Studies on the conformational properties of CP-10(42-55), the hinge region of CP-10, using circular dichroism and RP-HPLC.

The conformational properties of CP-10(42-55), a peptide corresponding to the hinge region of CP-10, were investigated using circular dichroism spectroscopy and reverse-phase high-performance liquid chromatography (RP-HPLC). The circular dichroism studies indicated that CP-10(42-55) formed considerable secondary structure in the presence of hydrophobic solution environments including 50% acetonitrile, 50% trifluoroethanol and 200 mM sodium dodecyl sulfate, which comprised a mixture of alpha-helix and beta-sheet. The effect of temperature on the conformation of CP-10(42-55) was investigated between 5 and 40 degrees C, with very small changes in the spectra being observed. RP-HPLC was then used to investigate the effect of temperature on the conformation of CP-10(42-55) in the presence of a hydrophobic surface. Using a C18-adsorbent, CP-10(42-55) exhibited a conformational transition at 25 degrees C, which was associated with an increase in the chromatographic contact area and the binding affinity of the peptide for the stationary phase. In addition, near-planar bandbroadening behaviour indicated that conformational species interconverted with rapid rate constants compared with the chromatographic time scale. These results indicated that the conformational change at 25 degrees C in the RP-HPLC system most likely corresponds to the unfolding of an alpha-helical and/or beta-sheet structure to an extended coil structure. Therefore, the strong chemotactic properties of this peptide may be attributed to its ability to form considerable secondary structure in the presence of a hydrophobic environment.     

Effect of temperature on Ca-ATPase from sarcoplasmic reticulum membranes: ESR studies

Using spin-labeled fatty acid derivatives and maleimide, the effect of temperature on the structural state of various parts of the lipid bilayer of sarcoplasmic reticulum (SR) membranes and the segmental motion of the Ca-ATPase molecule were investigated. The mobility of the spin probes localized in the hydrophobic zone and the outer part of the SR membrane was shown to increase with a rise in temperature from 4 to 44 degrees C, the temperature of 20 degrees C being critical for these changes. In the presence of ATP, critical changes in the spin probe mobility occur at lower temperatures, while in the presence of ATP and Ca2+ they are observed at 20 degrees C for a spin probe localized in the outer part of the SR membrane. The mobility of a spin probe localized in the hydrophobic part of the membrane increases linearly with a rise in temperature. In the absence of ligands, the segmental motion of Ca-ATPase changes linearly within a temperature range of 10-30 degrees C. However, when ATP alone or ATP and Ca2+ are simultaneously added to the incubation mixture, the protein mobility undergoes critical changes at 20 degrees C. The Arrhenius plots for ATPase activity and Ca2+ uptake rate in SR membrane preparations also have a break at 20 degrees C. It is assumed that changes in the structural state of membrane lipids produce conformational changes in the Ca-ATPase molecule; the enzyme seems to be unsensitive to the structural state of the membrane lipid matrix in the absence of the ligands.     

Mismatch-induced DNA unbending upon duplex opening

A DNA duplex can be torn open at a specific position by introducing a branch or bulge to create an asymmetric three-way junction (TWJ). The opened duplex manifests a bent conformation (bending angle approximately 60 degrees , relative to the unopened form), which leads to a dramatic decrease in gel electrophoretic mobility. In the presence of a basepair mismatch at the opening position, the DNA backbone becomes less bent and assumes a distorted T-shaped structure, resulting in an increase in polyacrylamide gel electrophoretic mobility. Both conformational changes are confirmed using fluorescence resonance energy transfer experiments and found to be similar to the signature conformational changes of DNA duplex upon MutS protein binding. Our results imply that some structural rearrangements essential for mismatch recognition are achievable without protein interference. The gel electrophoretic mobility data for DNA TWJs with and without base mismatches correlates well with rotational diffusivity, computed by taking into account the conformational change of TWJ induced by base mismatch.     

