Use of amide 1H-nmr titration shifts for studies of polypeptide conformation

This paper shows that backbone amide proton titration shifts in polypeptide chains are a very sensitive manifestation of intramolecular hydrogen bonding between carboxylate groups and backbone amide protons. The population of specific hydrogen-bonded structures in the ensemble of species that constitutes the conformation of a flexible nonglobular linear peptide can be determined from the extent of the titration shifts. As an illustration, an investigation of the molecular conformation of the linear peptide H-Gly-Gly-L -Glu-L -Ala-OH is described. The proposed use of amide proton titration shifts for investigating polypeptide conformation is based on 360-MHz ¹H-nmr studies of selected linear oligopeptides in H₂O solutions. It was found that only a very limited number of amide protons in a polypeptide chain show sizable intrinsic intration shifts arising from through-bond interactions with ionizable groups. These are the amide proton of the C-terminal amino acid residue, the amide protons of Asp and the residues following Asp, and possibly the amide proton of the residue next to the N-terminus. Since the intrinsic titration shifts are upfield, the downfield titration shifts arising from conformation-dependent through-space interactions, in particular hydrogen bonding between the amide protons and carboxylate groups, can readily be identified.     

Solid state NMR studies of hydrogen bonding in a citrate synthase inhibitor complex.

The ionization state and hydrogen bonding environment of the transition state analogue (TSA) inhibitor, carboxymethyldethia coenzyme A (CMX), bound to citrate synthase have been investigated using solid state NMR. This enzyme-inhibitor complex has been studied in connection with the postulated contribution of short hydrogen bonds to binding energies and enzyme catalysis: the X-ray crystal structure of this complex revealed an unusually short hydrogen bond between the carboxylate group of the inhibitor and an aspartic acid side chain [Usher et al. (1994) Biochemistry 33, 7753-7759]. To further investigate the nature of this short hydrogen bond, low spinning speed 13C NMR spectra of the CMX-citrate synthase complex were obtained under a variety of sample conditions. Tensor values describing the chemical shift anisotropy of the carboxyl groups of the inhibitor were obtained by simulating MAS spectra (233 +/- 4, 206 +/- 5, and 105 +/- 2 ppm vs TMS). Comparison of these values with our previously reported database and ab initio calculations of carbon shift tensor values clearly indicates that the carboxyl is deprotonated. New data from model compounds suggest that hydrogen bonds in a syn arrangement with respect to the carboxylate group have a pronounced effect upon the shift tensors for the carboxylate, while anti hydrogen bonds, regardless of their length, apparently do not perturb the shift tensors of the carboxyl group. Thus the tensor values for the enzyme-inhibitor complex could be consistent with either a very long syn hydrogen bond or an anti hydrogen bond; the latter would agree very well with previous crystallographic results. Two-dimensional 1H-13C heteronuclear correlation spectra of the enzyme-inhibitor complex were obtained. Strong cross-peaks were observed from the carboxyl carbon to proton(s) with chemical shift(s) of 22 +/- 5 ppm. Both the proton chemical shift and the intensity of the cross-peak indicate a very short hydrogen bond to the carboxyl group of the inhibitor, the C.H distance based upon the cross-peak intensity being 2.0 +/- 0.4 A. This proton resonance is assigned to Hdelta2 of Asp 375, on the basis of comparison with crystal structures and the fact that this cross-peak was absent in the heteronuclear correlation spectrum of the inhibitor-D375G mutant enzyme complex. In summary, our NMR studies support the suggestion that a very short hydrogen bond is formed between the TSA and the Asp carboxylate.     

Proton nuclear magnetic resonance study of an active pentapeptide fragment of ubiquitin

The aqueous solution conformation of Tyr-Asn-Ile-Gln-Lys (UB5) corresponding to positions 59-63 of the polypeptide, ubiquitin, has been investigated by proton NMR. Like the parent protein, UB5 induces nonspecifically both T and B lymphocyte differentiation. The various NH and CH resonances of this pentapeptide have been assigned, and its solution conformation has been probed through a study of chemical shift variations with pH, temperature dependence of amide hydrogen chemical shifts, vicinal NH--C alpha H and C alpha H--C beta H2 coupling constant data, and amide hydrogen-exchange rates. The latter were measured in H2O by using a combination of transfer of solvent saturation and saturation recovery NMR experiments. The data are compatible with the assumption of a highly motile dynamic equilibrium among different conformations for this peptide. The various secondary amide hydrogens remain essentially exposed to the solvent. The temperature-dependence study of the amide hydrogen chemical shifts also did not reveal any strong internal hydrogen bonds. A rotamer population analysis of tyrosine and asparagine side chains suggests that two of the rotomers are predominantly populated for each of these residues. From these results, a picture emerges of the dynamic conformation of UB5 in aqueous solution.     

