Characterization of the mobility of various chemical groups in the purple membrane of halobacterium halobium by 13C, 31P and 2H solid state N M R

Lyophilized purple membrane sheets have been investigated by C-13- and P-31-cross polarization/magic angle spinning N M R spectroscopy. The high-resolution C-13 spectrum and its non-quaternary suppression version indicate fast protein side-chain motions but a rigid backbone structure on a time scale of roughly < 0.001 to 0.01 s. Three components of exchangeable hydrogen have been detected by deuterium N M R. The mean exchange time of the peptide hydrogens must be longer than 1 μs. The medium component is attributed to mobile side-chains. In addition a narrow line has been observed which is assigned to the residual hydration water.     

Collisional activation by MALDI tandem time-of-flight mass spectrometry induces intramolecular migration of amide hydrogens in protonated peptides

Considerable controversy exists in the literature as to the occurrence of intramolecular migration of amide hydrogens upon collisional activation of protonated peptides and proteins. This phenomenon has important implications for the application of CID as an experimental tool to obtain site-specific information about the incorporation of deuterium into peptides and proteins in solution. Using a unique set of peptides with their carboxyl-terminal half labeled with deuterium we have shown unambiguously that hydrogen (1H/2H) scrambling is such a dominating factor during low energy collisional activation of doubly protonated peptides that the original regioselective deuterium pattern of these peptides is completely erased (J?rgensen, T. J. D., G?rdsvoll, H., Ploug, M., and Roepstorff, P. (2005) Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation. J. Am. Chem. Soc.127, 2785-2793). Taking further advantage of this unique test system we have now investigated the influence of the charge state and collision energy on the occurrence of scrambling in protonated peptides. Our MALDI tandem time-of-flight experiments clearly demonstrate that complete positional randomization among all exchangeable sites (i.e. all N- and O-linked hydrogens) also occurs upon high energy collisional activation of singly protonated peptides. This intense proton/deuteron traffic precludes the use of MALDI tandem time-of-flight mass spectrometry to obtain reliable information on the specific incorporation pattern of deuterons obtained during exchange experiments in solution.     

Incoherent neutron scattering of copper azurin: a comparison with molecular dynamics simulation results

The low-frequency dynamics of copper azurin has been studied at different temperatures for a dry and deuterium hydrated sample by incoherent neutron scattering and the experimental results have been compared with molecular dynamics (MD) simulations carried out in the same temperature range. Experimental Debye-Waller factors are consistent with a dynamical transition at approximately 200 K which appears partially suppressed in the dry sample. Inelastic and quasielastic scattering indicate that hydration water modulates both vibrational and diffusive motions. The low-temperature experimental dynamical structure factor of the hydrated protein shows an excess of inelastic scattering peaking at about 3 meV and whose position is slightly shifted downwards in the dry sample. Such an excess is reminiscent of the “boson peak” observed in glass-like materials. This vibrational peak is quite well reproduced by MD simulations, although at a lower energy. The experimental quasielastic scattering of the two samples at 300 K shows a two-step relaxation behaviour with similar characteristic times, while the corresponding intensities differ only by a scale factor. Also, MD simulations confirm the two-step diffusive trend, but the slow process seems to be characterized by a decay faster than the experimental one. Comparison with incoherent neutron scattering studies carried out on proteins having different structure indicates that globular proteins display common elastic, quasielastic and inelastic features, with an almost similar hydration dependence, irrespective of their secondary and tertiary structure. Received: 12 October 1998 / Revised version: 19 February 1999 / Accepted: 1 March 1999     

Rapid Analysis of Protein Structure and Dynamics by Hydrogen/Deuterium Exchange Mass Spectrometry

An automated approach for the rapid analysis of protein structure has been developed and used to study acid-induced conformational changes in human growth hormone. The labeling approach involves hydrogen/deuterium exchange (H/D-Ex) of protein backbone amide hydrogens with rapid and sensitive detection by mass spectrometry (MS). Briefly, the protein is incubated for defined intervals in a deuterated environment. After rapid quenching of the exchange reaction, the partially deuterated protein is enzymatically digested and the resulting peptide fragments are analyzed by liquid chromatography mass spectrometry (LC-MS). The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. Additional analyses allow mapping of the free energy of folding on localized segments along the protein sequence affording unique dynamic and structural information. While amide H/D-Ex coupled with MS is recognized as a powerful technique for studying protein structure and protein–ligand interactions, it has remained a labor-intensive task. The improvements in the amide H/D-Ex methodology described here include solid phase proteolysis, automated liquid handling and sample preparation, and integrated data reduction software that together improve sequence coverage and resolution, while achieving a sample throughput nearly 10-fold higher than the commonly used manual methods.     

