Structure and dynamics of micelle-associated human immunodeficiency virus gp41 fusion domain

The N-terminal fusion domain of the HIV-1 gp41 envelope glycoprotein is responsible for initiating the fusion of viral and cellular membranes, leading to the subsequent infection of the host cell by HIV-1. We have investigated the backbone structure and dynamics of the 30 N-terminal residues of HIV-1 gp41 in membrane-mimicking environments using NMR spectroscopy and (15)N- and (15)N,(13)C,(2)H-labeled peptides. Similar (15)N-(1)H HSQC spectra were obtained in a variety of detergents, including SDS, DPC, mixed DPC/SDS, and LPPG micelles, indicating that the peptide structure is not strongly influenced by the type of detergent used. Detailed characterization was carried out in SDS micelles, where the long-term sample stability was found to be optimal. In addition to J-coupling and NOE restraints, a nearly complete set of backbone residual dipolar coupling restraints was recorded for the fusion domain-micelle complex aligned with respect to the magnetic field using a stretched polyacrylamide gel. Backbone amide (15)N spin relaxation and amide hydrogen exchange rates with the solvent were also measured. The ensemble of NMR structures reveals an uninterrupted alpha-helix for the least mobile residues (S(2) > 0.65), Ile-4 to Met-19, with transient helical character extending up to Ala-22. A 12-residue (Ile-4 to Ala-15) segment is fully shielded from solvent, with Gly-3 and Gly-16 found at micelle-solvent interfaces. Residues external to the micelle exhibit enhanced picosecond to nanosecond time scale dynamics relative to the residues buried in the micelle, and their mobility increases with the distance from the micelle.     

Carbon-13 NMR method for the detection of correlated hydrogen exchange at adjacent backbone peptide amides and its application to hydrogen exchange in five antiparallel beta strands within the hydrophobic core of Streptomyces subtilisin inhibitor (SSI)

A novel method for monitoring proton-deuteron (H/D) exchange at backbone amides is based on the observation of H/D isotope effects on the (13)C NMR signals from peptide carbonyls. The line shape of the carbonyl (13)C(i) signal is influenced by differential H/D occupancy at the two adjacent amides: the H(N)(i)(+1) (beta site) and the H(N)(i) (gamma site). At a carbon frequency of 75.4 MHz, the H --> D isotope shifts on the (13)C signal are about 5-7 Hz for exchange at the beta site and 2 Hz or less for exchange at the gamma site. Because the effects at the two sites are additive, the time dependence of the line shape of a particular carbonyl resonance can report not only the exchange rates at the individual sites but also the level of dual exchange. Therefore, the data can be analyzed to determine the rate (k(c)) and degree of correlated exchange (X(betagamma)) at the two sites. We have applied this approach to the investigation of the pH dependence of hydrogen exchange at several adjacent residues in Streptomyces subtilisin inhibitor (SSI). Two selectively labeled SSI proteins were produced: one with selective (13)C' labeling at all valyl residues and one with selective (13)C' labeling at all leucyl residues. This permitted the direct observation by one-dimensional (13)C NMR of selected carbonyl signals from residues with slowly exchanging amides at the i and i + 1 positions. The residues investigated were located in an alpha helix and in a five-stranded antiparallel beta sheet. Samples of the two labeled proteins were prepared at various pH values, and (13)C NMR spectra were collected at 50 degrees C prior to and at various times after transferring the sample from H(2)O to (2)H(2)O. Most of the slowly exchanging amides studied were intramolecular hydrogen-bond donors. In agreement with prior studies, the results indicated that the exchange rates of the amide hydrogens in proteins are governed not only by hydrogen bonding but also by other factors. For example, the amide hydrogen of Thr34 exchanges rapidly even though it is an intramolecular hydrogen-bond donor. Over nearly the whole pH range studied, the apparent rates of uncorrelated exchange (k(beta) and k(gamma)) were proportional to [OH(-)] and the apparent rates of correlated exchange at two adjacent sites (k(c)) were roughly proportional to [OH(-)](2). This enabled us to extract the pH-independent exchange rates (k(beta) degrees , k(gamma) degrees , and k(c) degrees ). In all cases in which correlated exchange could be measured, the observed sigmoidal pH dependence of X(betagamma) could be replicated roughly from the derived pH-independent rates.     

