Identification of rare partially unfolded states in equilibrium with the native conformation in an all beta-barrel protein

Human acidic fibroblast growth factor 1 (hFGF-1) is an all beta-barrel protein, and the secondary structural elements in the protein include 12 antiparallel beta-strands arranged into a beta-trefoil fold. In the present study, we investigate the stability of hFGF-1 by hydrogen-deuterium exchange as a function of urea concentration. Urea-induced equilibrium unfolding of hFGF-1 monitored by fluorescence and CD spectroscopy suggests that the protein unfolds by a two-state (native to denatured) mechanism. Hydrogen exchange in hFGF-1, under the experimental conditions used, occurs by the EX2 mechanism. In contrast to the equilibrium unfolding events monitored by optical probes, native state hydrogen exchange data show that the beta-trefoil architecture of hFGF-1 does not behave as a single cooperative unit. There are at least two structurally independent units with differing stabilities in hFGF-1. Beta-strands I, II, III, VI, VII, X, XI, and XII fit into the global unfolding isotherm. By contrast, residues in beta-strands IV, V, VIII, and IX exchange by the subfolding isotherm and could be responsible for the occurrence of high-energy partially unfolded state(s) in hFGF-1. There appears to be a broad continuum of stabilities among the four beta-strands (beta-strands IV, V, VIII, and IX) constituting the subglobal folding unit. The slow exchanging residues in hFGF-1 do not represent the folding nucleus of the protein.     

NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I—relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1)

Human fibroblast growth factor (hFGF-1) is a ∼ 17 kDa heparin binding cytokine. It lacks the conventional hydrophobic N-terminal signal sequence and is secreted through non-classical secretion routes. Under stress, hFGF-1 is released as a multiprotein complex consisting of hFGF-1, S100A13 (a calcium binding protein), and p40 synaptotagmin (Syt1). Copper (Cu²⁺) is shown to be required for the formation of the multiprotein hFGF-1 release complex (Landriscina et al. ,2001; Di Serio et al., 2008). Syt1, containing the lipid binding C2B domain, is believed to play an important role in the eventual export of the hFGF-1 across the lipid bilayer. In this study, we characterize Cu²⁺ and lipid interactions of the C2B domain of Syt1 using multidimensional NMR spectroscopy. The results highlight how Cu²⁺ appears to stabilize the protein bound to pS vesicles. Cu²⁺ and lipid binding interface mapped using 2D ¹H-¹⁵N heteronuclear single quantum coherence experiments reveal that residues in β-strand I contributes to the unique Cu²⁺ binding site in the C2B domain. In the absence of metal ions, residues located in Loop II and β-strand IV contribute to binding to unilamelar pS vesicles. In the presence of Cu²⁺, additional residues located in Loops I and III appear to stabilize the protein-lipid interactions. The results of this study provide valuable information towards understanding the molecular mechanism of the Cu²⁺-induced non-classical secretion of hFGF-1.     

Characterization of the structure and dynamics of a near-native equilibrium intermediate in the unfolding pathway of an all beta-barrel protein

The structure and dynamics of equilibrium intermediate in the unfolding pathway of the human acidic fibroblast growth factor (hFGF-1) are investigated using a variety of biophysical techniques including multidimensional NMR spectroscopy. Guanidinium hydrochloride (GdnHCl)-induced unfolding of hFGF-1 proceeds with the accumulation of a stable intermediate state. The transition from the intermediate state to the unfolded state(s) is cooperative without the accumulation of additional intermediate(s). The intermediate state induced maximally in 0.96 m GdnHCl is found to be obligatory in the folding/unfolding pathway of hFGF-1. Most of the native tertiary structure interactions are preserved in the intermediate state. (1)H-(15)N chemical shift perturbation data suggest that the residues in the C-terminal segment including those located in the beta-strands IX, X, and XI undergo the most discernible structural change(s) in the intermediate state in 0.96 m GdnHCl. hFGF-1 in the intermediate state (0.96 m GdnHCl) does not bind to its ligand, sucrose octasulfate. Limited proteolytic digestion experiments and hydrogen-deuterium exchange monitored by (15)N heteronuclear single quantum coherence (HSQC) spectra show that the conformational flexibility of the protein in the intermediate state is significantly higher than in the native conformation. (15)N spin relaxation experiments show that many residues located in beta-strands IX, X, and XI exhibit conformational motions in the micro- to millisecond time scale. Analysis of (15)N relaxation data in conjunction with the amide proton exchange kinetics suggests that residues in the beta-strands II, VIII, and XII possibly constitute the stability core of the protein in the near-native intermediate state.     

