediates. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

De novo structure generation using chemical shifts for proteins with high‐sequence identity but different folds

Proteins with high‐sequence identity but very different folds present a special challenge to sequence‐based protein structure prediction methods. In particular, a 56‐residue three‐helical bundle protein (GA⁹⁵) and an α/β‐fold protein (GB⁹⁵), which share 95% sequence identity, were targets in the CASP‐8 structure prediction contest. With only 12 out of 300 submitted server‐CASP8 models for GA⁹⁵ exhibiting the correct fold, this protein proved particularly challenging despite its small size. Here, we demonstrate that the information contained in NMR chemical shifts can readily be exploited by the CS‐Rosetta structure prediction program and yields adequate convergence, even when input chemical shifts are limited to just amide ¹H^N and ¹⁵N or ¹H^N and ¹H^α values.     

Synthesis and conformational analysis of salivary proline‐rich peptide P‐B

The 57‐amino acid human salivary polypeptide P‐B has been synthesized by the solid‐phase method using 9‐fluorenylmethoxycarbonyl (Fmoc) strategy. The circular dichroism (CD) spectroscopy, Fourier‐transform infrared spectroscopy (FTIR) and molecular modeling methods have been used for conformational studies of P‐B. Examination of the CD spectra of P‐B showed the content of the secondary structure to be independent of temperature over the range 0–60 °C at pH = 7 as well as over the pH range of 2–12 at 37 °C. P‐B adopts predominantly unordered structure with locally appearing β‐turns. The cumulative results obtained using the CD and FTIR spectroscopic techniques indicate the percentage of the polyproline type‐II (PPII) helix being as low as about 10%. Similarly, the molecular dynamics (MD) simulations reveal only a short PPII helix in the C‐terminal fragment of the peptide (Pro⁵¹–Pro⁵⁴), which constitutes 7%. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

The effect of calcium and magnesium on the interaction between β‐lactoglobulin and carotenoids from sea buckthorn berries

β‐Lactoglobulin has been shown to interact with carotenoids from sea buckthorn berries. However, previously, no studies have taken into account the effect of calcium and magnesium on the β‐lactoglobulin–carotenoids complex. This study aims to determine the effect of calcium and magnesium on the interaction between β‐lactoglobulin and carotenoids from sea buckthorn berries extract, during heating from the perspective of deepening interaction mechanisms as prerequisites for micro‐ and nanoencapsulation. Phase diagram, intrinsic fluorescence spectra, quenching experiments and synchronous spectra were employed to acquire information regarding the conformation of protein in the presence of calcium chloride and magnesium chloride. Intrinsic fluorescence data showed that, between 25°C and 60°C, the presence of calcium chloride in the complex favoured the movement of tryptophan residues to domains located at the protein–water interface, while magnesium chloride favoured the burial of tryptophan residues. Higher temperatures generated blue shifts regardless of which salt was present, suggesting exposure of tryptophan residues to the hydrophobic core of the protein. Extrinsic fluorescence intensity of the non‐heat‐treated complex with magnesium chloride was significantly higher (P < 0.01) than of the complex with calcium chloride, suggesting that 1‐anilino‐8‐naphtalenesulphonic acid was bound to a higher proportion of the β‐lactoglobulin–carotenoids complex. Calcium chloride increased extrinsic fluorescence to a greater extent than magnesium chloride at temperatures above 70°C and was related to small structural changes induced by preheating β‐lactoglobulin.     

Synthetic peptide TPLVTLFK (octarphin) reduces the corticosterone production by rat adrenal cortex through nonopioid β‐endorphin receptor

The synthetic peptide octarphin (TPLVTLFK) corresponding to the sequence 12–19 of β‐endorphin, a selective agonist of nonopioid β‐endorphin receptor, was labeled with tritium to a specific activity of 29 Ci/mmol. [³H]Octarphin was found to bind to high‐affinity naloxone‐insensitive binding sites on membranes isolated from rat adrenal cortex (K_d = 35.7 ± 2.3 nM, B_max = 41.0 ± 3.6 pmol/mg protein). The binding specificity study revealed that these binding sites were insensitive not only to naloxone but to α‐endorphin, γ‐endorphin, [Met⁵]enkephalin, and [Leu⁵]enkephalin as well. At the same time, the [³H]octarphin‐specific binding with adrenal cortex membranes was inhibited by unlabeled β‐endorphin (K_i = 32.9 ± 3.8 nM). Octarphin at concentrations of 10^?9–10^?6 M was found to inhibit the adenylate cyclase activity in adrenocortical membranes, whereas intranasal injection of octarphin at doses of 5 and 20 µg/rat was found to reduce the secretion of corticosterone from the adrenals to the bloodstream. Thus, octarphin decreases the adrenal cortex functional activity through the high affinity binding to nonopioid receptor of β‐endorphin. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.