Role of arginine 67 in the stabilization of chymotrypsin inhibitor 2: examination of amide proton exchange rates and denaturation thermodynamics of an engineered protein

We have examined the contribution to protein stability of an interaction involving a charged hydrogen bond from an arginyl side chain (Arg67) in the serine proteinase inhibitor chymotrypsin inhibitor 2 (CI-2), by replacing this side chain with an alanyl residue by protein engineering. Using nuclear magnetic resonance spectroscopy (NMR), we have examined the effect of this mutation on the hydrogen-deuterium exchange rates of several backbone amide protons in the native and engineered proteins at 50 degrees C. These exchange rates provide a localized probe at multiple discrete sites throughout the protein and from comparison of native and mutant exchange rates allow calculation of the difference in free energy of exchange (delta delta Gex) resulting from the mutation. The results show that for the majority of amides observed this mutation results in delta delta Gex of ca. 1.7 kcal mol-1 over the whole CI-2 molecule. However, for two relatively exposed amide protons the exchange rates are found to be far less perturbed, implying that local unfolding mechanisms predominate for these protons. Direct measurement of the stability of both proteins to denaturation by guanidinum hydrochloride shows that the interaction contributes 1.4 kcal mol-1 to the stability of the molecule. This value is comparable to those obtained from the NMR exchange measurements and indicates that the exchange processes reflect the differences in stability between the native and mutant proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and dynamics of a detergent-solubilized membrane protein: measurement of amide hydrogen exchange rates in M13 coat protein by 1H NMR spectroscopy

The coat protein of bacteriophage M13 is inserted into the inner membrane of Escherichia coli where it exists as an integral membrane protein during the reproductive cycle of the phage. The protein sequence consists of a highly hydrophobic 19-residue central segment flanked by an acidic 20-residue N-terminus and a basic 11-residue C-terminus. We have measured backbone amide hydrogen exchange of the protein solubilized in perdeuteriated sodium dodecyl sulfate using 1H nuclear magnetic resonance (NMR) spectroscopy. Direct proton exchange-out measurements in D2O at 24 degrees C were used to follow the exchange of the slowest amides in the protein. Multiple exponential fitting of the exchange data showed that these amides (29 +/- 3 at pH 4.5) exchanged in two kinetic sets with exchange rates [(1.2 +/- 0.4) x 10(-4) s-1 and (4.1 +/- 1.2) x 10(-7) s-1] that differed by more than 100-fold, the slower kinetic set being retarded 10(5)-fold relative to poly(DL-alanine). The exchange rate constant for the slowest set of amides exhibited an unusual pD dependence, being proportional to [OD-]1/2. It is shown that this is an artifact of the multiple exponential fitting of the data, and a new method of presentation of exchange data as a function of pD is introduced. Steady-state saturation-transfer techniques were also used to measure exchange. These methods showed that 15-20 amides in the protein are very stable at 55 degrees C and that about 30 amides have exchange rates retarded by at least 10(5)-fold at 24 degrees C. Saturation-transfer studies also showed that the pH dependence of exchange in the hydrophilic termini was unusual. This is explained as being due to long-range electrostatic effects arising both from the protein itself and also from the anionic detergent molecules. Hydrogen exchange studies on the products of proteinase K digestion of the protein localized the slowly exchanging amides to the hydrophobic core of the protein. Relaxation [Henry, G.D., Weiner, J.H., & Sykes, B.D. (1986) Biochemistry 25, 590-598] and solid-state NMR experiments [Leo, G.C., Colnago, L.A., Valentine, K.G., & Opella, S.J. (1987) Biochemistry 26, 854-862] have previously shown that the majority of the protein backbone is rigid on the picosecond to microsecond time scale, except for the extreme ends of the molecule which are mobile.(ABSTRACT TRUNCATED AT 400 WORDS)     

Amide proton exchange rates of a bound pepsin inhibitor determined by isotope-edited proton NMR experiments

From a series of isotope-edited proton NMR spectra, amide proton exchange rates were measured at 20 degrees C, 30 degrees C, and 40 degrees C for a tightly bound 15N-labeled tripeptide inhibitor of porcine pepsin (IC50 = 1.7 X 10(-) M). Markedly different NH exchange rates were observed for the three amide protons of the bound inhibitor. The P1 NH exchanged much more slowly than the P2 NH and P3 NH. These results are discussed in terms of the relative solvent accessibility in the active site and the role of the NH protons of the inhibitor for hydrogen bonding to the enzyme. In this study a useful approach is demonstrated for obtaining NH exchange rates on ligands bound to biomacromolecules, the knowledge of which could be of potential utility in the design of therapeutically useful nonpeptide enzyme inhibitors from peptide leads.     