Structural events during the refolding of an all beta-sheet protein

The refolding kinetics of the 140-residue, all beta-sheet, human fibroblast growth factor (hFGF-1) is studied using a variety of biophysical techniques such as stopped-flow fluorescence, stopped-flow circular dichroism, and quenched-flow hydrogen exchange in conjunction with multidimensional NMR spectroscopy. Urea-induced unfolding of hFGF-1 under equilibrium conditions reveals that the protein folds via a two-state (native <--> unfolded) mechanism without the accumulation of stable intermediates. However, measurement of the unfolding and refolding rates in various concentrations of urea shows that the refolding of hFGF-1 proceeds through accumulation of kinetic intermediates. Results of the quenched-flow hydrogen exchange experiments reveal that the hydrogen bonds linking the N- and C-terminal ends are the first to form during the refolding of hFGF-1. The basic beta-trefoil framework is provided by the simultaneous formation of beta-strands I, IV, IX, and X. The other beta-strands comprising the beta-barrel structure of hFGF-1 are formed relatively slowly with time constants ranging from 4 to 13 s.     

Identification and characterization of an equilibrium intermediate in the unfolding pathway of an all beta-barrel protein

The guanidinium hydrochloride (GdnHCl)-induced unfolding of an all beta-sheet protein, the human acidic fibroblast growth factor (hFGF-1), is studied using a variety of biophysical techniques including multidimensional NMR spectroscopy. The unfolding of hFGF-1 in GdnHCl is shown to involve the formation of a stable equilibrium intermediate. Size exclusion chromotagraphy using fast protein liquid chromatography shows that the intermediate accumulates maximally at 0.96 m GdnHCl. 1-Anilinonapthalene 8-sulfonate binding, one-dimensional (1)H NMR, and limited proteolytic digestion experiments suggest that the intermediate has characteristics resembling a molten globule state. Chemical shift perturbation and hydrogen-deuterium exchange monitored by (1)H-(15)N heteronuclear single quantum coherence spectra reveal that profound structural changes in the intermediate state (in 0.96 m GdnHCl) occur in the C-terminal, heparin binding region of the protein molecule. Additionally, results of the stopped flow fluorescence experiments suggest that the kinetic refolding of hFGF-1 proceeds through the accumulation of an intermediate at low concentrations of the denaturant. To our knowledge, the present study is the first report wherein an equilibrium intermediate is characterized in detail in an all beta-barrel protein.     

Comparison of the structure of human recombinant short form stromelysin by multidimensional heteronuclear NMR and X-ray crystallography

Summary Stromelysin-1 is a matrix metalloprotease that has been implicated in a number of degenerative diseases. Here we present the refined NMR solution structure of the catalytic domain of stromelysin-1 complexed with a small inhibitor and compare it to the X-ray crystal structure of the same complex. The structures are similar in global fold and show an unusual bottomless S1' subsite. There are differences, however, in the least well defined regions, Phe⁸³-Ile⁸⁹, His²²⁴-Phe²³² and Pro²⁴⁹-Pro²⁵⁰, reflecting the lack of NOE data and large B-factors. The region His²²⁴-Phe²³² contains residues of the Sl' subsite and, consequently, small differences are observed in this subsite. Hydrogen-bond data show that, in contrast to the crystal structure, the solution structure lacks a hydrogen bond between the amide of Tyr²²³ and the carbonyl of the P3' residue. Analysis of bound water shows two tightly bound water molecules both in the solution and the crystal structure; neither of these waters are in the inhibitor binding site.Abbreviations MMP matrix metalloendoprotease - HMQC heteronuclear multiple quantum coherence - HSQC heteronuclear single quantum coherence - NOE nuclear Overhauser enhancement - NOESY NOE spectroscopy - PFG pulsed field gradient - sfSTR stromelysin-1 (EC 3.4.24.17), truncated at residue 255 The coordinates of the NMR solution structure (file name 2SRT) and the X-ray crystal structure (file name 1SLN) have been deposited in the Brookhaven Protein Data Bank.     

