Investigations of pH-dependent dynamic properties of OmpG-16SL,an outer membrane protein G mutant by ATR-FTIR spectroscopy

In this paper, the dynamic properties of outer membrane protein G mutant (OmpG-16SL) are investigated with ATR-FTIR spectroscopy. While OmpG-WT has 14 β-strands in its structure, the mutant is designed to have 16 β-strands with the intention of creating an enlarged pore. Loop L6 is elongated by introducing six residues, two of which are negatively charged. The solvent accessibility of the OmpG-16SL mutant is compared with WT and a previously reported mutant OmpG-16S by tracking the ¹H/²H exchange kinetics in acidic and neutral buffer conditions. The exchange kinetics and dynamics in the fast and slow exchange phases are separately investigated using the 2DCOS technique, which enables the tracking of the structural changes at each phase of the exchange process. The results suggest that the mutant OmpG-16SL is equally exposed to buffer in both acidic and neutral pH conditions. Additionally, the time range in the fast phase is very short - one-tenth of that for WT - and most of the exchange is completed in this phase. This fast exchange within minutes is also indicative of the presence of highly flexible and/or unstructured regions. In all, the fast exchange rates independent of the buffer pH justify the assumption that there is an altered interaction among the charged residues, which leads to a steadily-open pore. The role of the side-chain interactions within the pore and between the loops involving the loop L6 is also discussed.     

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: III. The influence of heme removal

Two main types of conformational fluctuations, local and global, are characteristic of the native protein structure and are detectable by hydrogen exchange. The probability of such fluctuations changes to a different degree during hemoglobin (Hb) oxygenation, changes in pH, and splitting of the intersubunit contracts. For comparison with the effect of heme removal, the rate of the hydrogen-deuterium (H-D) exchange of peptide H atoms (PHs) of human apoHb was studied by IR spectroscopy at pH 5.5–9.0 and temperatures of 10–38°C. The removal of heme increased the H-D exchange rate for 80% of Hb PHs with the exchange retardation factor P ～ 10²-10⁸. For the majority of PHs, the probability of local fluctuations depended weakly on the temperature; changes in enthalpy upon such local conformational transitions were ΔH _op ^o = 0–15 kcal/M. Global fluctuations, displaying a stronger temperature dependence, did not arise with an increase in temperature to 38°C at pH 7.0, although apoHb began slowly denaturing and aggregating under these conditions. Destabilization of the apoHb structure with a concurrent decrease in pH to 5.5 and temperature to 10°C intensified global fluctuations in the native protein structure with ΔH _op ^o < 0. The mechanism underlying the overall intensification of local fluctuations upon the heme removal, the specific features of apoHb heat denaturation under conditions close to those of in vivo Hb self-assembly, and the analogies between low-temperature global fluctuations and cold denaturation of globular proteins are discussed.     

NMR Solution Structure and Biophysical Characterization of Vibrio harveyi Acyl Carrier Protein A75H: EFFECTS OF DIVALENT METAL IONS*

Bacterial acyl carrier protein (ACP) is a highly anionic, 9 kDa protein that functions as a cofactor protein in fatty acid biosynthesis. Escherichia coli ACP is folded at neutral pH and in the absence of divalent cations, while Vibrio harveyi ACP, which is very similar at 86% sequence identity, is unfolded under the same conditions. V. harveyi ACP adopts a folded conformation upon the addition of divalent cations such as Ca²⁺ and Mg²⁺ and a mutant, A75H, was previously identified that restores the folded conformation at pH 7 in the absence of divalent cations. In this study we sought to understand the unique folding behavior of V. harveyi ACP using NMR spectroscopy and biophysical methods. The NMR solution structure of V. harveyi ACP A75H displays the canonical ACP structure with four helices surrounding a hydrophobic core, with a narrow pocket closed off from the solvent to house the acyl chain. His-75, which is charged at neutral pH, participates in a stacking interaction with Tyr-71 in the far C-terminal end of helix IV. pH titrations and the electrostatic profile of ACP suggest that V. harveyi ACP is destabilized by anionic charge repulsion around helix II that can be partially neutralized by His-75 and is further reduced by divalent cation binding. This is supported by differential scanning calorimetry data which indicate that calcium binding further increases the melting temperature of V. harveyi ACP A75H by ∼20 °C. Divalent cation binding does not alter ACP dynamics on the ps-ns timescale as determined by ¹⁵N NMR relaxation experiments, however, it clearly stabilizes the protein fold as observed by hydrogen-deuterium exchange studies. Finally, we demonstrate that the E. coli ACP H75A mutant is similarly unfolded as wild-type V. harveyi ACP, further stressing the importance of this particular residue for proper protein folding.     