A 1H NMR determination of the solution conformation of a synthetic peptide analogue of calcium-binding site III of rabbit skeletal troponin C

NMR techniques have been used to determine the structure in solution of acetyl (Asp 105) skeletal troponin C (103-115) amide, one of a series of synthetic peptide analogues of calcium-binding site III of rabbit skeletal troponin C [Marsden et al. (1988) Biochemistry 27, 4198-4206]. The NMR measurements include 1H-1H nuclear Overhauser enhancements and gadolinium-induced 1H relaxation measurements. The former yield short-range internuclear distances (less than 4 A); the latter, once properly corrected for chemical exchange, yield longer range metal to proton distances (5-10 A). These measurements were then used as pseudo potential energy restraints in energy minimization and molecular dynamics calculations to determine the solution structure. Further information was provided by NMR coupling constants, amide proton exchange rates, and the temperature dependences of amide proton chemical shifts. The solution structure of the peptide analogue is very similar to that of the calcium-binding loop in the protein, the root-mean-square deviation between the backbone atoms being approximately 1.1 A.     

Hydrogen--deuterium exchange kinetics of the amide protons of oxytocin studied by nuclear magnetic resonance

The contribution of intramolecular hydrogen bonding to the solution structure of oxytocin was evaluated by study of amide hydrogen exchange rates in D2O by Fourier transform 1H NMR spectroscopy. Resolution enhancement filtering was employed in the determination of individual pseudo-first-order rate constants. Apparent barriers to exchange of 0.5 and 0.6 kcal mol-1 were measured for Asn5 and Cys6 peptide NH, respectively. The slowing is best explained by steric hindrance to solvent access in the case of Asn5, while for the Cys6 participation in a weak intramolecular hydrogen bond is possible. Fourfold acceleration of base-catalyzed exchange was observed for Tyr2 NH; it is proposed that this is the result of electronic effects induced by hydrogen bonding of Cys1 C=0, either to Cys6 NH or to the N-terminal amino group. Exchange proceeds near the random coil limit for each of the remaining residues. Comparison with exchange data for the model tripeptide N-acetyl-L-prolyl-L-leucylglycinamide demonstrates no evidence of noncovalent association of the tocin ring with the tripeptide tail of the hormone.     

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.     

A globular protein with slower amide proton exchange from an alpha helix than from antiparallel beta sheets

In proteinase inhibitor IIA from bull seminal plasma, which is a small globular protein with 57 amino acid residues, measurements of individual amide proton exchange rates by two-dimensional correlated 1H NMR spectroscopy (COSY) showed that the exchange was slowest for some hydrogen bonded amide groups in an alpha-helix. This contrasts with all other proteins which were so far studied in detail, where the slowest exchange rates were observed for hydrogen bonded amide protons in antiparallel beta-sheets.     

Comparison of the structure and dynamics of chicken gizzard and rabbit cardiac tropomyosins: 1H NMR spectroscopy and measurement of amide hydrogen exchange rates

