Unfolding and refolding of bovine serum albumin at acid pH: ultrasound and structural studies.

Serum albumin is the most abundant protein in the circulatory system. The ability of albumins to undergo a reversible conformational transition, observed with changes in pH, is conserved in distantly related species, suggesting for it a major physiological role possibly related to the transport of small molecules including drugs. We have followed changes of bovine serum albumin (BSA) in volume by densimetry and in adiabatic compressibility during its conformational transition from pH 7-2, using ultrasound measurements. In parallel, circular dichroism was measured. The volume and adiabatic compressibility decrease from pH 4 to 2. The change in ellipticity shows a decrease over the same pH range from 70% to 40% of its alpha-helix content. Sorbitol, at concentrations from 0 to 2 M, led to the progressive restoration of BSA volume and compressibility values, as well as a substantial recovery of its original alpha-helix content. This finding implies that the compressibility variation observed reflects the conformational changes during the transition. The mutual interactions of the mechanical properties and structural features of BSA reported here are important in biotechnology for research in material sciences and for the design and the development of new, tailor-made drug carriers.

Environmentally induced reversible conformational switching in the yeast cell adhesion protein alpha-agglutinin.

The yeast cell adhesion protein alpha-agglutinin is expressed on the surface of a free-living organism and is subjected to a variety of environmental conditions. Circular dichroism (CD) spectroscopy shows that the binding region of alpha-agglutinin has a beta-sheet-rich structure, with only approximately 2% alpha-helix under native conditions (15-40 degrees C at pH 5.5). This region is predicted to fold into three immunoglobulin-like domains, and models are consistent with the CD spectra as well as with peptide mapping and site-specific mutagenesis. However, secondary structure prediction algorithms show that segments comprising approximately 17% of the residues have high alpha-helical and low beta-sheet potential. Two model peptides of such segments had helical tendencies, and one of these peptides showed pH-dependent conformational switching. Similarly, CD spectroscopy of the binding region of alpha-agglutinin showed reversible conversion from beta-rich to mixed alpha/beta structure at elevated temperatures or when the pH was changed. The reversibility of these changes implied that there is a small energy difference between the all-beta and the alpha/beta states. Similar changes followed cleavage of peptide or disulfide bonds. Together, these observations imply that short sequences of high helical propensity are constrained to a beta-rich state by covalent and local charge interactions under native conditions, but form helices under non-native conditions.

Interaction of alpha-agglutinin and a-agglutinin, Saccharomyces cerevisiae sexual cell adhesion molecules.

alpha-Agglutinin and a-agglutinin are complementary cell adhesion glycoproteins active during mating in the yeast Saccharomyces cerevisiae. They bind with high affinity and high specificity: cells of opposite mating types are irreversibly bound by a few pairs of agglutinins. Equilibrium and surface plasmon resonance kinetic analyses showed that the purified binding region of alpha-agglutinin interacted similarly with purified a-agglutinin and with a-agglutinin expressed on cell surfaces. At 20 degrees C, the K(D) for the interaction was 2 x 10(-9) to 5 x 10(-9) M. This high affinity was a result of a very low dissociation rate ( approximately 2.6 x 10(-4) s(-1)) coupled with a low association rate (= 5 x 10(4) M(-1) s(-1)). Circular-dichroism spectroscopy showed that binding of the proteins was accompanied by measurable changes in secondary structure. Furthermore, when binding was assessed at 10 degrees C, the association kinetics were sigmoidal, with a very low initial rate. An induced-fit model of binding with substantial apposition of hydrophobic surfaces on the two ligands can explain the observed affinity, kinetics, and specificity and the conformational effects of the binding reaction.

Buried charged surface in proteins.

BACKGROUND: The traditional picture of charged amino acids in globular proteins is that they are almost exclusively on the outside exposed to the solvent. Buried charges, when they do occur, are assumed to play an essential role in catalysis and ligand binding, or in stabilizing structure as, for instance, helix caps. RESULTS: By analyzing the amount and distribution of buried charged surface and charges in proteins over a broad range of protein sizes, we show that buried charge is much more common than is generally believed. We also show that the amount of buried charge rises with protein size in a manner which differs from other types of surfaces, especially aromatic and polar uncharged surfaces. In large proteins such as hemocyanin, 35% of all charges are greater than 75% buried. Furthermore, at all sizes few charged groups are fully exposed. As an experimental test, we show that replacement of the buried D178 of muconate lactonizing enzyme by N stabilizes the enzyme by 4.2 degrees C without any change in crystallographic structure. In addition, free energy calculations of stability support the experimental results. CONCLUSIONS: Nature may use charge burial to reduce protein stability; not all buried charges are fully stabilized by a prearranged protein environment. Consistent with this view, thermophilic proteins often have less buried charge. Modifying the amount of buried charge at carefully chosen sites may thus provide a general route for changing the thermophilicity or psychrophilicity of proteins.