The hydration of amides in helices; a comprehensive picture from molecular dynamics,IR, and NMR

We examined the hydration of amides of alpha(3)D, a simple, designed three-helix bundle protein. Molecular dynamics calculations show that the amide carbonyls on the surface of the protein tilt away from the helical axis to interact with solvent water, resulting in a lengthening of the hydrogen bonds on this face of the helix. Water molecules are bonded to these carbonyl groups with partial occupancy ( approximately 50%-70%), and their interaction geometries show a large variation in their hydrogen bond lengths and angles on the nsec time scale. This heterogeneity is reflected in the carbonyl stretching vibration (amide I' band) of a group of surface Ala residues. The surface-exposed amides are broad, and shift to lower frequency (reflecting strengthening of the hydrogen bonds) as the temperature is decreased. By contrast, the amide I' bands of the buried (13)C-labeled Leu residues are significantly sharper and their frequencies are consistent with the formation of strong hydrogen bonds, independent of temperature. The rates of hydrogen-deuterium exchange and the proton NMR chemical shifts of the helical amide groups also depend on environment. The partial occupancy of the hydration sites on the surface of helices suggests that the interaction is relatively weak, on the order of thermal energy at room temperature. One unexpected feature that emerged from the dynamics calculations was that a Thr side chain subtly disrupted the helical geometry 4-7 residues N-terminal in sequence, which was reflected in the proton chemical shifts and the rates of amide proton exchange for several amides that engage in a mixed 3(10)/alpha/pi-helical conformation.     

Intramolecular hydrogen bonds and conformation of the lincomycin molecule in organic solvents

IR spectra (1600-1800 and 3000-3650 cm-1) of lincomycin base solutions in inert (CCl4 and C2Cl4), proton acceptor (dioxane, dimethylsulfoxide and triethyl amine) and proton donor (CHCl3, CD3OD and D2O) solvents were studied. Analysis of the concentration and temperature changes in the spectra revealed that association in lincomycin in the inert solvents was due to intramolecular hydrogen linkage involving amide and hydroxyl groups. Disintegration of the associates after the solution dilution and temperature rise was accompanied by formation of intramolecular bonds stabilizing the stable conformation structure of the lincomycin molecule. The following hydrogen linkage in the conformation was realized: NH...N (band v NH...N at 3340 cm-1), OH...O involving the hydroxyl at C-7 and O atoms in the D-galactose ring (band v OH...O at 3548 cm-1), a chain of the hydrogen bonds OH...OH...OH in the lincomycin carbohydrate moiety (band v OH...O at 3593 cm-1 and v OH of the end hydroxyl group at 3625 cm-1). Bonds NH and C-O of the amide group were located in transconformation. Group C-O did not participate in the intramolecular hydrogen linkage.     

Molecular modelling of secondary and tertiary structures of hyaluronan,compared with electron microscopy and NMR data. Possible sheets and tubular structures in aqueous solution

Electron microscopy shows that hyaluronan (HA) forms sheets and tube-like structures in solution. Molecular modelling by Tartu plastic space-filling atomic models revealed that hydroxymethyl and carboxylate groups of HA anti-parallel chains can be joined by H-bonds. Using these bonds, HA molecules can be modelled as sheets and tubules. These tertiary structures have three kinds of lateral contact: (1) antiparallel chains stacked by hydrophobic patches; (2) parallel chains joined by both stacking interactions and H-bonds; and (3) crossing chains joined by H-bonds and stacking interactions. Sheet and tubular structures may explain some viscoelastic and biological properties of HA.     