Hydrogen/deuterium exchange and mass spectrometric analysis of a protein containing multiple disulfide bonds: Solution structure of recombinant macrophage colony stimulating factor-beta (rhM-CSFbeta)

Studies with the homodimeric recombinant human macrophage colony-stimulating factor beta (rhM-CSFbeta), show for the first time that a large number (9) of disulfide linkages can be reduced after amide hydrogen/deuterium (H/D) exchange, and the protein digested and analyzed successfully for the isotopic composition by electrospray mass spectrometry. Analysis of amide H/D after exchange-in shows that in solution the conserved four-helix bundle of (rhM-CSFbeta) has fast and moderately fast exchangeable sections of amide hydrogens in the alphaA helix, and mostly slow exchanging sections of amide hydrogens in the alphaB, alphaC, and alphaD helices. Most of the amide hydrogens in the loop between the beta1 and beta4 sheets exhibited fast or moderately fast exchange, whereas in the amino acid 63-67 loop, located at the interface of the two subunits, the exchange was slow. Solvent accessibility as measured by H/D exchange showed a better correlation with the average depth of amide residues calculated from reported X-ray crystallographic data for rhM-CSFalpha than with the average B-factor. The rates of H/D exchange in rhM-CSFbeta appear to correlate well with the exposed surface calculated for each amino acid residue in the crystal structure except for the alphaD helix. Fast hydrogen isotope exchange throughout the segment amino acids 150-221 present in rhM-CSFbeta, but not rhM-CSFalpha, provides evidence that the carboxy-terminal region is unstructured. It is, therefore, proposed that the anomalous behavior of the alphaD helix is due to interaction of the carboxy-terminal tail with this helical segment.     

Analysis of proton exchange kinetics with time-dependent exchange rate

Mass spectrometry is used to probe the kinetics of hydrogen–deuterium exchange in lysozyme in pH 5, 6 and 7.4. An analysis based on a Verhulst growth model is proposed and effectively applied to the kinetics of the hydrogen exchange. The data are described by a power-like function which is based on a time-dependence of the exchange rate. Experimental data ranging over many time scales is considered and accurate fits of a power-like function are obtained. Results of fittings show correlation between faster hydrogen–deuterium exchange and increase of pH. Furthermore a model is presented that discriminates between easily exchangeable hydrogens (located in close proximity to the protein surface) and those protected from the exchange (located in the protein interior). A possible interpretation of the model and its biological significance are discussed.     

2H nuclear magnetic resonance of exchange-labeled gramicidin in an oriented lyotropic nematic phase

Lyotropic nematic liquid-crystalline phases, such as that formed by potassium laurate/decanol/KCl/water, are found to accept readily large amphiphilic solute molecules. Since these phases spontaneously orient in high magnetic fields, it becomes possible to obtain NMR spectra of biologically interesting solutes in an oriented axially symmetric environment. The amide hydrogens of the peptide backbone of gramicidin D (Dubos) were exchanged for deuterium, and the gramicidin was incorporated into a lyotropic nematic phase made with deuteriated buffer in place of water. 2H NMR spectra of oriented, exchange-labeled gramicidin were then obtained. The strong water signal from the deuteriated buffer was eliminated by using selective excitation and a polynomial subtraction procedure. The 2H NMR spectra at high temperature consist of twelve major quadrupolar doublets. The splittings observed are largely independent of temperature, suggesting a highly rigid backbone structure. Two of the doublets, which are chemically shifted relative to the others, show stronger temperature dependence. These two probably arise from the exchangeable amino hydrogens on the tryptophan indole moieties of the peptide. While we cannot yet assign all of the doublets, the spectra and nuclear magnetic relaxation data are consistent with a rigid slightly distorted beta LD6.3 helix undergoing axially symmetric reorientation about the director of the liquid-crystalline phase. The correlation time for the axially symmetric reorientation is determined by relaxation measurements to be about 10(-7) s.     