Dynameomics: A Consensus View of the Protein Unfolding/Folding Transition State Ensemble across a Diverse Set of Protein Folds

The Dynameomics project aims to simulate a representative sample of all globular protein metafolds under both native and unfolding conditions. We have identified protein unfolding transition state (TS) ensembles from multiple molecular dynamics simulations of high-temperature unfolding in 183 structurally distinct proteins. These data can be used to study individual proteins and individual protein metafolds and to mine for TS structural features common across all proteins. Separating the TS structures into four different fold classes (all proteins, all-α, all-β, and mixed α/β and α + β) resulted in no significant difference in the overall protein properties. The residues with the most contacts in the native state lost the most contacts in the TS ensemble. On average, residues beginning in an α-helix maintained more structure in the TS ensemble than did residues starting in β-strands or any other conformation. The metafolds studied here represent 67% of all known protein structures, and this is, to our knowledge, the largest, most comprehensive study of the protein folding/unfolding TS ensemble to date. One might have expected broad distributions in the average global properties of the TS relative to the native state, indicating variability in the amount of structure present in the TS. Instead, the average global properties converged with low standard deviations across metafolds, suggesting that there are general rules governing the structure and properties of the TS.     

1H-15N-NMR studies of bacteriorhodopsin Halobacterium halobium. Conformational dynamics of the four-helical bundle.

Series of uniformly and selectively 15N-labeled bacteriorhodopsins of Halobacterium halobium (strain ET 1001) were obtained and a 1H-15N-NMR study was performed in methanol/chloroform (1:1) and 0.1 M NH4CHOO, medium which mimics that in the membrane in vivo. Less than half of the cross-peaks expected from the amino acid sequence of uniformly 15N-labeled bacteriorhodopsin were observed, using heteronuclear 1H-15N coherence spectroscopy. In order to assign the observed cross-peaks, a selective 15N-labeling of amino acid residues (Tyr, Phe, Trp, Lys, Gly, Leu, Val or Ile) was carried out and 1H-15N-NMR spectra of bacteriorhodopsin and its fragments C1 (residues (72-231), C2 (residues 1-71), B1 (residues 1-155) and BP2 (residues 163-231) were investigated. By this procedure, all observed 1H-15N cross-peaks of the entire bacteriorhodopsin were found to belong to the transmembrane segments A, B and G. The cross-peaks from four (C, D, E and F) helical bundles (79-189 residues) were missed. These results clearly indicate that dynamic processes occur in the four helice bundle. The significance of this, in respect to bacteriorhodopsin functioning, is discussed.     

Rapid screening for structural integrity of expressed proteins by heteronuclear NMR spectroscopy.

A simple and rapid method based on 15N labeling and 1H-15N heteronuclear single quantum coherence spectroscopy is presented to directly assess the structural integrity of overexpressed proteins in crude Escherichia coli extracts without the need for any purification. The method is demonstrated using two different expression systems and two different proteins, the B1 immunoglobulin-binding domain of streptococcal protein G (56 residues) and human interleukin-1 beta (153 residues). It is shown that high quality 1H-15N correlation spectra, recorded in as little as 15 min and displaying only cross-peaks arising from the overexpressed protein of interest, can be obtained from crude E. coli extracts.     