1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: the amide proton environment

The exchangeable amide protons of hyaluronic acid (HA) oligosaccharides and a higher-molecular-weight segment dissolved in H2O at pH 2.5 or 5.5 were examined by H NMR spectroscopy at 250 MHz. The HA segment preparation showed a single amide resonance, near the chemical shift for the amide proton of the monosaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-GlcNAc). Smaller HA oligosaccharides showed two or three separate amide proton resonances, corresponding in relative peak area to interior or end GlcNAc residues. The interior GlcNAc amide resonance occurred at the same chemical shift as the single resonance of the HA segment. For the end GlcNAc residues, linkage to D-glucuronopyranose (GlcUA) through C1 resulted in an upfield shift relative to the beta-anomer of GlcNAc, whereas linkage through C3 resulted in a downfield shift relative to the corresponding anomer of GlcNAc. These chemical-shift perturbations appeared to be approximately offsetting in the case of linkage at both positions. The amide proton vicinal coupling constant (ca. 9 Hz) was found to be essentially independent of chain length, residue position, or solution pH. These data favor a nearly perpendicular orientation for the acetamido group with respect to the sugar ring, little affected by linkage of GlcNAc to GlcUA. No evidence for the existence of a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. The amide proton resonances for chondroitin, chondroitin 4-sulfate, and dermatan sulfate were compared to that of HA. The chemical shifts of these resonances deviated no more than 0.1 ppm from that of HA. A small dependence on the identity of the adjacent uronic acid residue was noted, based on the observation of two resonances for dermatan sulfate.     

Conserved hydrogen-bond motifs of membrane transporters and receptors

Membrane transporters and receptors often rely on conserved hydrogen bonds to assemble transient paths for ion transfer or long-distance conformational couplings. For transporters and receptors that use proton binding and proton transfer for function, inter-helical hydrogen bonds of titratable protein sidechains that could change protonation are of central interest to formulate hypotheses about reaction mechanisms. Knowledge of hydrogen bonds common at sites of potential interest for proton binding could thus inform and guide studies on functional mechanisms of protonation-coupled membrane proteins. Here we apply graph-theory approaches to identify hydrogen-bond motifs of carboxylate and histidine sidechains in a large data set of static membrane protein structures. We find that carboxylate-hydroxyl hydrogen bonds are present in numerous structures of the dataset, and can be part of more extended H-bond clusters that could be relevant to conformational coupling. Carboxylate-carboxyamide and imidazole-imidazole hydrogen bonds are represented in comparably fewer protein structures of the dataset. Atomistic simulations on two membrane transporters in lipid membranes suggest that many of the hydrogen bond motifs present in static protein structures tend to be robust, and can be part of larger hydrogen-bond clusters that recruit additional hydrogen bonds.     

Structural analysis of d(GCAATTGC)2 and its complex with berenil by nuclear magnetic resonance spectroscopy

The structures of d(GCAATTGC)2 and its complex with berenil in solution were analyzed by two-dimensional 1H NMR spectroscopy. Intra- and internucleotide nuclear Overhauser effect (NOE) connectivities demonstrate that the octanucleotide duplex is primarily in the B conformation. Binding with berenil stabilizes the duplex with respect to thermal denaturation by about 10 degrees C, based on the appearance of the imino proton signals. The berenil-d(GCAATTGC)2 system is in fast exchange on the NMR time scale. The two-dimensional NMR data reveal that berenil binds in the minor groove of d(GCAATTGC)2. The aromatic drug protons are placed within 5 A of the H2 proton of both adenines, the H1', H5', and H5" of both thymidines, and the H4', H5', and H5" of the internal guanosine. The amidine protons on berenil are also close to the H2 proton of both adenines. The duplex retains an overall B conformation in the complex with berenil. At 18 degrees C, NOE contacts at longer mixing times indicate the presence of end-to-end association both in the duplex alone and also in its complex with berenil. These intermolecular contacts either vanished or diminished substantially at 45 degrees C. Two molecular models are proposed for the berenil-(GCAATTGC)2 complex; one has hydrogen bonds between the berenil amidine protons and the carbonyl oxygen, O2, of the external thymines, and the other has hydrogen bonds between the drug amidine protons and the purine nitrogen, N3, of the internal adenines. Quantitative analysis of the NOE data favors the second model.     