Analysis of oligomeric stability of insulin analogs using hydrogen/deuterium exchange mass spectrometry

Insulin analog products for subcutaneous injection are prepared as solutions in which insulin analog molecules exist in several oligomeric states. Oligomeric stability can affect their onset and duration of action and has been exploited in designing them. To investigate the oligomeric stability of insulin analog products having different pharmacokinetics, we performed hydrogen/deuterium exchange mass spectrometry (HDX/MS), which is a rapid method to analyze dynamic aspects of protein structures. Two rapid-acting analogs (lispro and glulisine) incorporated deuteriums more and faster than recombinant human insulin, whereas a long-acting analog (glargine) and two intermediate-acting preparations (protamine-containing formulations) incorporated them less and more slowly. Kinetic analysis revealed that the number of slowly exchanged hydrogens (D(s)) (k<0.01 min(-1)) accounted for the difference in HDX reactivity among analogs. Furthermore, we found correlations between HDX kinetics and pharmacokinetics reported previously. Their maximum serum concentration (C(max)) was linearly correlated with D(s) (r=0.88) and the number of maximum exchangeable hydrogens (D(∞)) (r=0.89). The maximum drug concentration time (t(max)) was also correlated with reciprocals of D(s) and D(∞) (r=0.86 and r=0.96, respectively). Here we demonstrate the ability of HDX/MS to evaluate oligomeric stability of insulin analog products.     

Hydrogen exchange/electrospray ionization mass spectrometry studies of structural features of proteins and protein/protein interactions

The rate at which amide hydrogens located at the peptide backbone in protein/protein complexes undergo hydrogen/deuterium exchange is highly dependent on whether the amide groups participate in binding. Here, a new mass spectrometric method is presented in which this effect is utilized for the characterization of protein/ligand binding sites. The information obtained is which region within the protein participates in binding. The method includes hydrogen/deuterium exchange of receptor and ligand protein amide protons, binding, and back exchange. After this procedure those backbone amide groups that participate in protein binding are protected from back exchange and therefore still deuterated. These regions were then identified by peptic proteolysis, fast microbore high-performance liquid chromatography separation, and electrospray ionization mass spectrometry. The approach has been applied to the investigation of structural features of insulin-like growth factor I (IGF-I) and the interaction of insulin-like growth factor I with IGF-I binding protein 1. The data show that the approach can provide information on the location of the hydrophobic core of IGF-1 and on two regions that are mainly involved in binding to IGF-I binding protein 1. The data are consistent with results obtained with other approaches. The amount of sample required for one experiment is in the subnanomolar range.     

Determinants of protein hydrogen exchange studied in equine cytochrome c.

The exchange of a large number of amide hydrogens in oxidized equine cytochrome c was measured by NMR and compared with structural parameters. Hydrogens known to exchange through local structural fluctuations and through larger unfolding reactions were separately considered. All hydrogens protected from exchange by factors greater than 10(3) are in defined H-bonds, and almost all H-bonded hydrogens including those at the protein surface were measured to exchange slowly. H-exchange rates do not correlate with H-bond strength (length) or crystallographic B factors. It appears that the transient structural fluctuation necessary to bring an exchangeable hydrogen into H-bonding contact with the H-exchange catalyst (OH(-)-ion) involves a fairly large separation of the H-bond donor and acceptor, several angstroms at least, and therefore depends on the relative resistance to distortion of immediately neighboring structure. Accordingly, H-exchange by way of local fluctuational pathways tends to be very slow for hydrogens that are neighbored by tightly anchored structure and for hydrogens that are well buried. The slowing of buried hydrogens may also reflect the need for additional motions that allow solvent access once the protecting H-bond is separated, although it is noteworthy that burial in a protein like cytochrome c does not exceed 4 angstroms. When local fluctuational pathways are very slow, exchange can become dominated by a different category of larger, cooperative, segmental unfolding reactions reaching up to global unfolding.     

Hydrogen/deuterium exchange and aggregation of a polyvaline and a polyleucine alpha-helix investigated by matrix-assisted laser desorption ionization mass spectrometry

