Folding kinetics of staphylococcal nuclease studied by tryptophan engineering and rapid mixing methods

To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side-chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophan residues in an apolar environment. The kinetics of folding was observed by continuous and stopped-flow fluorescence measurements over refolding times ranging from 100 micros to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophan residues in the beta-barrel and the N-terminal part of the alpha-helical domain become partially shielded from the solvent at an early stage (<1 ms), indicating that this region undergoes a rapid collapse. For some variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophan residues are buried only during the late stages of folding. Other variants exhibit a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophan residues into close contact with intramolecular quenchers.     

High‐resolution structures of collagen‐like peptides [(Pro‐Pro‐Gly)4‐Xaa‐Yaa‐Gly‐(Pro‐Pro‐Gly)4]: Implications for triple‐helix hydration and Hyp(X) puckering

Structures of (Pro‐Pro‐Gly)₄‐Xaa‐Yaa‐Gly‐(Pro‐Pro‐Gly)₄ (ppg9‐XYG) where (Xaa, Yaa) = (Pro, Hyp), (Hyp, Pro) or (Hyp, Hyp) were analyzed at high resolution using synchrotron radiation. Molecular and crystal structures of these peptides are very similar to those of the (Pro‐Pro‐Gly)₉ peptide. The results obtained in this study, together with those obtained from related compounds, indicated the puckering propensity of the Hyp in the X position: (1) Hyp(X) residues involved in the Hyp(X):Pro(Y) stacking pairs prefer the down‐puckering conformation, as in ppg9‐OPG, and ppg9‐OOG; (2) Hyp(X) residues involved in the Hyp(X):Hyp(Y) stacking pairs prefer the up‐puckering conformation if there is no specific reason to adopt the down‐puckering conformation. Water molecules in these peptide crystals are classified into two groups, the 1st and 2nd hydration waters. Water molecules in the 1st hydration group have direct hydrogen bonds with peptide oxygen atoms, whereas those in the 2nd hydration group do not. Compared with globular proteins, the number of water molecules in the 2nd hydration shell of the ppg9‐XYG peptides is very large, likely due to the unique rod‐like molecular structure of collagen model peptides. In the collagen helix, the amino acid residues in the X and Y positions must protrude outside of the triple helix, which forces even the hydrophobic side chains, such as Pro, to be exposed to the surrounding water molecules. Therefore, most of the waters in the 2nd hydration shell are covering hydrophobic Pro side chains by forming clathrate structures. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 361–372, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Structural analysis of linear hydroxyproline-bound O-glycans of Chlamydomonas reinhardtii--conservation of the inner core in Chlamydomonas and land plants

Linear hydroxyproline-bound O-glycans of the unicellular green alga Chlamydomonas reinhardtii were isolated from outer cell wall glycoproteins and their structure elucidated by chemical and spectroscopic methods. They consist exclusively of arabinose and galactose, the latter in the furanose form, unusual for plants. The first two arabinoses linked to hydroxyproline have the same anomeric configuration and linkage to each other as do the arabinosides isolated from land plants, suggesting that these two steps of hydroxyproline-O-glycosylation have been conserved during evolution. In a concomitant analysis of Hyp-bound O-tri- and tetraarabinans isolated from Arabidopsis thaliana, we were able to unambiguously identify the natural trans-4-l-Hyp-bound O-glycans and the artificially produced d-allo forms and to identify diagnostic signals in NMR spectra of these compounds for the first time.     

Fluorescence and 13C NMR determination of side-chain and backbone dynamics of synthetic melittin and melittin analogues in isotropic solvents

