Superoxide dismutase from Thermus aquaticus. Isolation and characterisation of manganese and apo enzymes.

Superoxide dismutase has been isolated and characterised from the extreme thermophile Thermus aquaticus. The pure enzyme is a reddish-purple manganese-containing protein with a molecular weight of approximately 80000 +/- 5000. Combination of gel electrophoresis in dodecylsulphate and amino acid analysis shows that it is composed of four identical subunit polypeptide chains consisting of approximately 186 amino acids. The tetrameric protein contains two atoms of manganese. A stable manganese-free apoprotein has been prepared by treatment with EDTA in 8 M urea at acidic pH. The apoprotein regains the tetrameric structure in the absence of manganese but is inactive. Reconstitution of active Mn-enzyme was achieved byaddition of Mm2+ apoprotein in 8 M urea at acid pH. Reconstitution was monitored by absorption spectroscopy, manganese analysis and regain of activity and by these criteria the reconstituted enyzme with two atoms Mn per mole is indistinguishable from the native enzyme. The enhanced stability of the thermophile apoenzyme and Mn-enzyme is of advantage for studies of the structure and mechanism of action of superoxide dismutase. The N-terminal amino acid sequence to the 40th residue of the submit was determined by automated Edman degradation. The sequence has a close resemblance to that of the dimeric Mn-enzyme from another thermophile, Bacillus stearothermophilus.     

Protonation-mediated structural flexibility in the F conjugation regulatory protein, TraM

TraM is essential for F plasmid-mediated bacterial conjugation, where it binds to the plasmid DNA near the origin of transfer, and recognizes a component of the transmembrane DNA transfer complex, TraD. Here we report the 1.40 A crystal structure of the TraM core tetramer (TraM58-127). TraM58-127 is a compact eight-helical bundle, in which the N-terminal helices from each protomer interact to form a central, parallel four-stranded coiled-coil, whereas each C-terminal helix packs in an antiparallel arrangement around the outside of the structure. Four protonated glutamic acid residues (Glu88) are packed in a hydrogen-bonded arrangement within the central four-helix bundle. Mutational and biophysical analyses indicate that this protonated state is in equilibrium with a deprotonated tetrameric form characterized by a lower helical content at physiological pH and temperature. Comparison of TraM to its Glu88 mutants predicted to stabilize the helical structure suggests that the protonated state is the active form for binding TraD in conjugation.     

The distal C-terminal region of the KcsA potassium channel is a pH-dependent tetramerization domain

The intracellular C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is a 40-residue-long segment that natively adopts a helical bundle conformation with 4-fold symmetry. A hallmark of KcsA behavior is pH-induced conformational change, which leads to the opening of the channel at acidic pH. Previous studies have reached conflicting conclusions as to the role of the CTD in this transition. Here, we investigate the involvement of this domain in pH-mediated channel opening by NMR using a soluble peptide corresponding to residues 128-160 of the CTD (CTD34). At neutral pH, CTD34 exhibits concentration-dependent spectral changes consistent with oligomer formation. We prove this slowly tumbling species to be a tetramer with a dissociation constant of (2.0±0.5)×10(-)(11)?M(3) by NMR and sedimentation equilibrium experiments. Whereas monomeric CTD34 is only mildly helical, secondary chemical shifts prove that the tetrameric species adopts a tight native-like helical bundle conformation. The tetrameric species undergoes pH-dependent dissociation, and CTD34 is fully monomeric below pH?5.0. The structural basis for this phenomenon is the destabilization of the tetrameric CTD34 by protonation of residue H145 in the monomeric form of the peptide. We conclude that (i) the CTD34 peptide is independently capable of forming a tetrameric helical bundle, and (ii) this structurally significant conformational shift is modulated by the effects of solution pH on residue H145. Therefore, the involvement of this domain in the pH gating of the channel is strongly suggested.     