The membrane-associated pulmonary surfactant protein C (SP-C), containing a polyvaline alpha-helix, and a synthetic SP-C analogue with a polyleucine helix (SP-C(Leu)) were studied by hydrogen/deuterium exchange matrix-assisted laser desorption ionization (MALDI) mass spectrometry. SP-C, but not SP-C(Leu), formed abundant amyloid fibrils under experimental conditions. In CD(3)OD/D(2)O, 91:9 (v/v), containing 2 mM ammonium acetate, SP-C(Leu) and SP-C exchanged 40% of their exchangeable hydrogens within 1 min. This corresponds to exchange of labile side-chain hydrogen atoms, hydrogens on the N- and C-terminal heteroatoms, and amide hydrogen atoms in the unstructured N-terminal regions. After approximately 300 h, four exchangeable hydrogen atoms in SP-C(Leu) and 10 in SP-C remained unexchanged. During this time period the ion current corresponding to singly charged SP-C decreased to <10% of the initial value due to the formation of insoluble aggregates that are not detected by MALDI mass spectrometry. In contrast, the ion current for SP-C(Leu) was maintained over this time period, although the peptides were incubated together. In combination, hydrogen/deuterium exchange and aggregation data indicate that the polyleucine peptide refolds into a helix after opening, while the unfolded polyvaline peptide forms insoluble beta-sheet aggregates rather than refolding into a helix. The SP-C helix, but not the SP-C(Leu) helix, is thus in a metastable state, which may contribute to the recently observed tendency of SP-C and its precursor to misfold and aggregate in vivo.     

Proton magnetic resonance study of the influence of heme 2,4 substituents on the exchange rates of labile protons in the heme pocket of myoglobin.

Four exchangeable protons with large hyperfine shifts are assigned in the heme pocket of sperm whale met-cyano myoglobin reconstituted with heme possessing acetyl groups, ethyl groups, bromines, and hydrogens at the 2,4 position, using both relaxation and chemical-shift data. The four protons arise from the ring NH's of the proximal (F8), distal (E7), and FG2 histidines, and the peptide NH of His F8. The similarity of all chemical shifts to those of the native protein as well as the invariance of the relaxation rates of the distal histidyl ring NH dictate essentially the same structure for the heme cavity of both native and reconstituted proteins. The exchange rates with bulk water of the four labile proteins in each modified protein were determined by saturation-transfer and line width methods. All four labile protons were found to have the same exchange rate as in the native protein for acetyl and ethyl 2,4 substituents; the two resolved labile protons in the derivative with 2,4 bromine were also unchanged. The reconstituted protein with hydrogens at the 2,4 position exhibited slower exchange rates for three of the four protons, indicating an increased dynamic stability of the heme pocket in the absence of bulky 2,4 substituents.     

The presequence of a chimeric construct dictates which of two mechanisms are utilized for translocation across the thylakoid membrane: evidence for the existence of two distinct translocation systems.

The translocation of plastocyanin across the thylakoid membrane in Pisum sativum has been studied in reconstitution assays and using chimeric constructs. The reconstitution assays demonstrate that plastocyanin translocation is absolutely dependent on the presence of a stromal factor(s) and nucleotide triphosphates (NTPs), whereas neither element is required for the translocation of the 23 or 16 kDa proteins of the oxygen-evolving complex. Previous studies had revealed that the transthylakoidal delta pH is essential for translocation of the 23 and 16 kDa proteins but unnecessary for plastocyanin translocation. The basis for these mechanistic differences has been tested by analysing the translocation of a chimeric construct consisting of the presequence of the 23 kDa protein linked to the mature plastocyanin sequence. This construct is efficiently imported into thylakoids in the absence of stromal extracts or NTPs and translocation across the thylakoid membrane within intact chloroplasts is totally inhibited by the uncoupler nigericin: the translocation requirements are thus identical to those of the pre-23 kDa protein and diametrically opposite to those of pre-plastocyanin. Transport across the thylakoid membrane of a second fusion protein, consisting of the presequence of the 16 kDa protein linked to mature plastocyanin, is also dependent on a delta pH. The data suggest that two distinct systems are involved in the translocation of proteins across the thylakoid membrane, with each system recognizing specific signals within the presequences of a subset of lumenal protein precursors.     

Hydration effect on low-frequency protein dynamics observed in simulated neutron scattering spectra

Hydration effects on protein dynamics were investigated by comparing the frequency dependence of the calculated neutron scattering spectra between full and minimal hydration states at temperatures between 100 and 300 K. The protein boson peak is observed in the frequency range 1-4 meV at 100 K in both states. The peak frequency in the minimal hydration state shifts to lower than that in the full hydration state. Protein motions with a frequency higher than 4 meV were shown to undergo almost harmonic motion in both states at all temperatures simulated, whereas those with a frequency lower than 1 meV dominate the total fluctuations above 220 K and contribute to the origin of the glass-like transition. At 300 K, the boson peak becomes buried in the quasielastic contributions in the full hydration state but is still observed in the minimal hydration state. The boson peak is observed when protein dynamics are trapped within a local minimum of its energy surface. Protein motions, which contribute to the boson peak, are distributed throughout the whole protein. The fine structure of the dynamics structure factor is expected to be detected by the experiment if a high resolution instrument (<∼20 μeV) is developed in the near future.     