Elucidation of the nature of the conformational changes of the EF-interhelical loop in bacteriorhodopsin and of the helix VIII on the cytoplasmic surface of bovine rhodopsin: a time-resolved fluorescence depolarization study

The conformation of the AB-loop and EF-loop of bacteriorhodopsin and of the fourth cytoplasmic loop (helix VIII) of bovine rhodopsin were assessed by a combination of time-resolved fluorescence depolarization and site-directed fluorescence labeling. The fluorescence anisotropy decays were measured employing a tunable Ti:sapphire laser/microchannel plate based single-photon counting apparatus with picosecond time resolution. This method allows measurement of the diffusional dynamics of the loops directly on a nanosecond time-scale. We implemented the method to study model peptides and two-helix systems representing sequences of bacteriorhodopsin. Thus, we systematically analyzed the anisotropic behavior of four different fluorescent dyes covalently bound to a single cysteine residue on the protein surface and assigned the anisotropy decay components to the modes of motion of the protein and its segments. We have identified two mechanisms of loop conformational changes in the functionally intact proteins bacteriorhodopsin and bovine rhodopsin. First, we found a surface potential-dependent transition between two conformational states of the EF-loop of bacteriorhodopsin, detected with the fluorescent dye bound to position 160. A transition between the two conformational states at 150mM KCl and 20 degrees C requires a surface potential change that corresponds to Deltasigma approximately -1.0e(-)/bacteriorhodopsin molecule. We suggest, that the surface potential-based switch of the EF-loop is the missing link between the movement of helix F and the transient surface potential change detected during the photocycle of bacteriorhodopsin. Second, in the visual pigment rhodopsin, with the fluorescent dye bound to position 316, a particularly striking pH-dependent conformational change of the fourth loop on the cytoplasmic surface was analyzed. The loop mobility increased from pH 5 to 8. The midpoint of this transition is at pH 6.2 and correlates with the midpoint of the pH-dependent equilibrium between the active metarhodopsin II and the inactive metarhodopsin I state.     

The conformational dynamics of the tetramer hemoglobin molecule as revealed by hydrogen exchange. III. Influence of the heme removal

Two main types of conformational fluctuations--local and global are characteristic of the native protein structure and revealed by hydrogen exchange. The probability of those fluctuations changes to a different extent upon hemoglobin oxygenation, changing of pH, splitting of the intersubunit contacts. To compare with the influence of the heme removal the rate of the H-D exchange of the peptide NH atoms of the human apoHb was studied at the pH range 5.5-9.0 and temperature 10-38 degrees C by the IR spectroscopy. The removal of the heme increases the rate of the H-D exchange of the 80% peptide NH atoms with the factor retardation of the exchange rate (P) in the range approximately 10(2)-10(8). For the most of the peptide NH atoms the probability of the local fluctuations weakly depends on the temperature, the enthalpy changes upon all such local conformational transitions deltaH(op) degrees are 0-15 kcal/M. Characterized by the stronger temperature dependence the global fluctuations are not arised upon the temperature increases up to 38 degrees C at pH 7.0 inspite of in these conditions the slow denaturation and aggregation of apoHb begin to occur. Upon the destabilization of the apoHb structure by the simultaneous decreasing of pH to 5.5 and temperature to 10 degrees C the global fluctuations of the apoHb native structure described by deltaH(op)o < 0 begin to intensify. The mechanism of the overall intensification of the local fluctuations upon the heme removal, the peculiarity of the heat denaturation of apoHb in conditions, close to that existing upon the selfassembly of Hb in vivo, and analogy between low temperature global fluctuations and cold denaturation of globular proteins are discussed.     

Bacteriorhodopsin retains the conformation of Val69-Gly72 fragment during functioning

Effect of the monoclonal antibody (MAb) 5B6 produced to the solubilized preparation of bacteriorhodopsin on the protein photocycle was studied to examine conformational rearrangements on the surface of functioning bacteriorhodopsin molecule. Using the methods of solid phase enzyme immunoassay, peptide phage display, and 1H NMR spectroscopy, we demonstrated that the epitope recognized by MAb 5B6 is the Val69-Pro-Phe-Gly72 fragment of the protein, with the aromatic ring of Phe71 and the methyl groups of Val69 participating in the binding. MAb 5B6 exerted no significant effect on the photocycle of bacteriorhodopsin solubilized in Triton X-100 at pH 6.2 and 7.4, which suggested that, when functioning, bacteriorhodopsin retains the conformation and position of its Val69-Pro-Phe-Gly72 fragment.     