N-terminus of the photosystem II manganese stabilizing protein: effects of sequence elongation and truncation

The importance of the N-terminal domain of manganese stabilizing protein in binding to photosystem II has been previously demonstrated [Eaton-Rye and Murata (1989) Biochim. Biophys. Acta 977, 219-226; Odom and Bricker (1992) Biochemistry 31, 5616-5620]. In this paper, we report results from a systematic study of functional and structural consequences of N-terminal elongation and truncation of manganese stabilizing protein. Precursor manganese stabilizing protein is the unprocessed wild-type protein, which carries an N-terminal extension of 84 amino acids in the form of its chloroplastic signal peptide. Despite its increased size, this protein is able to reconstitute O(2) evolution activity to levels observed with the mature, processed protein, but it also binds nonspecifically to PSII. Truncation of wild-type manganese stabilizing protein by site-directed mutagenesis to remove three N-terminal amino acids, resulting in a mutant called DeltaG3M, causes no loss of activity reconstitution, but this protein also exhibits nonspecific binding. Further truncation of the wild-type protein by ten N-terminal amino acids, producing DeltaE10M, limits binding of manganese stabilizing protein to 1 mol/mol of photosystem II and decreases activity reconstitution to about 65% of that obtained with the wild-type protein. Because two copies of wild type normally bind to photosystem II, amino acids in the domain (4)K-(10)E must be involved in the binding of one copy of manganese stabilizing protein to photosystem II. Spectroscopic analysis (CD and UV spectra) reveals that N-terminal elongation and deletion of manganese stabilizing protein influence its overall conformation, even though secondary structure content is not perturbed. Our data suggest that the solution structure of manganese stabilizing protein attains a more compact solution structure upon removal of N-terminal amino acids.     

Expression,purification, and inhibition of in vitro proteolysis of human AMPD2 (isoform L) recombinant enzymes

AMP deaminase (AMPD) is a multigene family in higher eukaryotes whose three members encode tetrameric isoforms that catalyze the deamination of AMP to IMP. AMPD polypeptides share conserved C-terminal catalytic domains of approximately 550 amino acids, whereas divergent N-terminal domains of approximately 200-330 amino acids may confer isoform-specific properties to each enzyme. However, AMPD polypeptides are subject to limited N-terminal proteolysis during purification and subsequent storage at 4 degrees C. This presents a technical challenge to studies aimed at determining the structural and functional significance of these divergent sequences. This study describes the recombinant overexpression of three naturally occurring human AMPD2 proteins, 1A/2, 1B/2, and 1B/3, that differ by N-terminal extensions of 47-128 amino acids, resulting from the use of multiple promoters and alternative splicing events. A survey of protease inhibitors reveals that E-64 and leupeptin are able to maintain the subunit structure of each AMPD2 protein when they are included in extraction and storage buffers. Gel filtration chromatography of these three purified AMPD2 enzymes comprised of intact subunits reveals that each migrates faster than expected, resulting in observed molecular masses significantly greater than those predicted for native tetrameric structures. However, chemical crosslinking analysis indicates four subunits per AMPD2 molecule, confirming that these enzymes have a native tetrameric structure. These combined results suggest that AMPD2 N-terminal extensions may exist as extended structures in solution.     

Subunit interaction in extracellular superoxide dismutase: effects of mutations in the N-terminal domain.

Human extracellular superoxide dismutase (hEC-SOD) is a secreted tetrameric protein involved in protection against oxygen free radicals. Because EC-SOD is too large a protein for structural determination by multidimensional NMR, and attempts to crystallize the protein for X-ray structural determination have failed, the three-dimensional structure of hEC-SOD is unknown. This means that alternative strategies for structural studies are needed. The N-terminal domain of EC-SOD has already been studied using the fusion protein FusNN, comprised of the 49 N-terminal amino acids from hEC-SOD fused to human carbonic anhydrase (HCAII). The N-terminal domain in this fusion protein forms a well-defined three-dimensional structure, which probably contains alpha-helical elements and is responsible for the tetramerization of the protein. In this work, we have extended the studies, using site-directed mutagenesis in combination with size-exclusion chromatography, CD, and fluorescence spectroscopy, to investigate the nature of the tetrameric interaction. Our results show that the hydrophobic side of a predicted amphiphatic alpha-helix (formed by residues 14-32) in the N-terminal domain is essential for the subunit interaction.     