Thermodynamic stabilities of internal loops with GU closing pairs in RNA

Many internal loops that form tertiary contacts in natural RNAs have GU closing pairs; examples include the tetraloop receptor and P1 helix docking site in group I introns. Thus, thermodynamic parameters of internal loops with GU closing pairs can contribute to the prediction of both secondary and tertiary structure. Oligoribonucleotide duplexes containing small internal loops with GU closing pairs were studied by optical melting, one-dimensional imino proton NMR, and one-dimensional phosphorus NMR. The thermodynamic stabilities of asymmetric internal loops with GU closing pairs relative to those of loops with GC closing pairs may be explained by hydrogen bonds. In contrast, the free energy increments for symmetric internal loops of two noncanonical pairs with GU closing pairs relative to loops with GC closing pairs show much more sequence dependence. Imino proton and phosphorus NMR spectra suggest that some GA pairs adjacent to GU closing pairs may form an overall thermodynamically stable but non-A-form conformation.     

Redox-related conformational changes in Rhodobacter capsulatus cytochrome c2

WEFT-NOESY and transfer WEFT-NOESY NMR spectra were used to determine the heme proton assignments for Rhodobacter capsulatus ferricytochrome c2. The Fermi contact and pseudo-contact contributions to the paramagnetic effect of the unpaired electron in the oxidized state were evaluated for the heme and ligand protons. The chemical shift assignments for the 1H and 15N NMR spectra were obtained by a combination of 1H-1H and 1H-15N two-dimensional NMR spectroscopy. The short-range nuclear Overhauser effect (NOE) data are consistent with the view that the secondary structure for the oxidized state of this protein closely approximates that of the reduced form, but with redox-related conformational changes between the two redox states. To understand the decrease in stability of the oxidized state of this cytochrome c2 compared to the reduced form, the structural difference between the two redox states were analyzed by the differences in the NOE intensities, pseudo-contact shifts and the hydrogen-deuterium exchange rates of the amide protons. We find that the major difference between redox states, although subtle, involve heme protein interactions, orientation of the heme ligands, differences in hydrogen bond networks and, possible alterations in the position of some internal water molecules. Thus, it appears that the general destabilization of cytochrome c2, which occurs on oxidation, is consistent with the alteration of hydrogen bonds that result in changes in the internal dynamics of the protein.     

Conformational changes in the metal-binding sites of cardiac troponin C induced by calcium binding.

Isotope labeling of recombinant normal cardiac troponin C (cTnC3) with 15N-enriched amino acids and multidimensional NMR were used to assign the downfield-shifted amide protons of Gly residues at position 6 in Ca(2+)-binding loops II, III, and IV, as well as tightly hydrogen-bonded amides within the short antiparallel beta-sheets between pairs of Ca(2+)-binding loops. The amide protons of Gly70, Gly110, and Gly146 were found to be shifted significantly downfield from the remaining amide proton resonances in Ca(2+)-saturated cTnC3. No downfield-shifted Gly resonance was observed from the naturally inactive site I. Comparison of downfield-shifted amide protons in the Ca(2+)-saturated forms of cTnC3 and CBM-IIA, a mutant having Asp65 replaced by Ala, demonstrated that Gly70 is hydrogen bonded to the carboxylate side chain of Asp65. Thus, the hydrogen bond between Gly and Asp in positions 6 and 1, respectively, of the Ca(2+)-binding loop appears crucial for maintaining the integrity of the helix-loop-helix Ca(2+)-binding sites. In the apo- form of cTnC3, only Gly70 was found to be shifted significantly downfield with respect to the remaining amide proton resonances. Thus, even in the absence of Ca2+ at binding site II, the amide proton of Gly70 is strongly hydrogen bonded to the side-chain carboxylate of Asp65. The amide protons of Ile112 and Ile148 in the C-terminal domain and Ile36 in the N-terminal domain data-sheets exhibit chemical shifts consistent with hydrogen-bond formation between the pair of Ca(2+)-binding loops in each domain of Ca(2+)-saturated cTnC3.(ABSTRACT TRUNCATED AT 250 WORDS)     

A systematic evaluation of different hydrogen bonding patterns in unsymmetrical 1,n′-disubstituted ferrocenoyl peptides