Bacteriorhodopsin Retains the Conformation of Val69–Gly72 Fragment during Functioning

Effect of the monoclonal antibody (MAb) 5B6 produced to the solubilized preparation of bacteriorhodopsin on the protein photocycle was studied to examine conformational rearrangements on the surface of a functioning bacteriorhodopsin molecule. Using solid phase enzyme immunoassay, peptide phage display, and ¹H NMR spectroscopy, we demonstrated that the epitope recognized by MAb 5B6 is the Val69-Pro-Phe-Gly72 fragment of the protein, with the aromatic ring of Phe71 and the methyl groups of Val69 participating in the binding. MAb 5B6 exerted no significant effect on the photocycle of bacteriorhodopsin solubilized in Triton X-100 at pH 6.2 and 7.4 which suggested that, when functioning, bacteriorhodopsin retains the conformation and position of its Val69-Pro-Phe-Gly72 fragment.     

The role of the native lipids and lattice structure in bacteriorhodopsin protein conformation and stability as studied by temperature-dependent Fourier transform-infrared spectroscopy

We report the effect of partial delipidation and monomerization on the protein conformational changes of bacteriorhodopsin (bR) as a function of temperature. Removal of up to 75% of the lipids is known to have the lattice structure of the purple membrane, albeit as a smaller unit cell, whereas treatment by Triton monomerizes bR into micelles. The effects of these modifications on the protein secondary structure is analyzed by monitoring the protein amide I and amide II bands in the Fourier transform-infrared (FT-IR) spectra. It is found that removal of the first 75% of the lipids has only a slight effect on the secondary structure at physiological temperature, whereas monomerizing bR into micelles alters the secondary structure considerably. Upon heating, the bR monomer is found to have a very low thermal stability compared with the native bR with its melting point reduced from 97 to 65 degrees C, and the pre-melting transition in which the protein changes conformation in native bR at 80 degrees C could not be observed. Also, the N[bond]H to N[bond]D exchange of the amide II band is effectively complete at room temperature, suggesting that there are no hydrophobic regions that are protected from the aqueous medium, possibly explaining the low thermal stability of the monomer. On the other hand, 75% delipidated bR has its melting temperature close to that of the native bR and does have a pre-melting transition, although the pre-melting transition occurs at significantly higher temperature than that of the native bR (91 degrees C compared with 80 degrees C) and is still reversible. Furthermore, we have also observed that the reversibility of this pre-melting transition of both native and partially delipidated bR is time-dependent and becomes irreversible upon holding at 91 degrees C between 10 and 30 min. These results are discussed in terms of the lipid and lattice contribution to the protein thermal stability of native bR.     

FTIR analysis of the SII540 intermediate of sensory rhodopsin II: Asp73 is the Schiff base proton acceptor

Sensory rhodopsin II (SRII), a repellent phototaxis receptor found in Halobacterium salinarum, has several homologous residues which have been found to be important for the proper functioning of bacteriorhodopsin (BR), a light-driven proton pump. These include Asp73, which in the case of bacteriorhodopsin (Asp85) functions as the Schiff base counterion and proton acceptor. We analyzed the photocycles of both wild-type SRII and the mutant D73E, both reconstituted in Halobacterium salinarum lipids, using FTIR difference spectroscopy under conditions that favor accumulation of the O-like, photocycle intermediate, SII540. At both room temperature and -20 degrees C, the difference spectrum of SRII is similar to the BR-->O640 difference spectrum of BR, especially in the configurationally sensitive retinal fingerprint region. This indicates that SII540 has an all-trans chromophore similar to the O640 intermediate in BR. A positive band at 1761 cm-1 downshifts 40 cm-1 in the mutant D73E, confirming that Asp73 undergoes a protonation reaction and functions in analogy to Asp85 in BR as a Schiff base proton acceptor. Several other bands in the C=O stretching regions are identified which reflect protonation or hydrogen bonding changes of additional Asp and/or Glu residues. Intense bands in the amide I region indicate that a protein conformational change occurs in the late SRII photocycle which may be similar to the conformational changes that occur in the late BR photocycle. However, unlike BR, this conformational change does not reverse during formation of the O-like intermediate, and the peptide groups giving rise to these bands are partially accessible for hydrogen/deuterium exchange. Implications of these findings for the mechanism of SRII signal transduction are discussed.     