Oligomeric structure of a cathelicidin antimicrobial peptide in dodecylphosphocholine micelle determined by NMR spectroscopy

The broad spectrum of antibacterial activities of host defense cationic antimicrobial peptides (AMPs) arises from their ability to perturb membrane integrity of the microbes. The mechanisms are often thought to require assembly of AMPs on the membrane surface to form pores. However, three dimensional structures in the oligomeric form of AMPs in the context of lipid membranes are largely limited. Here, we demonstrate that a 22-residue antimicrobial peptide, termed VK22, derived from fowlicidin-1, a cathelicidin family of AMP from chicken oligomerizes into a predominantly tetrameric state in zwitterionic dodecylphosphocholine (DPC) micelles. An ensemble of NMR structures of VK22 determined in 200mM perdeuterated DPC, from 755 NOE constrains including 19 inter-helical NOEs, had revealed an assembly of four helices arranged in anti-parallel fashion. Hydrogen bonds, C(α)H-O=C types, and van der Waals interactions among the helical sub-units appear to be involved in the stabilization of the quaternary structures. The central region of the barrel shaped tetrameric bundle is non-polar with clusters of aromatic residues, whereas all the cationic residues are positioned at the termini. Paramagnetic spin labeled NMR experiments indicated that the tetrameric structure is embedded into micelles such that the non-polar region located inside the lipid acyl chains. Structure and micelle localization of a monomeric version, obtained from substitution of two Tyr residues with Ala, of the peptide is also compared. The mutated peptide VK22AA has been found be localized at the surface of the micelles. The tetrameric structure of VK22 delineates a small water pore that can be larger in the higher order oligomers. As these results provide structural insights, at atomic resolution, into the oligomeric states of a helical AMP in lipid environment, the structural details may be further utilized for the design of novel self-assembled membrane protein mimics.     

Nucleoside conformations : 12. An infrared study of the polymorphism of guanine nucleosides in the solid state

IR spectroscopy was used to define the previous observations of Shimanouchi et al. on the polymorphism of guanosine in the solid state. It is shown that form II is guanosine in its crystal form while form I is the helical tetrameric gel. This work was extended to 8-bromoguanosine which also shows polymorphism. It is shown that one of the forms [1] is also that of the helical gel.     

The monomeric, tetrameric, and fibrillar organization of Fib: the dynamic building block of the bacterial linear motor of Spiroplasma melliferum BC3

Spiroplasmas belong to the class Mollicutes, representing the minimal, free-living, and self-replicating forms of life. Spiroplasmas are helical wall-less bacteria and the only ones known to swim by means of a linear motor (rather than the near-universal rotary bacterial motor). The linear motor follows the shortest path along the cell's helical membranal tube. The motor is composed of a flat monolayered ribbon of seven parallel fibrils and is believed to function in controlling cell helicity and motility through dynamic, coordinated, differential length changes in the fibrils. The latter cause local perturbations of helical symmetry, which are essential for net directional displacement in environments with a low Reynolds number. The underlying fibrils' core building block is a circular tetramer of the 59-kDa protein Fib. The fibrils' differential length changes are believed to be driven by molecular switching of Fib, leading consequently to axial ratio and length changes in tetrameric rings. Using cryo electron microscopy, diffractometry, single-particle analysis of isolated ribbons, and sequence analyses of Fib, we determined the overall molecular organization of the Fib monomer, tetramer, fibril, and linear motor of Spiroplasma melliferum BC3 that underlies cell geometry and motility. Fib appears to be a bidomained molecule, of which the N-terminal half is apparently a globular phosphorylase. By a combination of reversible rotation and diagonal shift of Fib monomers, the tetramer adopts either a cross-like nonhanded conformation or a ring-like handed conformation. The sense of Fib rotation may determine the handedness of the linear motor and, eventually, of the cell. A further change in the axial ratio of the ring-like tetramers controls fibril lengths and the consequent helical geometry. Analysis of tetramer quadrants from adjacent fibrils clearly demonstrates local differential fibril lengths.     