Automated extraction of backbone deuteration levels from amide H/2H mass spectrometry experiments

A Fourier deconvolution method has been developed to explicitly determine the amount of backbone amide deuterium incorporated into protein regions or segments by hydrogen/deuterium (H/D) exchange with high-resolution mass spectrometry. Determination and analysis of the level and number of backbone amide exchanging in solution provide more information about the solvent accessibility of the protein than do previous centroid methods, which only calculate the average deuterons exchanged. After exchange, a protein is digested into peptides as a way of determining the exchange within a local area of the protein. The mass of a peptide upon deuteration is a sum of the natural isotope abundance, fast exchanging side-chain hydrogens (present in MALDI-TOF H/2H data) and backbone amide exchange. Removal of the components of the isotopic distribution due to the natural isotope abundances and the fast exchanging side-chains allows for a precise quantification of the levels of backbone amide exchange, as is shown by an example from protein kinase A. The deconvoluted results are affected by overlapping peptides or inconsistent mass envelopes, and evaluation procedures for these cases are discussed. Finally, a method for determining the back exchange corrected populations is presented, and its effect on the data is discussed under various circumstances.     

Plastocyanin binding to photosystem I as a function of the charge state of the metal ion: effect of metal site conformation.

The binding of Ag- and Cd-substituted plastocyanin to reduced photosystem 1 of spinach has been studied through the rotational correlation time of plastocyanin measured by the technique of perturbed angular correlation of gamma-rays (PAC). Ag and Cd are used as models for native Cu(I) and Cu(II), respectively. A dissociation constant of 5 microM was found for Ag-plastocyanin, whereas the dissociation constant was at least 24 times higher for Cd-plastocyanin. PAC was further used to characterize the structure of the metal site of Cd- and Ag-plastocyanin. The Cd spectra are characteristic of a planar configuration of one cysteine and two histidines. However, the spectra show an unusual peak broadening and a high degree of internal motion, interpreted as motion of one of the histidines within the plane. (111)Ag decays to (111)Cd, followed by the emission of two gamma-rays used for the PAC experiment. The (111)Ag PAC spectra indicate that one of the coordinating histidines has a different position in the Ag protein than in the Cd protein but that the decay of Ag to Cd causes a relaxation of the position of this histidine to the position in the Cd protein within 20 ns. Binding of Ag-plastocyanin to photosystem I stabilized the Ag metal site structure so that no relaxation was observed on a time scale of 100 ns. This stabilization of the Ag structure upon binding indicates that the metal site structure is involved in regulating how the dissociation constant for plastocyanin depends on the charge of the metal ion.     

Hydrogen-bonded structure of the complex N-linked fetuin glycopeptide

The conformation of the N-linked complex glycopeptide of fetuin was examined with hydrogen-exchange techniques. The glycopeptide molecule contains eight acetamido hydrogens stemming from five N-acetylglucosamine residues and three N-acetylneuraminic acid residues and also one from the remaining sugar-peptide linkage. The hydrogen-exchange rates of these secondary amides were compared with small molecule model compounds having identical primary structures at their exchangeable hydrogen sites. Differences between the model rates and glycopeptide rates therefore cannot be accounted for by primary structure effects but reflect conformational features of the glycopeptide. Two glycopeptide hydrogens exhibit significantly hindered exchange; the rest exchange at the model rates. Removal of the three N-acetylneuraminic acid residues from terminal positions on the three branches of the glycopeptide removes the slowed hydrogens. The remaining ones continue to exchange at the model rate. These results indicate that two of the eight sugar acetamido hydrogens are involved in intramolecular hydrogen bonds. A likely structure includes two hydrogen bonds between the three N-acetylneuraminic acid residues. These two hydrogens, slowed to a moderate degree, reflect a preferred conformation stabilized by about 1 kcal/mol in free energy. The solution conformation of the glycopeptide suggested by these results is one that is partially ordered and can be easily modulated, owing to the relatively small amount of energy stabilizing the preferred conformation.     