1H and 13C NMR spectroscopic studies of the ferriheme resonances of three low-spin complexes of wild-type nitrophorin 2 and nitrophorin 2(V24E) as a function of pH

The ferriheme resonances of the low-spin (S = 1/2) complexes of wild-type (wt) nitrophorin 2 (NP2) and its heme pocket mutant NP2(V24E) with imidazole (ImH), histamine (Hm), and cyanide (CN⁻) as the sixth ligand have been investigated by NMR spectroscopy as a function of pH (4.0–7.5). For the three wt NP2 complexes, the ratio of the two possible heme orientational isomers, A and B, remains almost unchanged (ratio of A:B approximately 1:6 to 1:5) over this wide pH range. However, strong chemical exchange cross peaks appear in the nuclear Overhauser effect spectroscopy/exchange spectroscopy (NOESY/EXSY) spectra for the heme methyl resonances at low pH (pH* 4.0–5.5), which indicate chemical exchange between two species. We have shown these to be two different exogenous ImH or Hm orientations that are denoted B and B′, with the ImH plane nearly parallel and perpendicular to the ImH plane of the protein-provided His57, respectively. The wt NP2–CN complex also shows EXSY cross peaks due to chemical exchange, which is shown to be a result of interchange between two ruffling distortions of the heme. The same ruffling distortion interchange is also responsible for the ImH and Hm chemical exchange. For the three NP2(V24E) ligand complexes, no EXSY cross peaks are observed, but the A:B ratios change dramatically with pH. The fact that heme favors the A orientation highly for NP2(V24E) at low pH as compared with wt NP2 is believed to be due to the steric effect of the V24E mutation. The existence of the B′ species at lower pH for wt NP2 complexes and the increase in A heme orientation at lower pH for NP2(V24E) are believed to be a result of a change in structure near Glu53 when it is protonated at low pH. ¹H{¹³C} heteronuclear multiple quantum coherence (HMQC) spectra are very helpful for the assignment of heme and nearby protein side chain resonances.     

Structure and dynamics of the M13 coat signal sequence in membranes by multidimensional high-resolution and solid-state NMR spectroscopy

The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a ¹⁵N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an α-helix content of approximately 42%. The α-helix comprises an uninterrupted hydrophobic stretch of ≤12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15–75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled ¹⁵N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of ¹⁵N solid-state NMR spectroscopy (10⁻⁴ seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed. © 1997 Wiley-Liss Inc.     

Infrared spectroscopic study of the structural and functional properties of the Na/H antiporter MjNhaP1 from Methanococcus jannaschii

In this study, structural, functional, and mechanistic properties of the Na⁺/H⁺ antiporter MjNhaP1 from Methanococcus jannaschii were analyzed by infrared spectroscopic techniques. Na⁺/H⁺ antiporters are generally responsible for the regulation of cytoplasmic pH and Na⁺ concentration. MjNhaP1 is active in the pH range between pH 6 and pH 6.5; below and above it is inactive.The secondary structure analysis on the basis of ATR-IR spectra provides the first insights into the structural changes between inactive (pH 8) and active (pH 6) state of MjNhaP1. It results in decreased ordered structural elements with increasing the pH-value i.e. with inactivation of the protein. Analysis of temperature-dependent FTIR spectra indicates that MjNhaP1 in the active state exhibits a much higher unfolding temperature in the spectral region assigned to α-helical segments. In contrast, the temperature-induced structural changes for β-sheet structure are similar for inactive and active state. Consequently, this structure element is not the part of the activation region of the protein. The surface accessibility of the protein was analyzed by following the extent of H/D exchange. Due to higher content of unordered structural elements a higher accessibility for amide protons is observed for the inactive as compared to the active state of MjNhaP1. Altogether, the results present the active state of MjNhaP1 as the state with ordered structural elements which exhibit high thermal stability and increased hydrophobicity.     