Repressor--operator interaction in the lac operon. II. Observations at the tyrosines and tryptophans

This paper shows that ¹⁹F-nuelear magnetic resonance spectroscopy on 3-fluoro-tyrosine and 5-fluorotryptophan-substituted wild-type lactose operon repressors from Escherichia coli can be used to examine the interactions with lac operator DNA.A survey of inducer and salt concentration effects on the repressor-operator complex is presented. The data lead us to a scheme for the interactions between the repressor, operator and inducer, in both binary and ternary complexes, that accommodate the results published by others.The complex between the tetrameric repressor and one 36 base-pair operator DNA fragment results in the simultaneous broadening of the resonances from all four N-terminal DNA binding domains. The actual contacts made by these binding domains are similar but probably not identical.The binding of the inducer molecule to the tetrameric repressor results in an allosteric change that can be monitored by the increased intensity of the resonances from individual tyrosine residues in the N-terminal binding domain. This increased N-terminal tyrosine resonance intensity in the complex is transmitted to repressor subunits that have not yet bound an inducer molecule.     

Charge repulsion in the conformational stability of melittin.

Electrostatic repulsion between positively charged groups has been suggested to be critical in determining the conformation of melittin. To clarify the role of repulsive forces, we prepared a series of succinylated melittins, an acetylated melittin, and a synthetic melittin mutant, with various degrees of charge repulsion. The conformation of the melittin derivatives was examined by far-UV circular dichroism under various conditions of pH and salt at 20 degrees C. The stability of the tetrameric helical state was found to be dependent on the net charge of the peptides. The charge repulsive forces destabilized the helical state of intact melittin by 600 cal/(charge.mol of tetramer). This value was close to the corresponding one (450 cal/(charge.mol)) obtained for the acidic molten globule of horse cytochrome c [Goto, Y., & Nishikiori, S. (1991) J. Mol. Biol. 222, 679-686], which has a molecular weight and a net charge comparable to those of the tetrameric melittin. Small-angle X-ray scattering of the tetrameric melittin and the molten globule of cytochrome c showed that the two states are also comparable to each other in the radius of gyration. These results suggest that the contribution of electrostatic repulsion to the conformational stability of melittin is similar to that of the molten globule.     

Characterization of monomeric L1 metallo-beta -lactamase and the role of the N-terminal extension in negative cooperativity and antibiotic hydrolysis

The L1 metallo-beta-lactamase from Stenotrophomonas maltophilia is unique among this class of enzymes because it is tetrameric. Previous work predicted that the two regions of important intersubunit interaction were the residue Met-140 and the N-terminal extensions of each subunit. The N-terminal extension was also implicated in beta-lactam binding. Mutation of methionine 140 to aspartic acid results in a monomeric L1 beta-lactamase with a greatly altered substrate specificity profile. A 20-amino acid N-terminal deletion mutant enzyme (N-Del) could be isolated in a tetrameric form but demonstrated greatly reduced rates of beta-lactam hydrolysis and different substrate profiles compared with that of the parent enzyme. Specific site-directed mutations of individual N terminus residues were made (Y11S, W17S, and a double mutant L5A/L8A). All N-terminal mutant enzymes were tetramers and all showed higher K(m) values for ampicillin and nitrocefin, hydrolyzed ceftazidime poorly, and hydrolyzed imipenem more efficiently than ampicillin in contrast to wild-type L1. Nitrocefin turnover was significantly increased, probably because of an increased rate of breakdown of the intermediate species due to a lack of stabilizing forces. K(m) values for monomeric L1 were greatly increased for all antibiotics tested. A model of a highly mobile N-terminal extension in the monomeric enzyme is proposed to explain these findings. Tetrameric L1 shows negative cooperativity, which is not present in either the monomer or N-terminal deletion enzymes, suggesting that the cooperative effect is mediated via N-terminal intersubunit interactions. These data indicate that while the N terminus of L1 is not essential for beta-lactam hydrolysis, it is clearly important to its activity and substrate specificity.     

Phosphoglycerate mutase from Streptomyces coelicolor A3(2): purification and characterization of the enzyme and cloning and sequence analysis of the gene.

The enzyme 3-phosphoglycerate mutase was purified 192-fold from Streptomyces coelicolor, and its N-terminal sequence was determined. The enzyme is tetrameric with a subunit Mr of 29,000. It is 2,3-bisphosphoglycerate dependent and inhibited by vanadate. The gene encoding the enzyme was cloned by using a synthetic oligonucleotide probe designed from the N-terminal peptide sequence, and the complete coding sequence was determined. The deduced amino acid sequence is 64% identical to that of the phosphoglycerate mutase of Saccharomyces cerevisiae and has substantial identity to those of other phosphoglycerate mutases.     