The side chain and backbone mobilities of chicken gizzard tropomyosin (TM) and its nonpolymerizable derivative have been investigated by H NMR spectroscopy and amide hydrogen exchange kinetics and compared to those of rabbit cardiac TM and its nonpolymerizable derivative. Analysis of the 300-MHz H NMR spectra of native chicken gizzard and rabbit cardiac TMs and their nonpolymerizable derivatives showed that the line widths of the aromatic and histidine residues were within a factor of 2 for all four proteins, demonstrating that the side chain mobility of these residues is similar in the different TMs. Direct proton exchange-out kinetics were determined in D2O in the pD range 1.5-3.0 at 25 degrees C by H NMR spectroscopy. Multiple exponential fitting of the exchange data indicated the presence in gizzard TM of at least three kinetically distinct classes of amide hydrogens at pD 1.7 with average population sizes of 147, 74, and 61, whose rates were retarded by a factor of 10, 10(3), and 10(5), respectively, relative to the random-coil peptide poly(DL-alanine). Measurement of the direct exchange kinetics of both rabbit cardiac and nonpolymerizable gizzard TMs showed that their rate constants and population sizes were within experimental error of those for the gizzard protein, except that the fast exchanging class for cardiac TM was increased in size while that of the nonpolymerizable gizzard TM was reduced, relative to that for gizzard TM. Comparison of the exchange-out kinetics for the cardiac and gizzard proteins at pH 2.0 and 55 degrees C, where only the two slowly exchanging amide hydrogen sets are measured, again demonstrated the similarity of their kinetic parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Equilibrium hydrogen exchange reveals extensive hydrogen bonded secondary structure in the on-pathway intermediate of Im7

The four-helical immunity protein Im7 folds through an on-pathway intermediate that has a specific, but partially misfolded, hydrophobic core. In order to gain further insight into the structure of this species, we have identified the backbone hydrogen bonds formed in the ensemble by measuring the amide exchange rates (under EX2 conditions) of the wild-type protein and a variant, I72V. In this mutant the intermediate is significantly destabilised relative to the unfolded state (deltadeltaG(ui) = 4.4 kJ/mol) but the native state is only slightly destabilised (deltadeltaG(nu) = 1.8 kJ/mol) at 10 degrees C in 2H2O, pH* 7.0 containing 0.4 M Na2SO4, consistent with the view that this residue forms significant non-native stabilising interactions in the intermediate state. Comparison of the hydrogen exchange rates of the two proteins, therefore, enables the state from which hydrogen exchange occurs to be identified. The data show that amides in helices I, II and IV in both proteins exchange slowly with a free energy similar to that associated with global unfolding, suggesting that these helices form highly protected hydrogen-bonded helical structure in the intermediate. By contrast, amides in helix III exchange rapidly in both proteins. Importantly, the rate of exchange of amides in helix III are slowed substantially in the Im7* variant, I72V, compared with the wild-type protein, whilst other amides exchange more rapidly in the mutant protein, in accord with the kinetics of folding/unfolding measured using chevron analysis. These data demonstrate, therefore, that local fluctuations do not dominate the exchange mechanism and confirm that helix III does not form stable secondary structure in the intermediate. By combining these results with previously obtained Phi-values, we show that the on-pathway folding intermediate of Im7 contains extensive, stable hydrogen-bonded structure in helices I, II and IV, and that this structure is stabilised by both native and non-native interactions involving amino acid side-chains in these helices.     

Hydrogen exchange shows peptide binding stabilizes motions in Hck SH2.

Src-homology-2 domains are small, 100 amino acid protein modules that are present in a number of signal transduction proteins. Previous NMR studies of SH2 domain dynamics indicate that peptide binding decreases protein motions in the pico- to nanosecond, and perhaps slower, time range. We suggest that amide hydrogen exchange and mass spectrometry may be useful for detecting changes in protein dynamics because hydrogen exchange rates are relatively insensitive to the time domains of the dynamics. In the present study, hydrogen exchange and mass spectrometry were used to probe hematopoietic cell kinase SH2 that was either free or bound to a 12-residue high-affinity peptide. Hydrogen exchange rates were determined by exposing free and bound SH2 to D(2)O, fragmenting the SH2 with pepsin, and determining the deuterium level in the peptic fragments. Binding generally decreased hydrogen exchange along much of the SH2 backbone, indicating a widespread reduction in dynamics. Alterations in the exchange of the most rapidly exchanging amide hydrogens, which was detected following acid quench and analysis by mass spectrometry, were used to locate differences in low-amplitude motion when SH2 was bound to the peptide. In addition, the results indicate that hydrogen exchange from the folded form of SH2 is an important process along the entire SH2 backbone.     