Investigations of the molecular mechanism of metal-induced Abeta (1-40) amyloidogenesis

Transition-metal ions (Cu2+ and Zn2+) play critical roles in the Abeta plaque formation. However, precise roles of the metal ions in the Abeta amyloidogenesis have been controversial. In this study, the molecular mechanism of the metal-induced Abeta oligomerization was investigated with extensive metal ion titration NMR experiments. Upon additions of the metal ions, the N-terminal region (1-16) of the Abeta (1-40) peptide was selectively perturbed. In particular, polar residues 4-8 and 13-15 were more strongly affected by the metal ions, suggesting that those regions may be the major binding sites of the metal ions. The NMR signal changes of the N-terminal region were dependent on the peptide concentrations (higher peptide concentrations resulted in stronger signal changes), suggesting that the metal ions facilitate the intermolecular contact between the Abeta peptides. The Abeta (1-40) peptides (>30 microM) were eventually oligomerized even at low temperature (3 degrees C), where the Abeta peptides are stable as monomeric forms without the metal ions. The real-time oligomerization process was monitored by 1H/15N HSQC NMR experiments, which provided the first residue-specific structural transition information. Hydrophobic residues 12-21 initially underwent conformational changes due to the intermolecular interactions. After the initial structural rearrangements, the C-terminal residues (32-40) readjusted their conformations presumably for effective oligomerization. Similar structural changes of the metal-free Abeta (1-40) peptides were also observed in the presence of the preformed oligomers, suggesting that the conformational transitions may be the general molecular mechanism of the Abeta (1-40) amyloidogenesis.     

Nucleotide binding induces global and local structural changes of myosin head in muscle fibres.

Thermal stability and internal dynamics of myosin heads in fiber bundles from rabbit psoas muscle has been studied by electron paramagnetic resonance (EPR) spectroscopy and differential scanning calorimetry (DSC). Using ADP, ATP and orthovanadate (V(i)), three intermediate states of the ATP hydrolysis cycle were simulated in glycerinated muscle fibers. DSC transitions contained three overlapping endotherms in each state. Deconvolution showed that the transition temperature of 58.4 degrees C was almost independent of the intermediate state of myosin, while nucleotide binding shifted the melting temperatures of 54.0 and 62.3 degrees C, and changed the enthalpies. These changes suggest global rearrangements of the internal structure in myosin head. In the presence of ADP and ADP plus V(i), the conventional EPR spectra showed changes in the ordering of the probe molecules, suggesting local conformational and motional changes in the internal structure of myosin heads. Saturation transfer EPR measurements reported increased rotational mobility of spin labels in the presence of ATP plus orthovanadate corresponding to a weakly binding state of myosin to actin.     

A structural analysis of the bent kinetoplast DNA from Crithidia fasciculata by high resolution chemical probing.

The chemical probes potassium permanganate (KMnO4) and diethylpyrocarbonate (DEPC) have been used to study the conformation of bent kinetoplast DNA from Crithidia fasciculata at different temperatures. Chemical reactivity data shows that the numerous short A-tracts of this bent DNA adopt a similar structure at 43 degrees C. This conformation appears to be very similar to the conformation of A-tracts in DNA exhibiting normal gel mobility. The A-tract structure detected by chemical probing is characterized by a high degree of base stacking on the thymine strand, and by an abrupt conformational change at the 3' end of the adenine strand. In general, no major alteration of this A-tract specific structure was detected between 4-53 degrees C. However, probing with KMnO4 revealed two unusual features of the C. fasciculata sequence that may contribute to the highly aberrant gel mobility of this DNA: 1) the B DNA/A-tract junction 5' dC/A3-6 3'. 5' dT3-6/G 3' is disproportionately represented and is conformationally distinct from other 5' end junctions, and 2) low temperature favors a novel strand-specific conformational distortion over a 20 base pair region of the bent kinetoplast DNA. Presence of the minor groove binding drug distamycin had little detectable effect on the A-tract conformation. However, distamycin did inhibit formation of the novel KMnO4 sensitive low temperature structure and partially eliminated the anomalous gel mobility of the kinetoplast DNA. Finally, we describe a simple and reproducible procedure for the production of an adenine-specific chemical DNA sequence ladder.     