CcdB at pH 4 Forms a Partially Unfolded State with a Dry Core

pH is an important factor that affects the protein structure, stability, and activity. Here, we probe the nature of the low-pH structural form of the homodimeric CcdB (controller of cell death B) protein. Characterization of CcdB protein at pH 4 and 300 K using circular dichroism spectroscopy, 8-anilino-1-naphthalene-sulphonate binding, and Trp solvation studies suggests that it forms a partially unfolded state with a dry core at equilibrium under these conditions. CcdB remains dimeric at pH 4 as shown by multiple techniques, such as size-exclusion chromatography coupled to multiangle light scattering, analytical ultracentrifugation, and electron paramagnetic resonance. Comparative analysis using two-dimensional ¹⁵N-¹H heteronuclear single-quantum coherence NMR spectra of CcdB at pH 4 and 7 suggests that the pH 4 and native state have similar but nonidentical structures. Hydrogen-exchange-mass-spectrometry studies demonstrate that the pH 4 state has substantial but anisotropic changes in local stability with core regions close to the dimer interface showing lower protection but some other regions showing higher protection relative to pH 7.     

Enhanced Unfolding of Bovine β-Lactoglobulin Structure Using Microwave Treatment: a Multi-Spectroscopic Study

Commercial whey protein hydrolysates containing bovine β-lactoglobulin (β-Lg) may have residual allergenicity due to the inaccessibility of some sequential epitopes to proteases. Microwave may enhance unfolding pathways in protein structure due to its non-thermal effects. This research compared the effects of microwave heating (MW) and conventional heating (CH) on the unfolding in the secondary and tertiary structures of β-Lg over a temperature range of 40-90 °C using circular dichroism (CD), fluorescence spectroscopy, and two dimensional (2D) ¹H nuclear magnetic resonance (NMR) spectroscopy. Above 50 °C, β-sheet and α-helical secondary structures decreased during MW and CH, with a higher decrease being observed during MW. The near-UV spectra of MW β-Lg showed lower intensity suggesting higher tertiary structure loss than in CH β-Lg at all temperatures. The fluorescence spectra of MW β-Lg showed increased exposure of tryptophan residues to solvent as compared to CH β-Lg and suggested greater unfolding in tertiary structure in MW β-Lg at 60 °C than in CH β-Lg at 70 °C. 2D ¹H NMR spectra confirmed more extensive H-D exchange in MW β-Lg explained by the exposure of β-sheets (C, G, and H) at 50 °C under microwave treatment, which are thermally resistant to H-D exchange up to 75 °C during conventional heating. These results revealed a substantial enhancing effect of microwave treatment on the thermal unfolding and exposure of buried amide groups in β-Lg compared to conventional heating. Microwave processing could be a promising alternative to produce hydrolysates with lower allergenicity and improved bioactivity through structure modification.     

Interaction of monolayers of concanavalin A with mono- and polysaccharides A study by means of infrared-attenuated total reflectance spectroscopy