The dynamics in isotopic solvents of selectively 13C labeled synthetic melittin and three analogues have been investigated by using NMR and fluorescence techniques both separately and in combination. In conjunction with the "model-free" approach to interpretation of NMR relaxation data [Lipari, G., & Szabo, A. (1982) J. Am. Chem. Soc. 104, 4546-4570], the availability of steady-state fluorescence anisotropy and lifetime data augment T1, T2, and NOE data to provide quantitative information about fluorophore dynamics in these peptides. A method is presented for using combined fluorescence and NMR data to obtain technique- and model-independent values for parameters describing local motion of 13C-labeled fluorophores in peptides and proteins. The dynamics of melittin and melittin analogues are found to be consistent with structural characteristics inferred from CD, fluorescence, and NMR spectral information presented in the preceding paper (Weaver et al., 1989). In particular, the mobility of the random coil peptide monomers is shown to be quite similar, while side-chain as well as peptide backbone motion in the aggregated or oligomeric species differs markedly among the analogues. For melittin itself, experimentally determined overall rotational correlation times for the monomer and tetramer agree very well with values predicted on the basis of solvent-accessible protein surface area. The local dynamics of selectively 13C-labeled Trp-19 and Gly-12 residues of melittin are also found to be consistent with peptide structure. In random coil melittin monomer, a specific model for the motion indicates that the Trp side chain moves through an approximate angle of +/- 71 degrees about the beta-gamma bond with a correlation time of 159 +/- 24 ps. In melittin tetramer, the indole moiety is spatially more confined with a flip angle of +/- 37 degrees, yet demonstrates an increased rate of motion with a correlation time of 56 +/- 8 ps. The constrained mobility of the Trp-19 side chain is consistent with motional constraints inferred from the X-ray structure of melittin tetramer. These results show that protein side-chain motion, even of moieties as large as indole, can occur on the picosecond time scale and that these motions are reasonably similar to those inferred from molecular dynamics simulations.     

Structure and dynamics of LC8 complexes with KXTQT-motif peptides: swallow and dynein intermediate chain compete for a common site

The dynein light chain LC8 is an integral subunit of the cytoplasmic dynein motor complex that binds directly to and promotes assembly of the dynein intermediate chain (IC). LC8 interacts also with a variety of putative dynein cargo molecules such as Bim, a proapoptotic Bcl2 family protein, which have the KXTQT recognition sequence and neuronal nitric oxide synthase (nNOS), which has the GIQVD fingerprint but shares the same binding grooves at the LC8 dimer interface. The work reported here investigates the interaction of LC8 with IC and a putative cargo, Swallow, which share the KXTQT recognition sequence, and addresses the apparent paradox of how LC8, as part of dynein, mediates binding to cargo. The structures of Drosophila LC8 bound to peptides from IC and Swallow solved by X-ray diffraction show that the IC and Swallow peptides bind in the same grooves at the dimer interface. Differences in flexibility between bound and free LC8 were evaluated from hydrogen isotope exchange experiments using heteronuclear NMR spectroscopy. Peptide binding causes an increase in protection from exchange primarily in residues that interact directly with the peptide, such as the beta-strand intertwined at the interface and the N-terminal end of helix alpha2. There is considerably more protection upon Swallow binding, consistent with tighter binding relative to IC. Comparison with the LC8/nNOS complex shows how both the GIQVD and KXTQT fingerprints are recognized in the same groove. The similar structures of LC8/IC and LC8/Swa and the tighter binding of Swallow call into question the role for LC8 as a cargo adaptor protein, and suggest that binding of LC8 to Swallow serves another function, possibly that of a dimerization engine, which is independent of its role in dynein.     

Differential binding of ICln in platelets to integrin-derived activating and inhibitory peptides

The capacity of platelets to form a thrombus is mediated by integrin α_IIbβ₃. The cytoplasmic tail of α_IIb contains a highly conserved motif, ⁹⁸⁹KVGFFKR⁹⁹⁵, which plays a critical role in regulating integrin activation and acts as a recognition site for various intracellular proteins, e.g. CIB1, PP1, ICln and RN181. Previously, we demonstrated that a cell-permeable integrin-derived activating (IDA) peptide, KVGFFKR, induces platelet activation, whereas an integrin-derived inhibitory (IDI) peptide, KVGAAKR, is antithrombotic. To elucidate the molecular mechanism underlying these opposite effects we investigate the affinity of known integrin α_IIb binding proteins for the two immobilized peptides in dependence on the activation state of platelets by means of peptide-affinity chromatography, blotting techniques and protein:peptide docking studies.Our results provide a model for the inhibition of ICln interaction with the integrin in activated platelets by the IDI-peptide. Thus, ICln:IDI-peptide interaction profiles can have a pivotal purpose in the search for consensus pharmacophores specifically inhibiting ICln function in platelets potentially leading to the development of integrin-derived antithrombotic drugs.     