β‐amyloid model core peptides: Effects of hydrophobes and disulfides

The mechanism by which a disordered peptide nucleates and forms amyloid is incompletely understood. A central domain of β‐amyloid (Aβ21–30) has been proposed to have intrinsic structural propensities that guide the limited formation of structure in the process of fibrillization. In order to test this hypothesis, we examine several internal fragments of Aβ, and variants of these either cyclized or with an N‐terminal Cys. While Aβ21–30 and variants were always monomeric and unstructured (circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMRS)), we found that the addition of flanking hydrophobic residues in Aβ16–34 led to formation of typical amyloid fibrils. NMR showed no long‐range nuclear overhauser effect (nOes) in Aβ21–30, Aβ16–34, or their variants, however. Serial ¹H‐¹⁵N‐heteronuclear single quantum coherence spectroscopy, ¹H‐¹H nuclear overhauser effect spectroscopy, and ¹H‐¹H total correlational spectroscopy spectra were used to follow aggregation of Aβ16–34 and Cys‐Aβ16–34 at a site‐specific level. The addition of an N‐terminal Cys residue (in Cys‐Aβ16–34) increased the rate of fibrillization which was attributable to disulfide bond formation. We propose a scheme comparing the aggregation pathways for Aβ16–34 and Cys‐Aβ16–34, according to which Cys‐Aβ16–34 dimerizes, which accelerates fibril formation. In this context, cysteine residues form a focal point that guides fibrillization, a role which, in native peptides, can be assumed by heterogeneous nucleators of aggregation.     

Native-like beta-hairpin retained in the cold-denatured state of bovine beta-lactoglobulin.

Bovine beta-lactoglobulin is denatured by increased temperature (heat denaturation) and by decreased temperature (cold-denaturation) in the presence of 4 M urea at pH 2.5. We characterized the structure of the cold-denatured state of beta-lactoglobulin using circular dichroism (CD), small-angle X-ray scattering (SAXS) and heteronuclear nuclear magnetic resonance (NMR). CD and SAXS indicated that the cold-denatured state, in comparison with the highly denatured state induced by urea, is rather compact, retaining some secondary structure, but no tertiary structure. The location of the residual structures in the cold-denatured state and their stability were characterized by 1H/2H exchange combined with heteronuclear NMR. The results indicated that the residues adjacent to the disulfide bond (C106-C119) connecting beta-strands G and H had markedly high protection factors, suggesting the presence of a native-like beta-hairpin stabilized by the disulfide bond. Since this beta-hairpin is conserved between different conformational states, including the kinetic refolding intermediate, it should be of paramount importance for the folding and stability of beta-lactoglobulin. On the other hand, the non-native alpha-helix suggested for the folding intermediate was not detected in the cold-denatured state. The 1H/2H exchange experiments showed that the protection factors of a mixture of the native and cold-denatured states is strongly biased by that of the labile cold-denatured state, consistent with a two-process model of the exchange.     

Proline Substitution of Dimer Interface β-strand Residues as a Strategy for the Design of Functional Monomeric Proteins

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.     

Characterization of the backbone dynamics of an anti-digoxin antibody VL domain by inverse detected 1H–15N NMR: Comparisons with X-ray data for the Fab

The dynamic behavior of the polypeptide backbone of a recombinant anti-digoxin antibody V_L domain has been characterized by measurements of ¹⁵N T₁ and T₂ relaxation times, ¹H–¹⁵N NOE values, and ¹H–²H exchange rates. These data were acquired with 2D inverse detected heteronuclear ¹H–¹⁵N NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R₂ (= 1/T₂). All characterized residues display low-amplitude picosecond timescale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R₂ exchange contributions, which reflect motions on the microsecond to millisecond timescale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R₂ exchange contributions. The measured ¹H–²H exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B-factors derived from X-ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X-ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of β-strands A, B, and G. However, on a residue-specific level, correlations among the various NMR data, and between the NMR and X-ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the V_L domain, including the three hypervariable loops, undergo dynamic changes upon V_L:V_H association and/ or complexation with digoxin. Overall, the 26–10 V_L domain exhibits relatively low flexibility on the ps–ns timescale. The possible functional consequences of this result are considered. © 1993 Wiley-Liss, Inc.     