The effects of pH, Mn²⁺ and Ca²⁺ and urea denaturation on the interaction of monolayers of concanavalin A on saline with the polysaccharide dextran B-1355 and the monosaccharides methyl α-d-mannopyranoside and d-galactose have been investigated. Infrared absorption spectra of compressed monolayers of the protein and the protein-dextran complex coated on a germanium plate have been obtained by means of attenuated total reflectance spectroscopy. Except in one case of denaturation, the amide I absorption of concanavalin A peaked around 1631 cm^?1, indicating a predominance of the β-pleated sheet conformation, in agreement with its secondary structure in the solution and crystalline phases. The contribution to the absorbance of the concanavalin A-dextran films at 3300 cm^?1 due to absorption by the O-H stretching modes of the polysaccharide is a measure of its binding. Increasing the pH from 6.1 to 7.5 appreciably reduced the dextran binding, at pH 9.3 the binding was zero. Adding 1 mM Mn²⁺ and Ca²⁺ to the subphase at pH 7.5 restored both the dextran binding and the affinity of concanavalin A for methyl α-d-mannopyranoside to that of the native protein at pH 6.1. At this latter pH, the weak binding of dextran to monolayers of demetallized concanavalin A (apo-concanavalin A) was also restored to that for the native molecule by the addition of these divalents. This indicates the requirement of concanavalin A for these ions to maintain the integrity of the saccharide-binding site. The loss of dextran binding with urea denaturation was also observed. These results parallel those for solutions of the protein, indicating the validity of the monolayer system for the study of these interactions.     

A Source of Error in the Chromatographic Study of S-Sulfate Labeled Mucous Glycoproteins Secreted by the Gill Epithelium of Mytilus Epulis

HPLC combined with [³⁵S]-sulfate/[³H]-glucosamine radlolabellng were employed to study the synthesis and secretion of mucous glycoproteins. The secreted radiolabeled glycoproteins were separated from the medium by precipitation with a mixture of trichloroacetic-phosphotungstic acids (TCA/PTA). The redissolved glycoproteins were chromatographed on an anion exchange protein column at varying pH of the mobile phase and fractions were collected for liquid scintillation counting. Varying the pH of the mobile phase from pH 3 to 7 resulted in a decrease of glycoprotein bound [³⁵S] from 69.5 to 0.5% of the total recovered [³⁵S]-sulfate with the remainder recovered as free [³⁵S]-sulfate. The [³H]-labeled glycoprotein recovered under the uV peaks at this pH range was 99.5%. When high performance size exclusion chromatography was performed the change in mobile phase pH did not affect the 100% recovery of either [³⁵S]-or [³⁵H]-labels under the uV peaks. No free [³⁵S]-sulfate was obtained when [³⁵S]-labeled glycoproteins were separated from the medium using dialysis. These data suggest that the standard method of TCA/PTA precipitation of [³⁵S]-labeled glycoproteins may cleave the [³⁵S]-sulfate ester linkages to the oligosaccharide chains. The [³⁵S]-sulfate may then rebind to the macromolecule by a relatively strong noncovalent bond. This may prove critical in anion exchange protein HPLC studies.     

Saturation transfer electron paramagnetic resonance spectroscopy of spin labeled tobacco mosaic virus protein.

The coat protein of Tobacco Mosaic Virus is covalently labeled with a maleimide spin label at the single SH-group of the protein. Saturation transfer electron paramagnetic resonance spectroscopy, a technique that is sensitive to very slow molecular motion with rotational correlation times τ_c in the range 10^?7 to 10^?3 sec, shows the dissociation of large oligomers of spin labeled protein with τ_c～10^?4 sec at pH 5.5 to smaller oligomers at higher pH.     

Human Nucleotide Excision Repair Protein XPA: 1H NMR and CD Solution Studies of a Synthetic Peptide Fragment Corresponding to the Zinc-Binding Domain (101–141)