The synthesis and spectroscopic characterization of 21 l,l′-disubstituted ferrocenoyl peptides of the general formula [Fe(C₅H₄-CO-Aal-OR) (C₅H₄-CO-Aa2-OR′)] is reported, with Aal and Aa2 being different amino acids. The one-pot synthesis from activated ferrocene-l,l′-dicarboxylic acid and two different amino acid esters gives the unsymmetrical ferrocenoyl peptides in yields between 27% and 42%, which can be easily separated from their symmetrical byproducts by column chromatography. All new compounds are comprehensively characterized by mass spectrometry (El and FAB, including high-resolution EI-MS), ¹H and ¹³C NMR, and UV/Vis spectroscopy. CD spectroscopy in conjunction with ¹H NMR is used to elucidate the solution structures. Using the achiral glycine (Gly) as Aal permits to determine qualitatively the structure-determining influence of the different amino acids Aa2. Helically chiral structures in ferrocene amino acids in this study are stabilized by hydrogen bonds. If one hydrogen bond partner is systematically moved away by the introduction of methylene groups, then indeed the strength of the hydrogen bond decreases as indicated by ¹H NMR chemical shifts of the amide protons and the strength of characteristic CD bands. As proline (Pro) is the only naturally accuring secondary amino acid it cannot contribute any amide proton to intra-strand hydrogen bonding. DFT calculations on the compound [Fe(C₅H₄-CO-Gly-OMe)(C₅H₄-CO-Pro-OMe)] with one achiral and one secondary amino acid were therefore performed to quantify the more subtle influence of the relative orientations of the ferrocene carbonyl groups and the cis-/trans-conformation of both amide bonds. Not unexpectedly, the conformations with both amide bonds in cis orientation are highest in energy. Surprisingly, the calculations suggest the presence of a low-energy conformation with a non-classical hydrogen bond between the proline ester carbonyl oxygen and a glycine H_α atom. However, a second conformation with no apparent intra-strand contacts but optimal positioning of all relevant groups is similar in energy. Although two conformations were observed in solution for this compound, the experimental data did not permit to assign those two conformations.     

Imino proton NMR assignments and ion-binding studies on Escherichia coli tRNA3Gly

The imino region of the proton NMR spectrum of Escherichia coli tRNA3Gly has been assigned mainly by sequential nuclear Overhauser effects between neighbouring base pairs and by comparison of assignments of other tRNAs. The effects of magnesium, spermine and temperature on the 1H and 31P NMR spectra of this tRNA were studied. Both ions affect resonances close to the G15 . C48 tertiary base pair and in the ribosylthymine loop. The magnesium studies indicate the presence of an altered tRNA conformer at low magnesium concentrations in equilibrium with the high magnesium form. The temperature studies show that the A7 . U66 imino proton (from a secondary base pair) melts before some of the tertiary hydrogen bonds and that the anticodon stem does not melt sequentially from the ends. Correlation of the ion effects in the 1H and 31P NMR spectra has led to the tentative assignment of two 31P resonances not assigned in the comparable 31P NMR spectrum of yeast tRNAPhe. 31P NMR spectra of E. coli tRNA3Gly lack resolved peaks corresponding to peaks C and F in the spectra of E. coli tRNAPhe and yeast tRNAPhe. In the latter tRNAs these peaks have been assigned to phosphate groups in the anticodon loop. Ion binding E. coli tRNA3Gly and E. coli tRNAPhe had different effects on their 1H NMR spectra which may reflect further differences in their charge distribution and conformation.     

NMR study of thymulin, a lymphocyte differentiating thymic nonapeptide. Conformational states of free peptide in solution

The nonapeptide less than Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (formerly called serum thymic factor) is a factor produced by the thymic epithelium, which needs a zinc ion to express its immunoregulatory properties. We report here on 1H and 13C NMR investigation of the conformational properties of the free peptide in aqueous medium and in dimethyl sulfoxide-d6 solution by a combination of homo- and heteronuclear one- and two-dimensional experiments. The various resonances have been assigned in a straightforward manner on the basis of 1H,1H COSY spectroscopy for the recognition of the proton spin systems; two-dimensional NOESY spectra with the correlation peaks across amide bonds and for the amino acid sequence assignment; amide bonds and for the amino acid sequence assignment; 13C,1H COSY experiments using selective polarization transfer from 1H- to 13C-nucleus via the 13C,1H long-range couplings for the attribution of the carboxyl and carbonyl groups; and 13C,1H COSY experiments with selective polarization transfer via the 13C,1H direct couplings for the assignment of all the aliphatic carbons. Other experiments such as pH-dependent chemical shifts, combined use of multiple and selective proton-decoupled 1H and 13C NMR spectra, the temperature and the concentration dependence of the proton shifts of the amide resonances, the solvent dependences of peptide carbonyl carbon resonances, and comparison of the spectra with three different analogues were performed. In aqueous solution, the data are compatible with the assumption of a highly mobile dynamic equilibrium among different conformations, whereas in dimethyl sulfoxide-d6, a more rigid structure is found involving three internal hydrogen bonds. These observations provide an insight into the conformational tendencies of this peptidic hormone in two different media.     