Hydrogen-deuterium (H/D) exchange mapping of Abeta 1-40 amyloid fibril secondary structure using nuclear magnetic resonance spectroscopy

We describe here details of the hydrogen-deuterium (H/D) exchange behavior of the Alzheimer's peptide Abeta(1)(-)(40), while it is a resident in the amyloid fibril, as determined by high-resolution solution NMR. Kinetics of H/D exchange in Abeta(1)(-)(40) fibrils show that about half the backbone amide protons exchange during the first 25 h, while the other half remain unexchanged because of solvent inaccessibility and/or hydrogen-bonded structure. After such a treatment for 25 h with D(2)O, fibrils of (15)N-enriched Abeta were dissolved in a mixture of 95% dimethyl sulfoxide (DMSO) and 5% dichloroacetic acid (DCA) and successive heteronuclear (1)H-(15)N HSQC spectra were collected to identify the backbone amides that did not exchange in the fibril. These studies showed that the N and C termini of the peptide are accessible to the solvent in the fibril state and the backbone amides of these residues are readily exchanged with bulk deuterium. In contrast, the residues in the middle of the peptide (residues 16-36) are mostly protected, suggesting that that many of the residues in this segment of the peptide are involved in a beta structure in the fibril. Two residues, G25 and S26, exhibit readily exchangeable backbone amide protons and therefore may be located on a turn or a flexible part of the peptide. Overall, the data substantially supports current models for how the Abeta peptide folds when it engages in the amyloid fibril structure, while also addressing some discrepancies between models.     

Destabilization of collagen structure by amides and detergents in solution

The effects of amides and detergents on collagen to gelatin transition have been studied at neutral pH. Simple amides denature the protein. The substitution of H-atoms by the alkyl groups at the nonpolar end of amide increases the effectiveness of the compounds in destabilizing the collagen structure whereas substitution of the H-atom at the polar amide end shows marginal effects on the collagen transition. The capabilities of these reagents to denature collagen are much less pronounced than their effects on denaturing globular proteins. Anionic detergents are found to destabilize collagen at very low concentrations (below their cmc values). In this respect, the effects of the detergents on collagen are comparable to the denaturing effects of the detergents on globular proteins. The effect of detergents increases with the increase in the length of the alkyl chain. The structure of the anion in the detergent is also important as seen from the lower potency of the sulfonate containing detergent compared to the sulfate containing detergent in denaturing collagen. Cationic and nonionic detergents do not denature collagen.     

Reduced temperature dependence of collective conformational opening in a hyperthermophile rubredoxin.

Spatially localized differences in the conformational dynamics of the rubredoxins from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophile Clostridium pasteurianum (Cp) are monitored via amide exchange measurements. As shown previously for the hyperthermophile protein, nearly all backbone amides of the Cp rubredoxin exhibit EX(2) hydrogen exchange kinetics with conformational opening rates of >1 s(-)(1). Significantly slower amide exchange is observed for Pf rubredoxin in the region surrounding the metal site and the proximal end of the three-stranded beta-sheet, while for the rest of the structure, the exchange rates at 23 degrees C are similar for both proteins. For the multiple-turn region comprising residues 14-32 in both rubredoxins, the uniformity of both the exchange rate constants and the values of the activation energy at the slowly exchanging sites is consistent with a model of solvent exposure via a subglobal cooperative conformational opening. In contrast to the common expectation of increased rigidity in the hyperthermophile proteins, below room temperature Pf rubredoxin exhibits a larger apparent flexibility in this multiple-turn region. The smaller enthalpy for the conformational opening process of this region in Pf rubredoxin reflects the much weaker temperature dependence of the underlying conformational equilibrium in the hyperthermophile protein.     

Photoactivation of rhodopsin causes an increased hydrogen-deuterium exchange of buried peptide groups.

A key step in visual transduction is the light-induced conformational changes of rhodopsin that lead to binding and activation of the G-protein transducin. In order to explore the nature of these conformational changes, time-resolved Fourier transform infrared spectroscopy was used to measure the kinetics of hydrogen/deuterium exchange in rhodopsin upon photoexcitation. The extent of hydrogen/deuterium exchange of backbone peptide groups can be monitored by measuring the integrated intensity of the amide II and amide II' bands. When rhodopsin films are exposed to D2O in the dark for long periods, the amide II band retains at least 60% of its integrated intensity, reflecting a core of backbone peptide groups that are resistant to H/D exchange. Upon photoactivation, rhodopsin in the presence of D2O exhibits a new phase of H/D exchange which at 10 degrees C consists of fast (time constant approximately 30 min) and slow (approximately 11 h) components. These results indicate that photoactivation causes buried portions of the rhodopsin backbone structure to become more accessible.     