Abstract

A peptide corresponding to residues 101–141 of the human nucleotide excision repair protein XPA was synthesized with an isoleucine substituted for L138 and its solution structure studied by circular dichroism and homonuclear ¹H NMR spectroscopy. The peptide, (XPA-41), contains a C₄?type zinc-binding motif, C105-(X)₂C108-(X)_l7?C126-(X)₂ C129, which XPA requires for damaged-DNA binding activity. The proton resonances of XPA-41without zinc (apoXPA-41) were assigned using homonuclear TOCSY, NOESY and DQF-COSY data and show the apo-zinc peptide is a random coil. The peptide was folded with the addition of 1.2 equivalents of ZnCl₂ in dilute solution at pH 4.0. Electrospray ionization mass spectroscopy illustrated an increase in the molecular weight of XPA-41 by 65 amu. Circular dichroism spectra of the zinc-folded peptide (zXPA-41) showed the acquisition of elements of secondary structure. Such a conclusion was confirmed with'H NMR data collected at 25°C, pH 6.3. Hα-secondary shifts and NOE patterns indicate that regions V102-C105 and G109-F112 form an anti-parallel β-sheet and residues N128-K137 form a nascent α-helix. Rapid exchange of most amide resonances between S115-C126 prohibited unambiguous assignment of all the proton resonances in this region. However, a 1.19 ppm downfield shift of the H^α resonance of T125 relative to the apo-zinc peptide, together with downfield shifted H^α resonances for the adjacent residues (P124 and L123), suggest a second β-sheet is present in the S115-C126 region. On the basis of structural similarities to GATA-1 (Science 267:438–446), a homology generated structure for zXPA-41 was made, using GATA-1 as the template, which satisfied all the observed NOEs. Using the hybrid homology-NMR based zXPA-41 structure and analogy to GATA-1, models for the role played by the zinc-binding core (101–141) of XPA in DNA damage recognition are proposed.     

Resonance assignment and structural analysis of acid denatured E. coli [U-15N]-glutaredoxin 3

Virtually complete sequence specific ¹H and ¹⁵N resonance assignments are presented for acid denatured reduced E. coli glutaredoxin 3. The sequential resonance assignments of the backbone rely on the combined use of 3D F₁-decoupled ROESY-¹⁵N-HSQC and 3D ¹⁵N-HSQC-(TOCSY-NOESY)-¹⁵N-HSQC using a single uniformly ¹⁵N labelled protein sample. The sidechain resonances were assigned from a 3D TOCSY-¹⁵N-HSQC and a homonouclear TOCSY spectrum. The presented assignment strategy works in the absence of chemical exchange peaks with signals from the native conformation and without ¹³C/¹⁵N double labelling. Chemical shifts, ³J(H, NH) coupling constants and NOEs indicate extensive conformational averaging of both backbone and side chains in agreement with a random coil conformation. The only secondary structure element persisting at pH 3.5 appears to be a short helical segment comprising residues 37 to 40.Abbreviations HSQC heteronuclear single quantum coherence - NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - NOESY two-dimensional NOE spectroscopy - ROE nuclear Overhauser effect in the rotating frame - ROESY two-dimensional ROE spectroscopy - TOCSY total correlation spectroscopy - TPPI time proportional phase incrementation Correspondence to: G. Otting     

1H and15N resonance assignments and secondary structure of the human thioredoxin C62A,C69A,C73A mutant

Summary The complete assignment of¹H and¹⁵N backbone resonances and near-complete¹H side-chain resonance assignments have been obtained for the reduced form of a mutant of human thioredoxin (105 residues) in which the three non-active site cysteines have been substituted by alanines: C62A, C69A, C73A. The assignments were made primarily on the basis of three-dimensional.¹⁵N-separated nuclear Overhauser and Hartmann-Hahn spectroscopy, in conjunction with two-dimensional homonuclear and heteronuclear correlation experiments. Based on comparisons of short-range and interstrand nuclear Overhauser effects, patterns of amide exchange, and chemical-shift differences, the structure appears essentially unchanged from that of the previously determined solution structure of the native protein [Forman-Kay. J.D. et al. (1991)Biochemistry, 30, 2685–2698). An assay for thioredoxin shows that the C62A, C69A, C73A mutant retains activity. The assignment of the spectrum for this mutant of human thioredoxin constitutes the basis for future studies aimed at comparing the details of the active-site conformation in the reduced and oxidized forms of the protein.     