Refolding of a small all beta-sheet protein proceeds with accumulation of kinetic intermediates

The refolding kinetics of Cobrotoxin (CBTX), a small all beta-sheet protein is investigated using a variety of biophysical techniques including quenched-flow hydrogen-deuterium (H/D) exchange in conjunction with two-dimensional NMR spectroscopy. Urea-induced equilibrium unfolding of CBTX follows a two-state mechanism with no distinct intermediates. The protein is observed to fold very rapidly within 250 ms. Both the refolding and the unfolding limbs of the chevron plot of CBTX show a prominent curvature suggesting the accumulation of kinetic intermediates. Quenched-flow H/D exchange data suggest the presence of a broad continuum of kinetic intermediates between the unfolded and native states of the protein. Comparison of the native state hydrogen exchange data and the results of the quenched-flow H/D exchange experiments, reveals that the residues constituting the folding core of CBTX are not a subset of the slow exchange core. To our knowledge, this is the first report wherein the refolding of a small all beta-sheet protein is shown to be a multi-step process involving the accumulation of kinetic intermediates.     

An Analysis of the Amino Acid Clustering Pattern in (α/β)8 Barrel Proteins

The (α/β)₈ barrel proteins, in spite of having a common fold, do not show any sequence similarity. In order to understand the factors which are responsible for maintaining the common fold, the three-dimensional structures of 36 (α/β)₈ barrel proteins are analyzed for the presence of identical amino acid clusters or physicochemically similar clusters. The results reveal 14 identical amino acid clusters and a large number of physicochemically similar clusters. Further analysis of the similar clusters points to the conservation of secondary structures, the presence of pairs of residues occupying topologically equivalent secondary structures, and the presence of certain key residues which may play a vital role in directing and stabilizing the (α/β)₈ barrel fold.     

NMR analysis of partially folded states and persistent structure in the alpha subunit of tryptophan synthase: implications for the equilibrium folding mechanism of a 29-kDa TIM barrel protein

Structural insights into the equilibrium folding mechanism of the alpha subunit of tryptophan synthase (αTS) from Escherichia coli, a (βα)₈ TIM barrel protein, were obtained with a pair of complementary nuclear magnetic resonance (NMR) spectroscopic techniques. The secondary structures of rare high-energy partially folded states were probed by native-state hydrogen-exchange NMR analysis of main-chain amide hydrogens. 2D heteronuclear single quantum coherence NMR analysis of several ¹⁵N-labeled nonpolar amino acids was used to probe the side chains involved in stabilizing a highly denatured intermediate that is devoid of secondary structure. The dynamic broadening of a subset of isoleucine and leucine side chains and the absence of protection against exchange showed that the highest energy folded state on the free-energy landscape is stabilized by a hydrophobic cluster lacking stable secondary structure. The core of this cluster, centered near the N-terminus of αTS, serves as a nucleus for the stabilization of what appears to be nonnative secondary structure in a marginally stable intermediate. The progressive decrease in protection against exchange from this nucleus toward both termini and from the N-termini to the C-termini of several β-strands is best described by an ensemble of weakly coupled conformers. Comparison with previous data strongly suggests that this ensemble corresponds to a marginally stable off-pathway intermediate that arises in the first few milliseconds of folding and persists under equilibrium conditions. A second, more stable intermediate, which has an intact β-barrel and a frayed α-helical shell, coexists with this marginally stable species. The conversion of the more stable intermediate to the native state of αTS entails the formation of a stable helical shell and completes the acquisition of the tertiary structure.     

Solution structure of the phosphocarrier protein HPr from Bacillus subtilis by two-dimensional NMR spectroscopy.

The solution structure of the phosphocarrier protein, HPr, from Bacillus subtilis has been determined by analysis of two-dimensional (2D) NMR spectra acquired for the unphosphorylated form of the protein. Inverse-detected 2D (1H-15N) heteronuclear multiple quantum correlation nuclear Overhauser effect (HMQC NOESY) and homonuclear Hartmann-Hahn (HOHAHA) spectra utilizing 15N assignments (reported here) as well as previously published 1H assignments were used to identify cross-peaks that are not resolved in 2D homonuclear 1H spectra. Distance constraints derived from NOESY cross-peaks, hydrogen-bonding patterns derived from 1H-2H exchange experiments, and dihedral angle constraints derived from analysis of coupling constants were used for structure calculations using the variable target function algorithm, DIANA. The calculated models were refined by dynamical simulated annealing using the program X-PLOR. The resulting family of structures has a mean backbone rmsd of 0.63 A (N, C alpha, C', O atoms), excluding the segments containing residues 45-59 and 84-88. The structure is comprised of a four-stranded antiparallel beta-sheet with two antiparallel alpha-helices on one side of the sheet. The active-site His 15 residue serves as the N-cap of alpha-helix A, with its N delta 1 atom pointed toward the solvent to accept the phosphoryl group during the phosphotransfer reaction with enzyme I. The existence of a hydrogen bond between the side-chain oxygen atom of Tyr 37 and the amide proton of Ala 56 is suggested, which may account for the observed stabilization of the region that includes the beta-turn comprised of residues 37-40. If the beta alpha beta beta alpha beta (alpha) folding topology of HPr is considered with the peptide chain polarity reversed, the protein fold is identical to that described for another group of beta alpha beta beta alpha beta proteins that include acylphosphatase and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins.     