Interaction of a spin-labeled analog of ATP with myosin

By means of spin labeled analogs of ATP we have shown that conformational changes in myosin molecule induced by variation of temperature take place in the region of the active centre. In case of Mg-ATP and unmodified myosin conformation of the active centre changes monotonously with the change in temperature but after the modification of S1 thiol groups by N-ethylmaleimide on the temperature dependence curve of rotational mobility of the spin label a discontinuous is observed at 14-16 degrees C. It is also observed in case of K+-EDTA-ATP, or Ca2+-ATP and unmodified myosin. It is shown that the chemical analogs of Mg2+-paramagnetic ion Mn2+ are directly connected with the myosin active centre in the presence of ATP(ADP), i. e. a triple complex enzyme-bivalent cation-substrate is formed.     

Analysis of nucleotide myosin complexes in skeletal muscle fibres by DSC and EPR

The internal dynamics and thermal unfolding of fibre bundles prepared from rabbit psoas muscle has been studied in the presence of nucleotides by differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) spectroscopy. Using ADP, adenosine 5'-triphosphate (ATP), AMP.PNP and inorganic phosphate analogue orthovanadate (V(i)), AlF(4)(-) and BeF(3)(-), three intermediate states of the ATP hydrolysis cycle were simulated in glycerinated muscle fibres. In the main transition of the DSC pattern, three overlapping endotherms were detected in rigor, four in strongly as well as weakly binding state of myosin to actin. Deconvolution procedure showed that the transition temperature of 67.5 degrees C was the same for rigor and strong binding state of myosin. In contrast, nucleotide binding induced shift of the melting temperatures of 52 degrees C and 67.5 degrees C, appeared a new fourth peak at 74 and 77 degrees C and produced changes in the calorimetric enthalpies. The changes of the parameters of the peak functions suggest global rearrangements of the internal structure in myosin heads in the intermediate states. In the presence of ADP or ATP plus phosphate analogue orthovanadate or beryllium fluoride, aluminium fluoride, the conventional EPR spectra of spin-labeled muscle fibres showed large changes in the ordering of the probe molecules, and a new distribution of spin labels appeared. ATP plus orthovanadate induced the orientation disorder of myosin heads; the random population of spin labels gave evidence of large local conformational and motional changes in the internal structure of myosin heads. Saturation transfer EPR measurements reported increased rotational mobility of spin labels in the presence of ATP plus phosphate analogues corresponding to weakly binding state of myosin to actin.     

Conformational study of human serum albumin in pre-denaturation temperatures by differential scanning calorimetry, circular dichroism and UV spectroscopy

Thermal conformational changes of human serum albumin (HSA) in phosphate buffer, 10 mM at pH = 7 are investigated using differential scanning calorimetric (DSC), circular dichroism (CD) and UV spectroscopic methods. The results indicate that temperature increment from 25 degrees C to 55 degrees C induces reversible conformational changes in the structure of HSA. Conformational change of HSA are shown to be a three-step process. Interestingly, melting temperature of the last domain is equal to the maximum value of fever in pathological conditions, i.e. 42 degrees C. These conformational alterations are accompanied by a mild alteration of secondary structures. Study of HSA-SDS (sodium dodecyl sulphate) interaction at 45 degrees C and 35 degrees C reveals that SDS affects the HSA structure at least in three steps: the first two steps result in more stabilization and compactness of HSA structure, while the last one induces the unfolding of HSA. Since HSA has a more affinity for SDS at 45 degrees C compared to 35 degrees C, It is suggested that the net negative charge of HSA is decreased in fever, which results in the decrease of HSA-associated cations and plasma osmolarity, and consequently, heat removal via the increase in urine volume.     

A spectrofluorometric study of the environment of tryptophans in bacteriorhodopsin

The emission spectrum of intact purple membranes of Halobacterium halobium has a very short wavelength position (the main maximum at 314 nm) and can be fitted by two spectral components, one of which (component A) corresponds to the fluorescence of buried tryptophan residues located in a highly hydrophobic rigid environment (like the single tryptophan residue in azurin), the other (component I) being due to the emission of buried tryptophan residues located in a rather polar environment. Treatment of bacteriorhodopsin by NaBH4, fragmentation of the membranes and thermal formation of vesicles result in a decrease in the contribution of component A, an increase in that of component I and the appearance of spectral components corresponding to the emission of surface tryptophan residues. Temperature induces at least two distinct changes of the fluorescence parameters of the protein: one change occurs from 45 to 65 degrees C. the other from 65 to 90 degrees C. The spectral changes correlate with the peaks of heat sorption caused by thermal transitions in the purple membrane structure and conformational changes in the protein structure. Alkaline denaturation of bacteriorhodopsin registered by tryptophan fluorescence begins at pH > 11.0.     