Hydrogen exchange in native and denatured states of hen egg-white lysozyme.

The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30 degrees C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 10(5)-10(7) times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35 degrees C, by lowering the stability of the native state through selective cleavage of the Cys-6-Cys-127 disulfide cross-link (CM6-127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation (up to nearly 20-fold) relative to the predicted rates. The pattern of protection observed under these conditions does not reflect the behavior of the protein under strongly native conditions, suggesting that regions of native-like structure do not persist significantly in the denatured state of CM6-127 lysozyme. The pattern of exchange rates from the native protein at high temperature, pH 3.8 69 degrees C, resembles that of the acid-denatured state, suggesting that under these conditions the exchange kinetics are dominated by transient global unfolding. The rates of folding and unfolding under these conditions were determined independently by magnetization transfer NMR methods, enabling the intrinsic exchange rates from the denatured state to be deduced on the basis of this model, under conditions where the predominant equilibrium species is the native state. Again, in the case of most amides these rates showed only limited deviation from those predicted by a simple random coil model. This reinforces the view that these denatured states of lysozyme have little persistent residual order and contrasts with the behavior found for compact partially folded states of proteins, including an intermediate detected transiently during the refolding of hen lysozyme.     

Backbone dynamics of detergent-solubilized alamethicin from amide hydrogen exchange measurements.

Alamethicin is a 20 amino acid antibiotic peptide produced by the soil fungus Trichoderma viride. The peptide inserts into bacterial membranes and self-associates to form ion channels, but the details of this process are unknown. Residue-specific acid- and base-catalyzed exchange data were obtained for 16 of 18 backbone amides of alamethicin dissolved in sodium dodecyl sulfate micelles using high-resolution 2-dimensional heteronuclear nuclear magnetic resonance spectroscopy. To facilitate interpretation of the exchange data, we synthesized N-acetyl-alpha-aminoisobutyric acid-N'-methyl and N-acetyl-alanine-N'-methyl and measured the pD dependence of their hydrogen-deuterium exchange rates to determine the sequence-dependent inductive and steric effects of the alpha-aminoisobutyric acid residue. Intramolecular H-bonding in alamethicin was monitored through the exchange parameters kmin (minimum exchange rate) which indicate that the backbone is significantly more stable than the backbones of alanine-based helical peptides. Rapid exchange at Gly-11 suggests a highly local conformational flexibility in the middle of the peptide. Interactions with the detergent micelle were revealed by the exchange parameters pDmin (pD of minimum exchange) which suggest that the N-terminus of alamethicin interacts more strongly with the detergent micelle than does the C-terminus. A periodicity in pDmin difference data reveals that one surface of the helix interacts more strongly with the micelle. The surface consists of residues 1, 5, 9, 13, 16, and 20. The opposite face of the helix contains several polar residues (two glutamines and a glycine), suggesting that, on average, this face of the helix is directed toward the solvent. These results serve as a model for the interaction of the peptide with membranes containing anionic lipid. In combination with published molecular dynamics simulations [Gibbs et al. (1997) Biophys. J. 72, 2490-2495], the present results also offer insight into the mechanisms of hydrogen-deuterium exchange in helical peptides.     

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.     

Hydrogen exchange from the transbilayer hydrophobic peptide of glycophorin reconstituted in lipid bilayers

The hydrophobic transbilayer peptide of erythrocyte glycophorin has been purified following exchange of tritium into the backbone amides, and reconstituted in egg phosphatidylcholine micelles. Analysis of tritium exchange from the backbone amides of the membrane-reconstituted peptide shows that about two of the amides are virtually non-exchangeable, about 10 are slowed by factors of 10(7) relative to free amides in unstructured water soluble peptides and the remainder of the amides (about 20) have slowing factors of less than 1000. These classes of amides are proposed to reflect the stability of the peptide with respect to hydrogen bond breaking fluctuations and the accessibility of the amides to exchange catalysts in different regions of the bilayer.