Protein in sugar films and in glycerol/water as examined by infrared spectroscopy and by the fluorescence and phosphorescence of tryptophan

Sugars are known to stabilize proteins. This study addresses questions of the nature of sugar and proteins incorporated in solid sugar films. Infrared (IR) and Raman spectroscopy was used to examine trehalose and sucrose films and glycerol/water solvent. Proteins and indole-containing compounds that are imbedded in the sugar films were studied by IR and optical (absorption, fluorescence, and phosphorescence) spectroscopy. Water is able to move in the sugar films in the temperature range of 20-300 K as suggested by IR absorption bands of HOH bending and OH stretching modes that shift continuously with temperature. In glycerol/water these bands reflect the glass transition at approximately 160 K. The fluorescence of N-acetyl-L-tryptophanamide and tryptophan of melittin, Ca-free parvalbumin, and staphylococcal nuclease in dry trehalose/sucrose films remains broad and red-shifted over a temperature excursion of 20-300 K. In contrast, the fluorescence of these compounds in glycerol/water solvent shift to the blue as temperature decreases. The fluorescence of the buried tryptophan in Ca-bound parvalbumin in either sugar film or glycerol/water remains blue-shifted and has vibronic resolution over the entire temperature range. The red shift for fluorescence of indole groups exposed to solvent in the sugars is consistent with the motion of water molecules around the excited-state molecule that occurs even at low temperature, although the possibility of static complex formation between the excited-state molecule and water or other factors is discussed. The phosphorescence yield for protein and model indole compounds is sensitive to the matrix glass transition. Phosphorescence emission spectra are resolved and shift little in different solvents or temperature, as predicted by the small dipole moment of the excited triplet state molecule. The conclusion is that the sugar film maintains the environment present at the glass formation temperature for surface Trp and amide groups over a wide temperature excursion. In glycerol/water these groups reflect local changes in the environment as temperature changes.     

Common interruptions in the repeating tripeptide sequence of non-fibrillar collagens: sequence analysis and structural studies on triple-helix peptide models

Interruptions in the repeating (Gly-X1-X2)_n amino acid sequence pattern are found in the triple-helix domains of all non-fibrillar collagens, and perturbations to the triple-helix at such sites are likely to play a role in collagen higher-order structure and function. This study defines the sequence features and structural consequences of the most common interruption, where one residue is missing from the tripeptide pattern, Gly-X1-X2-Gly-AA₁-Gly-X1-X2, designated G1G interruptions. Residues found within G1G interruptions are predominantly hydrophobic (70%), followed by a significant amount of charged residues (16%), and the Gly-X1-X2 triplets flanking the interruption are atypical. Studies on peptide models indicate the degree of destabilization is much greater when Pro is in the interruption, GP, than when hydrophobic residues (GF, GY) are present, and a rigid Gly-Pro-Hyp tripeptide adjacent to the interruption leads to greater destabilization than a flexible Gly-Ala-Ala sequence. Modeling based on NMR data indicates the Phe residue within a GF interruption is located on the outside of the triple helix. The G1G interruptions resemble a previously studied collagen interruption GPOGAAVMGPO, designated G4G-type, in that both are destabilizing, but allow continuation of rod-like triple helices and maintenance of the single residue stagger throughout the imperfection, with a loss of axial register of the superhelix on both sides. Both kinds of interruptions result in a highly localized perturbation in hydrogen bonding and dihedral angles, but the hydrophobic residue of a G4G interruption packs near the central axis of the superhelix, while the hydrophobic residue of a G1G interruption is located on the triple-helix surface. The different structural consequences of G1G and G4G interruptions in the repeating tripeptide sequence pattern suggest a physical basis for their differential susceptibility to matrix metalloproteinases in type X collagen.     