The 2.0A resolution structure of the catalytic portion of a cyanobacterial membrane-bound manganese superoxide dismutase

Cyanobacteria are shown to be unique in containing membrane-bound manganese superoxide dismutases (MnSOD). They are homodimeric type 2 membrane proteins that protect this phototrophic organism against oxidative stress. We have determined, for the first time, the 2.0A resolution structure of the catalytic portion of the MnSOD from the filamentous cyanobacterium Anabaena PCC 7120. Within each subunit, both the N-terminal helical hairpin (His94 and His145) and the C-terminal alpha/beta domain (His232 and Asp228) contribute ligands to the catalytic manganese site. Together with a water or hydroxide ion (OH(x)) a five-coordinated trigonal bipyramidal geometry is formed, with OH(x) and His90 forming the axial ligands and manganese shifted out of the equatorial plane in the direction of OH(x). The ligands including OH(x) are tightly constrained by hydrogen bonding with surrounding residues either from the same monomer (Tyr98, Asn144, Trp194, Gln213, Val229, Trp230) or from the neighbouring subunit (Glu231, Tyr235). This underlines the important role of the symmetric dimeric structure of MnSODs in contributing elements to both the active site and the substrate funnel. The Mn cdots, three dots, centered Mn distance (18.4A) is bridged by the hydrogen-bonded His232 of one monomer with Glu231 of the other monomer. A detailed discussion of the structure, a comparison with known structures of soluble MnSODs as well as a model of the cyanobacterial membrane-bound MnSOD is presented.     

Head-to-head dimerization of parallel beta helices in a synthetic model of the linear gramicidins

In chloroform solution, the D ,L -alternating stereo-co-oligopeptide HCO-L -Phe-(D -Phe-L -Phe)₃-OMe (I) forms three major species, two of which are dimeric and one tetrameric. One of the two dimeric species gives a specific set of ¹H-nmr signals at 25°C; the other, together with the tetrameric species, gives another set of resonance signals. In a carbon tetrachloride or cyclohexane solution at 25°C, I forms virtually only the tetrameric species. From the nmr data, it can be shown that the dimeric and tetrameric species, that are in rapid equilibrium with each other in chloroform solutions, are a right-handed ↑↑β^5.6 helical dimer and the head-to-head (formyl-ends-to-formyl-ends) dimerization product of this dimer. It is suggested that the linear gramicidins may also form head-to-head dimers of parallel β helices, as observed for the model oligopeptide I.     

Structural analysis of the N-terminal domain of the human T-cell leukemia virus capsid protein

The N-terminal domain of the retroviral capsid (CA) protein is one of the least conserved regions encoded in the genome. Surprisingly, the three-dimensional structures of the CA from different genera exhibit alpha-helical structural features that are highly conserved. The N-terminal residues of the human immunodeficiency virus type 1 (HIV-1) and Rous sarcoma virus (RSV) capsid proteins form a beta-hairpin. To determine if this feature is conserved in the retroviral family, we cloned, expressed, purified, and solved the structure of a N-terminal 134 amino acid fragment (CA(134)) from the human T-cell leukemia virus type 1 (HTLV-I) using high resolution nuclear magnetic resonance (NMR) spectroscopy. The CA(134) fragment contains an N-terminal beta-hairpin and a central coiled-coil-like structure composed of six alpha-helices. The N-terminal Pro1 residue contacts Asp54 in the helical cluster through a salt bridge. Thus, the beta-hairpin is conserved and the helical cluster is structurally similar to other retroviral CA domains. However, although the same Asp residue defines the orientation of the hairpin in both the HTLV-1 and HIV-1 CA proteins, the HTLV-I hairpin is oriented away, rather than towards, the helical core. Significant differences were also detected in the spatial orientation and helical content of the long centrally located loop connecting the helices in the core. It has been proposed that the salt bridge allows the formation of a CA-CA interface that is important for the assembly of the conical cores that are characteristic of HIV-1. As HTLV-I forms spherical cores, the salt-bridge feature is apparently not conserved for this function although its role in determining the orientation of the beta-hairpin may be critical, along with the central loop. Comparison of three-dimensional structures is expected to elucidate the relationships between the retroviral capsid protein structure and its function.     