NMR spectroscopy of hydroxyl protons in aqueous solutions of peptides and proteins

Summary Hydroxyl groups of serine and threonine, and to some extent also tyrosine are usually located on or near the surface of proteins. NMR observations of the hydroxyl protons is therefore of interest to support investigations of the protein surface in solution, and knowledge of the hydroxyl NMR lines is indispensable as a reference for studies of protein hydration in solution. In this paper, solvent suppression schemes recently developed for observation of hydration water resonances were used to observe hydroxyl protons of serine, threonine and tyrosine in aqueous solutions of small model peptides and the protein basic pancreatic trypsin inhibitor (BPTI). The chemical shifts of the hydroxyl protons of serine and threonine were found to be between 5.4 and 6.2 ppm, with random-coil shifts at 4°C of 5.92 ppm and 5.88 ppm, respectively, and those of tyrosine between 9.6 and 10.1 ppm, with a random-coil shift of 9.78 ppm. Since these spectral regions are virtually free of other polypeptide¹H NMR signals, cross peaks with the hydroxyl protons are usually well separated even in homonuclear two-dimensional¹H NMR spectra. To illustrate the practical use of hydroxyl proton NMR in polypeptides, the conformations of the side-chain hydroxyl groups in BPTI were characterized by measurements of nuclear Overhauser effects and scalar coupling constants involving the hydroxyl protons. In addition, hydroxyl proton exchange rates were measured as a function of pH, where simple first-order rate processes were observed for both acid- and base-catalysed exchange of all but one of the hydroxyl-bearing residues in BPTI. For the conformations of the individual Ser, Thr and Tyr side chains characterized in the solution structure with the use of hydroxyl proton NMR, both exact coincidence and significant differences relative to the corresponding BPTI crystal structure data were observed.[/p]     

Rotational motion of cytochrome c oxidase in phospholipid vesicles

Natural abundance ¹³C and high field ¹H NMR spectroscopy are used to characterize the major coat protein of the filamentous bacteriophage fd in sodium dodecyl sulfate micelles. Chemical shift dispersion of protein resonances, slow and differential exchange rates of amide protons, and relaxation parameters of the alpha carbons of the protein indicate that the detergent solubilized coat protein has a stable native conformation. The structure of the coat protein in micelles differs from that found for typical globular proteins in solution in that parts of the peptide backbone exhibit rapid segmental motion.     

Complete 1H, 13C and 15N NMR assignments and secondary structure of the 269-residue serine protease PB92 from Bacillus alcalophilus

Summary The ¹H, ¹³C and ¹⁵N NMR resonances of serine protease PB92 have been assigned using 3D tripleresonance NMR techniques. With a molecular weight of 27 kDa (269 residues) this protein is one of the largest monomeric proteins assigned so far. The side-chain assignments were based mainly on 3D H(C)CH and 3D (H)CCH COSY and TOCSY experiments. The set of assignments encompasses all backbone carbonyl and CH_n carbons, all amide (NH and NH₂) nitrogens and 99.2% of the amide and CH_n protons. The secondary structure and general topology appear to be identical to those found in the crystal structure of serine protease PB92 [Van der Laan et al. (1992) Protein Eng., 5, 405–411], as judged by chemical shift deviations from random coil values, NH exchange data and analysis of NOEs between backbone NH groups.Abbreviations 2D/3D/4D two-/three-/four-dimensional - HSQC heteronuclear single-quantum coherence - HMQC heteronuclear multiple-quantum coherence - COSY correlation spectroscopy - TOCSY total correlation spectroscopy - NOE nuclear Overhauser enhancement (connectivity) - NOESY 2D NOE spectroscopy Experiment nomenclature (H(C)CH, etc.) follows the conventions used elsewhere [e.g. Ikura et al. (1990) Biochemistry, 29, 4659–4667].     

Use of the pH Memory Effect in Lyophilized Proteins to Achieve Preferential Methylation of α-Amino Groups

It is demonstrated that the pH memory effect can be used to control the ionization state of amino groups in lyophilized proteins and hence their chemical reactivity toward modifying reagents. When proteins were lyophilized from aqueous solutions at pH values between 6 and 7 and reacted in vacuo with iodomethane, the α-amino groups were found to be either preferentially or selectively trimethylated. Reaction with ¹³C-labeled iodomethane permitted detection and identification of individual trimethylated α-amino groups by ¹³C-NMR spectroscopy as distinct peaks in the spectral region between 52 and 57 ppm. There was adequate sensitivity to detect minor resonances of free α-amino groups arising from proteolysis of the major protein or from protein impurities. The resonances of the trimethylated α-amino groups in standard amino acids and peptides are sufficiently close to those in the derivatized protein to make a tentative identification of the N-terminal amino acid. It is also demonstrated that advantage can be taken of the pH memory effect to use the preferential ¹³C-methylation of amino groups to verify whether a protein has a free or blocked amino terminus.     