Ultrasonic study of melittin effects on phospholipid model membranes.

Low dose effects of melittin on dilute suspensions of dipalmitoylphosphatidylcholine multilamellar vesicles are investigated by studying the acoustic properties of the system. The temperature dependencies of sound velocity and absorption have been measured at 7.2 MHz in the temperature range of 20-55 degrees C, for different peptide/lipid molar ratios, R. The most pronounced effects were observed at R = 5 x 10(-3), in the vicinity of the pretransition, with a simultaneous increase in sound absorption and velocity. This indicates that melittin affects the polar head group region of the bilayer resulting in a decrease in mobility of the polar head groups. A nonmonotonic dependence of the main transition temperature, with an initial decrease followed by an increase as melittin is added, is interpreted as a consequence of a destabilizing action of the interfaces between mellitin-affected clusters and the unaffected phase.     

Simulation study on the disordered state of an Alzheimer's beta amyloid peptide Abeta(12 36) in water consisting of random-structural, beta-structural, and helical clusters

The monomeric Alzheimer's beta amyloid peptide, Abeta, is known to adopt a disordered state in water at room temperature, and a circular dichroism (CD) spectroscopy experiment has provided the secondary-structure contents for the disordered state: 70% random, 25% beta-structural, and 5% helical. We performed an enhanced conformational sampling (multicanonical molecular dynamics simulation) of a 25-residue segment (residues 12-36) of Abeta in explicit water and obtained the conformational ensemble over a wide temperature range. The secondary-structure contents calculated from the conformational ensemble at 300 degrees K reproduced the experimental secondary-structure contents. The constructed free-energy landscape at 300 degrees K was not plain but rugged with five clearly distinguishable clusters, and each cluster had its own characteristic tertiary structure: a helix-structural cluster, two beta-structural clusters, and two random-structural clusters. This indicates that the contribution from the five individual clusters determines the secondary-structure contents experimentally measured. The helical cluster had a similarity with a stable helical structure for monomeric Abeta in 2,2,2-trifluoroethanol (TFE)/water determined by an NMR experiment: The positions of helices in the helical cluster were the same as those in the NMR structure, and the residue-residue contact patterns were also similar with those of the NMR structure. The cluster-cluster separation in the conformational space indicates that free-energy barriers separate the clusters at 300 degrees K. The two beta-structural clusters were characterized by different strand-strand hydrogen-bond (H-bond) patterns, suggesting that the free-energy barrier between the two clusters is due to the H-bond rearrangements.     

Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1.

The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy. This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500. The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures. The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins. Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal. Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange. These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin.     

The role of interchain disulphide bridges in the conformational stability of human immunoglobulin G1 subclass. Hydrogen-deuterium exchange studies.

The hydrogen-deuterium exchange data of human immunoglobulin G1 (IgG1) are interpreted by assuming fast fluctuations of the protein conformation, through which the peptide groups become exposed to the solvent. The probability of solvent exposure of peptide hydrogens reflects a rather loose conformation for native IgG in comparison with other globular proteins. The probability of solvent exposure is greater than 10(-3) for half of the peptide groups, which shows that the conformational transitions by which these groups are exposed to the solvent are accompanied by changes in standard free energy less than 17 kJ/mol (4 kcal/mol). In the range of pH 6.2-8.45, at 25 degrees C no gross conformational changes are reflected in the hydrogen-deuterium exchange behaviour of the native, the reduced-nonalkylated-reassociated and the reduced-S-alkylated-reassociated IgG1. No difference could be detected in the conformational stability of the native and reoxidised reassociated IgG1 proteins. The lack of inter-subunit disulphide bridges in S-alkylated-reassociated molecules results in an increased conformational motility. This destabilization of protein conformation affects about 90% of the peptide groups covered by the measurements, and corresponds to changes in standard free energy of 8 kJ/mol on the average.