A CD2-based model of yeast alpha-agglutinin elucidates solution properties and binding characteristics.

We have previously shown that the Saccharomyces cerevisiae cell adhesion protein alpha-agglutinin has sequence characteristics of immunoglobulin-like proteins and have successfully modeled residues 200-325, based on the structure of immunoglobulin variable-type domains. Alignments matching residues 20-200 of alpha-agglutinin with domains I and II of members of the CD2/CD4 subfamily of the immunoglobulin superfamily showed > 80% conservation of key residues despite low sequence similarity overall. Three-dimensional models of two alpha-agglutinin domains constructed on the basis of these alignments were shown to conform to peptide mapping data and biophysical properties of alpha-agglutinin. In addition, the residue volume and surface accessibility characteristics of these models resembled those of the well-packed structures of related proteins. Residue-by-residue analysis showed that packing and accessibility anomalies were largely confined to glycosylated and protease-susceptible loop regions of the domains. Surface accessibility of hydrophobic residues was typical of proteins with extensive domain interactions, a finding compatible with the hydrodynamic properties of alpha -agglutinin and the hydrophobic nature of binding to its peptide ligand alpha-agglutinin. The procedures used to align the alpha-agglutinin sequence and test the quality of the model may be applicable to other proteins, especially those that resist crystallization because of extensive glycosylation.

Thermodynamic volume cycles for electron transfer in the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase.

Dilatometry is a sensitive technique for measuring volume changes occurring during a chemical reaction. We applied it to the reduction-oxidation cycle of cytochrome c oxidase, and to the binding of cytochrome c to the oxidase. We measured the volume changes that occur during the interconversion of oxidase intermediates. The numerical values of these volume changes have allowed the construction of a thermodynamic cycle that includes many of the redox intermediates. The system volume for each of the intermediates is different. We suggest that these differences arise by two mechanisms that are not mutually exclusive: intermediates in the catalytic cycle could be hydrated to different extents, and/or small voids in the protein could open and close. Based on our experience with osmotic stress, we believe that at least a portion of the volume changes represent the obligatory movement of solvent into and out of the oxidase during the combined electron and proton transfer process. The volume changes associated with the binding of cytochrome c to cytochrome c oxidase have been studied as a function of the redox state of the two proteins. The volume changes determined by dilatometry are large and negative. The data indicate quite clearly that there are structural alterations in the two proteins that occur on complex formation.

The refined X-ray structure of muconate lactonizing enzyme from Pseudomonas putida PRS2000 at 1.85 A resolution.

We report here the refined X-ray crystal structure of muconate lactonizing enzyme (MLE) from Pseudomonas putida PRS2000 at a resolution of 1.85 A with an R-factor of 16.8%. An enzyme from the beta-ketoadipate pathway, MLE catalyses the conversion of cis,cis-muconate to muconolactone. It is a homo-octamer, one monomer consisting of 373 amino acid residues. MLE has two large domains and a C-terminal subdomain: an alpha + beta domain, an alpha beta-barrel domain and a C-terminal meandering subdomain. The alpha beta-barrel domain is highly irregular. Its structure is (beta/alpha)7 beta, with the structural role of the last alpha-helix being replaced by both the C-terminal subdomain and part of the N-terminal domain. The fifth, seventh and eighth barrel strands are unusual because they have left-handed twist about their axes. The strand crossing angles also vary enormously, from +9 degrees to -69 degrees; the first and last strands, which close the barrel, cross at an angle of -69 degrees, making extensive strand-strand hydrogen bonding impossible. The first barrel strand is also unusual because it starts in the N-terminal domain and forms hydrogen bonds to the C-terminal subdomain beta-sheet as well as to its neighbouring strands in the barrel. It thus cements the whole protein together. As in other alpha beta-barrel proteins, the active site of MLE, present in each subunit is at the C-terminal ends of the barrel beta-strands. The active site cleft contains an essential manganese ion, is lined with charged and other polar residues, and contains many of the crystallographic water molecules. The manganese ion is octahedrally co-ordinated to three side-chain carboxylate groups and three water molecules, and is at the centre of a radiating web of ionic and hydrogen-bonding interactions. Additionally, two water molecules are buried in the centre of the barrel and two hydrophilic side-chains (Lys167 and Arg196) make both hydrophobic and hydrophilic packing interactions with much of the barrel interior. The barrel interior is thus also unusual because it is so hydrophilic; the dominating force appears to be the need to solvate the metal ion effectively. This might account for the irregularity of the barrel. The catalytic mechanism has been investigated by docking both substrate and product in the active site with the C-COO- of muconolactone superimposed on the corresponding atoms of cis,cis-muconate. In agreement with earlier kinetic and spectroscopic results, the manganese ion does not interact directly with substrate or product.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure of Saccharomyces cerevisiae alpha-agglutinin. Evidence for a yeast cell wall protein with multiple immunoglobulin-like domains with atypical disulfides.