Coloured peptides: synthesis,properties and use in preparation of peptide sub-library kits

Several methods were developed for the solid-phase synthesis (SPPS) of coloured peptides and peptide libraries. At first a bifunctional red compound, 4-(4-(N-ethyl-N-(3-(tert-butyloxycarbonyl)aminopropyl)amino)phenylazo)benzoic acid (Boc-EPAB), was coupled with chloromethyl resin to obtain a new solid support suitable for SPPS using Boc chemistry. Peptides synthesized on this coloured resin had the chromophore at their C-termini. N-terminally coloured peptides were synthesized on a traditional solid support, coupled with chromophoric carboxylic acid before cleavage. A model pentapeptide, Phe-Ala-Val-Leu-Gly, and its ten derivatives were synthesized and their properties studied. It was found that the presence of chromophores decreases the water solubility of peptides. However, insertion of solubilizing tags (penta-lysine sequences or polyoxyethyl chains) into the molecule of any coloured derivative resulted in enhancement of the solubility. The RP-HPLC hydrophobicity indexes (φ₀) of the coloured peptides were also determined because φ₀ values are closely related to their water solubility. A coloured pentapeptide library was synthesized using the portioning-mixing method. Each component of this library contained the red azo dye (EPAB) and the penta-lysine tag. Before the last coupling step the samples were not mixed. All of the 19 sub-libraries obtained after cleavage were readily soluble in water, giving intense red solutions. The effect of chromophore (EPAB) and/or penta-lysine solubilizing tag on the biological activity was also studied. Potencies of the bovine neurotensin 8–13 fragment and its different coloured and penta-lysine derivatives were compared in isolated longitudinal muscle strips of guinea pig ileum. It was shown that the hexapeptide with penta-lysine tag had almost the same activity as the 8–13 fragment itself. The activity of the EPAB-derivative was found to be rather low. However, the presence of the solubilizing tag in the coloured hexapeptide compensated the negative effect of the chromophore. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Organization and dynamics of tryptophan residues in erythroid spectrin: novel structural features of denatured spectrin revealed by the wavelength-selective fluorescence approach

We have investigated the organization and dynamics of the functionally important tryptophan residues of erythroid spectrin in native and denatured conditions utilizing the wavelength-selective fluorescence approach. We observed a red edge excitation shift (REES) of 4 nm for the tryptophans in the case of spectrin in its native state. This indicates that tryptophans in spectrin are localized in a microenvironment of restricted mobility, and that the regions surrounding the spectrin tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited state tryptophans. Interestingly, spectrin exhibits a REES of 3 nm even when denatured in 8 M urea. This represents the first report of a denatured protein displaying REES. Observation of REES in the denatured state implies that some of the structural and dynamic features of this microenvironment around the spectrin tryptophans are retained even when the protein is denatured. Fluorescence quenching data of denatured spectrin support this conclusion. In addition, we have deduced the organization and dynamics of the hydrophobic binding site of the polarity-sensitive fluorescent probe PRODAN that binds erythroid spectrin with high affinity. When bound to spectrin, PRODAN exhibits a REES of 9 nm. Because PRODAN binds to a hydrophobic site in spectrin, such a result would directly imply that this region of spectrin offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state fluorophore. The results of our study could provide vital insight into the role of tryptophans in the stability and folding of spectrin.     

Phosphatidylcholine structure determines cholesterol solubility and lipid polymorphism