The mechanism of fibril formation of a non-inhibitory serpin ovalbumin revealed by the identification of amyloidogenic core regions

Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys(73) and Cys(120) in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4-5B are highly β-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to β-strands, thereby forming sequential intermolecular linkages.     

The overexpression, purification and complete amino acid sequence of chorismate synthase from Escherichia coli K12 and its comparison with the enzyme from Neurospora crassa.

The enzyme chorismate synthase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroC gene and confirmed by determining the N-terminal amino acid sequence of the purified enzyme. The complete polypeptide chain consists of 357 amino acid residues and has a calculated subunit Mr of 38,183. Cross-linking and gel-filtration experiments show that the enzyme is tetrameric. An improved purification of chorismate synthase from Neurospora crassa is also described. Cross-linking and gel-filtration experiments on the N. crassa enzyme show that it is also tetrameric with a subunit Mr of 50,000. It is proposed that the subunits of the N. crassa enzyme are larger because they contain a diaphorase domain that is absent from the E. coli enzyme.     

Impact of N-terminal acetylation of α-synuclein on its random coil and lipid binding properties

N-Terminal acetylation of α-synuclein (aS), a protein implicated in the etiology of Parkinson's disease, is common in mammals. The impact of this modification on the protein's structure and dynamics in free solution and on its membrane binding properties has been evaluated by high-resolution nuclear magnetic resonance and circular dichroism (CD) spectroscopy. While no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in chemical shift perturbations of the first 12 residues of the protein that progressively decreased with the distance from the N-terminus. The directions of the chemical shift changes and small changes in backbone (3)J(HH) couplings are consistent with an increase in the α-helicity of the first six residues of aS, although a high degree of dynamic conformational disorder remains and the helical structure is sampled <20% of the time. Chemical shift and (3)J(HH) data for the intact protein are virtually indistinguishable from those recorded for the corresponding N-terminally acetylated and nonacetylated 15-residue synthetic peptides. An increase in α-helicity at the N-terminus of aS is supported by CD data on the acetylated peptide and by weak medium-range nuclear Overhauser effect contacts indicative of α-helical character. The remainder of the protein has chemical shift values that are very close to random coil values and indistinguishable between the two forms of the protein. No significant differences in the fibrillation kinetics were observed between acetylated and nonacetylated aS. However, the lipid binding properties of aS are strongly impacted by acetylation and exhibit distinct behavior for the first 12 residues, indicative of an initiation role for the N-terminal residues in an "initiation-elongation" process of binding to the membrane.     

Modifications to surfactant protein B structure and lipid interactions under respiratory distress conditions: consequences of tryptophan oxidation

These studies detail the altered structure-function relationships caused by oxidation of surfactant protein B (SP-B), a mode of damage thought to be important in acute respiratory distress syndrome (ARDS), a common and frequently fatal condition. An 18-residue fragment comprising the N-terminal helix of SP-B was investigated in oxidized and unmodified forms by solution and solid-state nuclear magnetic resonance (NMR), circular dichroism (CD), and molecular dynamics (MD) simulation. Taken together, the results indicate that tryptophan oxidation causes substantial disruptions in helical structure and lipid interactions. The structural modifications induced by tryptophan oxidation were severe, with a reduction in helical extent from approximately three helical turns to, at most, one turn, and were observed in a variety of solvent environments, including sodium dodecyl sulfate (SDS) micelles, dodecyl phosphocholine (DPC) micelles, and a 40% hexafluoro-2-propanol (HFIP) aqueous solution. The unmodified peptide takes on an orientation within lipid bilayers that is tilted approximately 30° away from an in-plane position. Tryptophan oxidation causes significant modifications to the peptide-lipid interactions, and the peptide likely shifts to a more in-plane orientation within the lipids. Interestingly, the character of the disruptions to peptide-lipid interactions caused by tryptophan oxidation was highly dependent on the charge of the lipid headgroup.