Interaction of γ-conglutin from Lupinus albus with model phospholipid membranes: Investigations on structure,thermal stability and oligomerization status

Interaction with model phospholipid membranes of lupin seed γ-conglutin, a glycaemia-lowering protein from Lupinus albus seeds, has been studied by means of Fourier-Transform infrared spectroscopy at p²H 7.0 and at p²H 4.5. The protein maintains the same secondary structure both at p²H 7.0 and at p²H 4.5, but at p²H 7.0 a higher ¹H/²H exchange was observed, indicating a greater solvent accessibility. The difference in T_m and T_D1/2 of the protein at the abovementioned p²H's has been calculated around 20 °C. Infrared measurements have been then performed in the presence of DMPG and DOPA at p²H 4.5. DMPG showed a little destabilizing effect while DOPA exerted a great stabilizing effect, increasing the T_m of γ-conglutin at p²H 4.5 of more than 20 °C. Since γ-conglutin at p²H 4.5 is in the monomeric form, the interaction with DOPA likely promotes the oligomerization even at p²H 4.5. Interaction between DMPG or DOPA and γ-conglutin has been confirmed by turbidity experiments with DMPC:DMPG or DOPC:DOPA SUVs. Turbidity data also showed high-affinity binding of γ-conglutin to anionic SUVs made up with DOPA. The molecular features outlined in this study are relevant to address the applicative exploitation and to delineate a deeper comprehension of the natural functional role of γ-conglutin.     

Structure of human salivary histatin 5 in aqueous and nonaqueous solutions

The solution structure of human salivary histatin 5 (D-S-H-A-K-R-H-H-G-Y-K-R-K-F-H-E-K-H-H-S-H-R-G-Y) was examined in water (pH 3.8) and dimethyl sulfoxide solutions using 500 MHz homo- and heteronuclear two-dimensional (2D) nmr. The resonance assignment of peptide backbone and side-chain protons was accomplished by 2D total correlated spectroscopy and nuclear Overhauser effect (NOE) spectroscopy. The high J values (≥7.4 Hz), absence of any characteristic NH-NH(i, i + 1) or C^αH-C^βH(i, i + 3) NOE connectivities, high dδ/dT values (≥0.004 ppm K⁻¹) and the fast ¹H/²H amide exchange suggest that histatin 5 molecules remain unstructured in aqueous solution at pH 3.8. In contrast, histatin 5 prefers largely α-helical conformation in dimethyl sulfoxide solution as evident from the J values (≤6.4 Hz), slow ¹H/²H exchange, low dδ/dT values (≤0.003 ppm K⁻¹) observed for amide resonances of residues 6–24, and the characteristic NH-NH(i, i + 1) and C^αH-C^βH(i, i +3) NOE connectivities. All backbone amide ¹⁵N-¹H connectivities fall within 6 ppm on the ¹⁵N scale in the 2D heteronuclear single quantum correlated spectrum, and the restrained structure calculations using DIANA suggest the prevalence of α-helical conformations stabilized by 19 (5 → 1) intramolecular backbone amide hydrogen bonds in polar aprotic medium such as dimethyl sulfoxide. The interside-chain hydrogen bonding and salt-bridge type interactions that normally stabilize the helical structure of linear peptides in aqueous solutions are not observed. Histatin 5, unlike other naturally occurring antimicrobial polypeptides such as magainins, defensins, and tachyplesins, does not adopt amphiphilic structure, precluding its insertion into microbial membranes and formation of ion channels across membranes. Electrostatic (ionic type) and hydrogen bonding interactions of the positively charged and polar residues with the head groups of microbial membranes or with a membrane-bound receptor could be the initial step involved in the mechanism of antimicrobial activity of histatins. © 1998 John Wiley & Sons, Inc. Biopoly 45: 51–67, 1998