In the present work, we demonstrate that phosphatidylcholine with (16:1)9 acyl chains undergoes polymorphic rearrangements in mixtures with 0.6-0.8 mol fraction cholesterol. Studies were performed using differential scanning calorimetry, X-ray diffraction, cryo-electron microscopy, 31P NMR static powder patterns and 13C MAS/NMR. Mixtures of phosphatidylcholine with (16:1)9 acyl chains and 0.6 mol fraction cholesterol, after being heated to 100 degrees C, can form an ordered array with periodicity 14 nm which may be indicative of a cubic phase. Our results indicate that the formation of highly curved bilayer structures, such as those required for membrane fusion, can occur in mixtures of cholesterol with certain phosphatidylcholines that do not form non-lamellar structures in the absence of cholesterol. We also determine the polymorphic behavior of mixtures of symmetric phosphatidylcholines with cholesterol. Species of phosphatidylcholine with (20:1)11, (22:1)13 or (24:1)15 acyl chains in mixtures with 0.6-0.8 mol fraction cholesterol undergo a transition to the hexagonal phase at temperatures 70-80 degrees C. This is not the case for phosphatidylcholine with (18:1)6 acyl chains which remains in the lamellar phase up to 100 degrees C when mixed with as much as 0.8 mol fraction cholesterol. Thus, the polymorphic behavior of mixtures of phosphatidylcholine and cholesterol is not uncommon and is dependent on the intrinsic curvature of the phospholipid. Crystals of cholesterol can be detected in mixtures of all of these phosphatidylcholines at sufficiently high cholesterol mole fraction. What is unusual about the formation of these crystals in several cases is that cholesterol crystals are present in the monohydrate form in preference to the anhydrous form. Furthermore, after heating to 100 degrees C and recooling, the cholesterol crystals are again observed to be in the monohydrate form, although pure cholesterol crystals require many hours to rehydrate after being heated to 100 degrees C. Both the nature of the acyl chain as well as the mole fraction cholesterol determine whether cholesterol crystals in mixtures with the phospholipids will be in the monohydrate or in the anhydrous form.     

A rapid and direct method for the determination of active site accessibility in proteins based on ESI-MS and active site titrations

We have developed an electrospray ionisation mass spectrometry (ESI-MS) technique that can be applied to rapidly determine the number of intact active sites in proteins. The methodology relies on inhibiting the protein with an active-site irreversible inhibitor and then using ESI-MS to determine the extent of inhibition. We have applied this methodology to a test system: a serine protease, subtilisin Carlsberg, and monitored the extent of inhibition by phenylmethylsulfonyl fluoride (PMSF), an irreversible serine hydrolase inhibitor as a function of the changes in immobilisation and hydration conditions. Two types of enzyme preparation were investigated, lyophilised enzymes and protein-coated microcrystals (PCMC).     

Localization and characterization of neuropeptide Y-like peptides in the brain and islet organ of the anglerfish (Lophius americanus)

Summary Results from a previous report demonstrate that more than one molecular form of neuropeptide Y-like peptide may be present in the islet organ of the anglerfish (Lophius americanus). Most of the neuropeptide Y-like immunoreactive material was anglerfish peptide YG, which is expressed in a subset of islet cells, whereas an additional neuropeptide Y-like peptide(s) was localized in islet nerves. To learn more about the neuropeptide Y-like peptides in islet nerves, we have employed immunohistochemical and biochemical methods to compare peptides found in anglerfish islets and brain. Using antisera that selectively react with either mammalian forms of neuropeptide Y or with anglerfish peptide YG, subsets of neurons were found in the brain that labelled with only one or the other of the antisera. In separate sections, other neurons that were labelled with either antiserum exhibited similar morphologies. Peptides from brains and islets were subjected to gel filtration and reverse-phase high performance liquid chromatography. Radioimmunoassays employing either the neuropeptide Y or peptide YG antisera were used to examine chromatographic eluates. Immunoreactive peptides having retention times of human neuropeptide Y and porcine neuropeptide Y were identified in extracts of both brain and islets. This indicates that peptides structurally similar to both of these peptides from the neuropeptide Y-pancreatic polypeptide family are expressed in neurons of anglerfish brain and nerve fibers of anglerfish islets. The predominant form of neuropeptide Y-like peptide in islets was anglerfish peptide YG. Neuropeptide Y-immunoreactive peptides from islet extracts that had chromatographic retention times identical to human neuropeptide Y and porcine neuropeptide Y were present in much smaller quantities. These results are consistent with the hypothesis that peptides having significant sequence homology with human neuropeptide Y and porcine neuropeptide Y are present in the nerve fibers that permeate the islet.     