Crystal structure of plastocyanin from a green alga, Enteromorpha prolifera

The crystal structure of the Cu-containing protein plastocyanin (Mr 10,500) from the green alga Enteromorpha prolifera has been solved by molecular replacement. The structure was refined by constrained-restrained and restrained reciprocal space least-squares techniques. The refined model includes 111 solvent sites. There is evidence for alternate conformers at eight residues. The residual is 0.12 for a data set comprising 74% of all observations accessible at 1.85 A resolution. The beta-sandwich structure of the algal plastocyanin is effectively the same as that of poplar leaf (Populus nigra var. italica) plastocyanin determined at 1.6 A resolution. The sequence homology between the two proteins is 56%. Differences between the contacts in the hydrophobic core create some significant (0.5 to 1.2 A) movements of the polypeptide backbone, resulting in small differences between the orientations and separations of corresponding beta-strands. These differences are most pronounced at the end of the molecule remote from the Cu site. The largest structural differences occur in the single non-beta strand, which includes the sole turn of helix in the molecule: two of the residues in a prominent kink of the poplar plastocyanin backbone are missing from the algal plastocyanin sequence, and there is a significant change in the position of the helical segment in relation to the beta-sandwich. Several other small but significant structural differences can be correlated with intermolecular contacts in the crystals. An intramolecular carboxyl-carboxylate hydrogen bond in the algal plastocyanin may be associated with an unusually high pKa. The dimensions of the Cu site in the two plastocyanins are, within the limits of precision, identical.     

Hydrogen exchange in nucleosides and nucleotides. Measurement of hydrogen exchange by stopped-flow and ultraviolet difference spectroscopy.

Time-dependent changes in the ultraviolet absorbance of the adenine chromophore are observed in the stopped-flow spectrophotometer when adenosine and its analogs are rapidly transferred from protium oxide to deuterium oxide. These absorbance changes are shown to result from hydrogen exchange in the exocyclic amino groups of the purine ribonucleosides by using derivatives of adenosine in which methyl groups replace exchangeable hydrogens and by showing that the general characteristics of hydrogen exchange in adenosine analogs agree with those found here. A study of the dependence of hydrogen-exchange rate constants on adenosine, AMP, and phosphate concentration showed there is a second-order dependence on AMP concentration which is primarily due to intermolecular catalysis by the phosphate group of the nucleotide. The deuterium oxide perturbation difference spectrum, obtained at equilibrium, was found to contain two components that result from blue shifts of the adenine chromophore absorbance: (1) a shift cause by the substitution of deuterium for protium in the ring (N1) nitrogen and exocyclic nitrogens, and (2) a shift associated with a change in the polarizability of the medium. Since the theory of solvent perturbation, which is used to measure the relative "exposure" of chromophores in macromolecules, assumes that the spectral shifts observed are solely due to (2) above, the use of deuterium oxide as a measure of chromophore exposure to perturbants the size of water must be reexamined.     

Abeta protofibrils possess a stable core structure resistant to hydrogen exchange

Protofibrils are transient structures observed during in vitro formation of mature amyloid fibrils and have been implicated as the toxic species responsible for cell dysfunction and neuronal loss in Alzheimer's disease (AD) and other protein aggregation diseases. To better understand the roles of protofibrils in amyloid assembly and Alzheimer's disease, we characterized secondary structural features of these heterogeneous and metastable assembly intermediates. We chromatographically isolated different size populations of protofibrils from amyloid assembly reactions of Abeta(1-40), both wild type and the Arctic variant associated with early onset familial AD, and exposed them to hydrogen-deuterium exchange analysis monitored by mass spectrometry (HX-MS). We show that HX-MS can distinguish among unstructured monomer, protofibrils, and fibrils by their different protection patterns. We find that about 40% of the backbone amide hydrogens of Abeta protofibrils are highly resistant to exchange with deuterium even after 2 days of incubation in aqueous deuterated buffer, implying a very stable, presumably H-bonded, core structure. This is in contrast to mature amyloid fibrils, whose equally stable structure protects about 60% of the backbone amide hydrogens over the same time frame. We also find a surprising degree of specificity in amyloid assembly, in that wild type Abeta is preferentially excluded from both protofibrils and fibrils grown from an equimolar mixture of wild type and Arctic mutant peptides. These and other data are interpreted and discussed in terms of the role of protofibrils in fibril assembly and in disease.