The foldon substructure of staphylococcal nuclease

To search for submolecular foldon units, the spontaneous reversible unfolding and refolding of staphylococcal nuclease under native conditions was studied by a kinetic native-state hydrogen exchange (HX) method. As for other proteins, it appears that staphylococcal nuclease is designed as an assembly of well-integrated foldon units that may define steps in its folding pathway and may regulate some other functional properties. The HX results identify 34 amide hydrogens that exchange with solvent hydrogens under native conditions by way of large transient unfolding reactions. The HX data for each hydrogen measure the equilibrium stability (ΔG_HX) and the kinetic unfolding and refolding rates (k_op and k_cl) of the unfolding reaction that exposes it to exchange. These parameters separate the 34 identified residues into three distinct HX groupings. Two correspond to clearly defined structural units in the native protein, termed the blue and red foldons. The remaining HX grouping contains residues, not well separated by their HX parameters alone, that represent two other distinct structural units in the native protein, termed the green and yellow foldons. Among these four sets, a last unfolding foldon (blue) unfolds with a rate constant of 6 × 10^− 6 s^− 1 and free energy equal to the protein's global stability (10.0 kcal/mol). It represents part of the β-barrel, including mutually H-bonding residues in the β4 and β5 strands, a part of the β3 strand that H-bonds to β5, and residues at the N-terminus of the α2 helix that is capped by β5. A second foldon (green), which unfolds and refolds more rapidly and at slightly lower free energy, includes residues that define the rest of the native α2 helix and its C-terminal cap. A third foldon (yellow) defines the mutually H-bonded β1-β2-β3 meander, completing the native β-barrel, plus an adjacent part of the α1 helix. A final foldon (red) includes residues on remaining segments that are distant in sequence but nearly adjacent in the native protein. Although the structure of the partially unfolded forms closely mimics the native organization, four residues indicate the presence of some nonnative misfolding interactions. Because the unfolding parameters of many other residues are not determined, it seems likely that the concerted foldon units are more extensive than is shown by the 34 residues actually observed.     

Position of residues in transmembrane peptides with respect to the lipid bilayer: A combined lipid NOEs and water chemical exchange approach in phospholipid bicelles

The model transmembrane peptide P16 (Ac-KKGLLLALLLLALLLALLLKKA-NH₂) was incorporated into small unaligned phospholipid bicelles, which provide a `native-like' lipid bilayer compatible with high-resolution solution NMR techniques. Using amide-water chemical exchange and amide-lipid cross-relaxation measurements, the interactions between P16 and bicelles were investigated. Distinctive intermolecular NOE patterns observed in band-selective 2D-NOESY spectra of bicellar solutions with several lipid deuteration schemes indicated that P16 is preferentially interacting with the `bilayered' region of the bicelle rather than with the rim. Furthermore, when amide-lipid NOEs were combined with amide-water chemical exchange cross-peaks of selectively ¹⁵N-labeled P16 peptides, valuable information was obtained about the position of selected residues relative to the membrane-water interface. Specifically, three main classes were identified. Class I residues lie outside the bilayer and show amide-water exchange cross-peaks but no amide-lipid NOEs. Class II residues reside in the bilayer-water interface and show both amide-water exchange cross-peaks and amide-lipid NOEs. Class III residues are embedded within the hydrophobic core of the membrane and show no amide-water exchange cross-peaks but strong amide-lipid NOEs.