Accessibility and dynamics of nitroxide side chains in T4 lysozyme measured by saturation recovery EPR

Long pulse saturation recovery electron paramagnetic resonance spectroscopy is applied to the investigation of spin-labeled side chains placed along a regular helix extending from 128 to 135 in T4 lysozyme. Under an argon atmosphere, analysis of the exponential saturation recovery curves gives the spin-lattice relaxation rates of the nitroxides, which depend on the nitroxide side-chain dynamics. In the presence of the fast-relaxing paramagnetic reagents O(2) or NiEDDA, global analysis of the saturation recovery provides the spin-lattice relaxation rates as well as the Heisenberg exchange rates of the nitroxide with the reagents. As previously shown with power saturation methods, such exchange rates are direct measures of the solvent accessibility of the nitroxide side chains in the protein structure. The periodic dependence of the spin-lattice relaxation rates and the exchange rates along the 128-135 sequence reveal the presence of the helical structure, demonstrating the use of these parameters in structure determination. In general, multiple exponentials are required to fit the saturation recovery data, thus identifying multiple states of the side chain. In one case, multiple conformations detected in the spectrum are not evident in the saturation recovery, suggesting rapid exchange on the timescale of spin-lattice relaxation.     

Identification of the novel bioactive peptides dRYamide-1 and dRYamide-2, ligands for a neuropeptide Y-like receptor in Drosophila

A number of bioactive peptides are involved in regulating a wide range of animal behaviors, including food consumption. Vertebrate neuropeptide Y (NPY) is a potent stimulator of appetitive behavior. Recently, Drosophila neuropeptide F (dNPF) and short NPF (sNPF), the Drosophila homologs of the vertebrate NPY, were identified to characterize the functions of NPFs in the feeding behaviors of this insect. Dm-NPFR1 and NPFR76F are the receptors for dNPF and sNPF, respectively; both receptors are G protein-coupled receptors (GPCRs). Another GPCR (CG5811; NepYR) was indentified in Drosophila as a neuropeptide Y-like receptor. Here, we identified 2 ligands of CG5811, dRYamide-1 and dRYamide-2. Both peptides are derived from the same precursor (CG40733) and have no significant structural similarities to known bioactive peptides. The C-terminal sequence RYamide of dRYamides is identical to that of NPY family peptides; on the other hand, dNPF and sNPF have C-terminal RFamide. When administered to blowflies, dRYamide-1 suppressed feeding motivation. We propose that dRYamides are related to the NPY family in vertebrates, similar to dNPF and sNPF.     

Structure, dynamics and mapping of membrane-binding residues of micelle-bound antimicrobial peptides by natural abundance C NMR spectroscopy

Worldwide bacterial resistance to traditional antibiotics has drawn much research attention to naturally occurring antimicrobial peptides (AMPs) owing to their potential as alternative antimicrobials. Structural studies of AMPs are essential for an in-depth understanding of their activity, mechanism of action, and in guiding peptide design. Two-dimensional solution proton NMR spectroscopy has been the major tool. In this article, we describe the applications of natural abundance ¹³C NMR spectroscopy that provides complementary information to 2D ¹H NMR. The correlation of ¹³Cα secondary shifts with both 3D structure and heteronuclear ¹⁵N NOE values indicates that natural abundance carbon chemical shifts are useful probes for backbone structure and dynamics of membrane peptides. Using human LL-37-derived peptides (GF-17, KR-12, and RI-10), as well as amphibian antimicrobial and anticancer peptide aurein 1.2 and its analog LLAA, as models, we show that the cross peak intensity plots of 2D ¹H-¹³Cα HSQC spectra versus residue number present a wave-like pattern (HSQC wave) where key hydrophobic residues of micelle-bound peptides are located in the troughs with weaker intensities, probably due to fast exchange between the free and bound forms. In all the cases, the identification of aromatic phenylalanines as a key membrane-binding residue is consistent with previous intermolecular Phe-lipid NOE observations. Furthermore, mutation of one of the key hydrophobic residues of KR-12 to Ala significantly reduced the antibacterial activity of the peptide mutants. These results illustrate that natural abundance heteronuclear-correlated NMR spectroscopy can be utilized to probe backbone structure and dynamics, and perhaps to map key membrane-binding residues of peptides in complex with micelles. ¹H-¹³Cα HSQC wave, along with other NMR waves such as dipolar wave and chemical shift wave, offers novel insights into peptide-membrane interactions from different angles.     