Binding mechanism of trans-N-caffeoyltyramine and human serum albumin: Investigation by multi-spectroscopy and docking simulation

trans-N-Caffeoyltyramine (TNC), which was isolated from the Cortex Lycii in our laboratory, is a phenolic amide compound with multiple pharmacological activities. The interaction between TNC and human serum albumin (HSA) was studied by Nuclear magnetic resonance (NMR) relaxation experiment, fluorescence spectroscopy, and docking simulation. NMR methodology is based on the analysis of selective and non-selective spin-lattice relaxation rate enhancements of TNC protons in the presence of the HSA. Result indicated that the interaction occurred between HSA and TNC, and changed the proton magnetic environment of TNC. Fluorescence spectroscopy confirmed that TNC displayed a strong capability to quench the fluorescence of HSA, and the acting forces for binding were hydrogen bonds and van der Waals forces. Furthermore, the circular dichroism, synchronous, and three-dimensional fluorescence spectra, which were employed to determine the conformation of protein, revealed that binding of TNC with HSA could induce conformational changes in HSA. In addition, the molecular modeling results exhibited that TNC mainly bonded to site I in sub-domain IIA of HSA.     

Hydrogen bonding and equilibrium protium-deuterium fractionation factors in the immunoglobulin G binding domain of protein G

Protium-deuterium fractionation factors (phi) were determined for more than 85% of the backbone amide protons in the IgG binding domains of protein G, GB1 and GB2, from NMR spectra recorded over a range of H2O/D2O solvent ratios. Previous studies suggest a correlation between phi and hydrogen bond strength; amide and hydroxyl groups in strong hydrogen bonds accumulate protium (phi < 1), while weak hydrogen bonds accumulate deuterium (phi > 1). Our results show that the alpha-helical residues have slightly lower phi values (1.03 +/- 0.05) than beta-sheet residues (1.12 +/- 0.07), on average. The lowest phi value obtained (0.65) does not involve a backbone amide but rather is for the interaction between two side chains, Y45 and D47. Fractionation factors for solvent-exposed residues are between the alpha-helix and beta-sheet values, on average, and are close to those for random coil peptides. Further, the difference in phiav between alpha-helix and solvent-exposed residues is small, suggesting that differences in hydrogen bond strength for intrachain hydrogen bonds and amide...water hydrogen bonds are also small. Overall, the enrichment for deuterium suggests that most backbone...backbone hydrogen bonds are weak.     

Conformational transition between native and reactive center cleaved forms of alpha 1-antitrypsin by Fourier transform infrared spectroscopy and small-angle neutron scattering

alpha 1-Antitrypsin (alpha 1-AT) is the best-characterized member of the serpin superfamily of plasma proteins. Protease inhibitor members of this family undergo a characteristic reactive-center cleavage during expression of their inhibitory activity. The physical basis of this transition in alpha 1-AT from the stressed native conformation to the more stable reactive center cleaved (split) form was studied by Fourier transform infrared (FT-IR) spectroscopy and neutron scattering. The FT-IR spectra show that, while split alpha 1-AT has three intense well-resolved components associated with the presence of antiparallel beta-sheet and alpha-helix conformations, the amide I band of native alpha 1-AT has only one intense component, associated with the presence of beta-sheet structure. 1H-2H exchange within the polypeptide backbone, studied by FT-IR and NMR spectroscopy, shows that the native form undergoes greater exchange than the split form. Under the same conditions, neutron scattering shows no differences in the radius of gyration RG of the native and the split forms. In contrast, in high concentrations of phosphate approaching those used for crystallization, the native form (unlike the split form) undergoes dimerization. These data indicate that the conformational transition largely involves localized secondary and tertiary structure rearrangements. We propose that the energetically stressed native alpha 1-AT structure is the consequence of a significantly reduced number of hydrogen bonds in secondary structure components and that reactive-site cleavage between Met358 and Ser359 is the key for the development of the fully hydrogen bonded more stable serpin structure.