Solid state NMR analysis of peptides in membranes: Influence of dynamics and labeling scheme

The functional state of a membrane-active peptide is often defined by its conformation, molecular orientation, and its oligomeric state in the lipid bilayer. These “static” structural properties can be routinely studied by solid state NMR using isotope-labeled peptides. In the highly dynamic environment of a liquid crystalline biomembrane, however, the whole-body fluctuations of a peptide are also of paramount importance, although difficult to address and most often ignored. Yet it turns out that disregarding such motional averaging in calculating the molecular alignment from orientational NMR-constraints may give a misleading, if not false picture of the system. Here, we demonstrate that the reliability of a simplified static or an advanced dynamic data analysis depends critically on the choice of isotope labeling scheme used. Two distinctly different scenarios have to be considered. When the labels are placed on the side chains of a helical peptide (such as a CD₃- or CF₃-group attached to the C^αC^β bond), their nuclear spin interaction tensors are very sensitive to motional averaging. If this effect is not properly accounted for, the helix tilt angle tends to be severely underestimated. At the same time, the analysis of labels in the side chains allows to extract valuable dynamical information about whole-body fluctuations of the peptide helix in the membrane. On the other hand, the alternative labeling scheme where ¹⁵N-labels are accommodated within the peptide backbone, will yield nearly correct helix tilt angles, irrespective as to whether dynamics are taken into account or not.     

Evenly tritium labeled peptides in study of peptide in vivo and in vitro biodegradation

Biologically active peptides evenly labeled with tritium were used for studying the in vitro and in vivo biodegradation of the peptides. Tritium-labeled peptides with a specific radioactivity of 50–150 Ci/mmol were obtained by high temperature solid phase catalytic isotope exchange (HSCIE) with spillover tritium. The distribution of the isotope label among all amino acid residues of these peptides allows the simultaneous determination of practically all possible products of their enzymatic hydrolysis. The developed analytical method includes extraction of tritium-labeled peptides from organism tissues and chromatographic isolation of individual labeled peptides from the mixture of degradation products. The concentrations of a peptide under study and the products of its biodegradation were calculated from the results of liquid scintillation counting. This approach was used for studying the pathways of biodegradation of the heptapeptide TKPRPGP (Selank) and the tripeptide PGP in blood plasma. The pharmacokinetics of Selank, an anxiolytic peptide, was also studied in brain tissues using the intranasal in vivo administration of this peptide. The concentrations of labeled peptides were determined, and the pentapeptide TKPRP, tripeptide TKP, and dipeptides RP and GP were shown to be the major products of Selank biodegradation. The study of the biodegradation of the heptapeptide MEHFPGP (Semax) in the presence of nerve cells showed that the major products of its biodegradation are the pentapeptide HFPGP and tripeptide PGP. The enkephalinase activity of blood plasma was studied with the use of evenly tritium labeled [Leu]enkephalin. A high inhibitory effect of Semax on blood plasma enkephalinases was shown to arise from its action on aminopeptidases. The method, based on the use of evenly tritium-labeled peptides, allows the determination of peptide concentrations and the activity of enzymes involved in their degradation on a μg scale of biological samples both in vitro and in vivo.     

Solid-state H and N NMR studies of side-chain and backbone dynamics of phospholamban in lipid bilayers: Investigation of the N27A mutation

Phospholamban (PLB) is an integral membrane protein regulating Ca²⁺ transport through inhibitory interaction with sarco(endo)plasmic reticulum calcium ATPase (SERCA). The Asn27 to Ala (N27A) mutation of PLB has been shown to function as a superinhibitor of the affinity of SERCA for Ca²⁺ and of cardiac contractility in vivo. The effects of this N27A mutation on the side-chain and backbone dynamics of PLB were investigated with ²H and ¹⁵N solid-state NMR spectroscopy in phospholipid multilamellar vesicles (MLVs). ²H and ¹⁵N NMR spectra indicate that the N27A mutation does not significantly change the side-chain or backbone dynamics of the transmembrane and cytoplasmic domains when compared to wild-type PLB. However, dynamic changes are observed for the hinge region, in which greater mobility is observed for the CD₃-labeled Ala24 N27A-PLB. The increased dynamics in the hinge region of PLB upon N27A mutation may allow the cytoplasmic helix to more easily interact with the Ca²⁺-ATPase; thus, showing increased inhibition of Ca²⁺-ATPase.