alpha-Agglutinin of Saccharomyces cerevisiae is a cell wall-associated protein that mediates cell interaction in mating. Although the mature protein includes about 610 residues, the NH2-terminal half of the protein is sufficient for binding to its ligand a-agglutinin. alpha-Agglutinin20-351, a fully active fragment of the protein, has been purified and analyzed. Circular dichroism spectroscopy, together with sequence alignments, suggest that alpha-agglutinin20-351 consists of three immunoglobulin variable-like domains: domain I, residues 20-104; domain II, residues 105-199; and domain III, residues 200-326. Peptide sequencing data established the arrangement of the disulfide bonds in alpha-agglutinin20-351. Cys97 is disulfide-bonded to Cys114, forming an interdomain bond between domains I and II. Cys202 is bonded to Cys300, in an atypical intradomain disulfide bond between the A and F strands of domain III. Cys227 and Cys256 have free sulfhydryls. Sequencing also showed that at least two of three potential N-glycosylation sites with sequence Asn-Xaa-Thr are glycosylated. At least one of three Asn-Xaa-Ser sequences is not glycosylated. No residues NH2-terminal to Ser282 were O-glycosylated, whereas Ser282, and all hydroxy amino acid residues COOH-terminal to this position were modified. Therefore O-glycosylated Ser and Thr residues cluster in the COOH-terminal region of domain III, and the O-glycosylation continues into a Ser/Thr-rich sequence that extends from domain III to the COOH-terminal of the full-length protein.

Homology modeling of an immunoglobulin-like domain in the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

The Saccharomyces cerevisiae adhesion protein alpha-agglutinin is expressed by cells of alpha mating type. On the basis of sequence similarities, alpha-agglutinin has been proposed to contain variable-type immunoglobulin-like (IgV) domains. The low level of sequence similarity to IgV domains of known structure made homology modeling using standard sequence-based alignment algorithms impossible. We have therefore developed a secondary structure-based method that allowed homology modeling of alpha-aggulutinin domain III, the domain most similar to IgV domains. The model was assessed and where necessary refined to accommodate information obtained by biochemical and molecular genetic approaches, including the positions of a disulfide bond, glycosylation sites, and proteolytic sites. The model successfully predicted surface exposure of glycosylation and proteolytic sites, as well as identifying residues essential for binding activity. One side of the domain was predicted to be covered by carbohydrate residues. Surface accessibility and volume packing analyses showed that the regions of the model that have greatest sequence dissimilarity from the IgV consensus sequence are poorly structured in the biophysical sense. Nonetheless, the utility of the model suggests that these alignment and testing techniques should be of general use for building and testing of models of proteins that share limited sequence similarity with known structures.

Volume changes of the molten globule transitions of horse heart ferricytochrome c: a thermodynamic cycle.

Volume changes among the unfolded (U), native (N), and molten globule (MG) conformations of horse heart ferricytochrome c have been measured. U to N (pH 2 to pH 7) was determined in the absence of added salt to be -136 +/- 5 mL/mol protein. U to MG (pH 2, no added salt to pH 2, 0.5 M KCl) yielded + 100 +/- 6 mL/mol. MG to N was broken into two steps, N to NClx at pH 7 by addition of buffered KCl to buffered protein lacking added salt (NClx = N interacting with an unknown number, X, of chloride ions), and MG to NClx by jumping MG at pH 2 in 0.5 M KCl to pH7 at the same salt concentration. The delta V of N to NClx was -30.9 +/- 1.4 mL/mol protein, whereas MG to NClx entailed a delta V of -235 +/- 6 mL/mol. Within experimental error, the results add up to zero for a complete thermodynamic cycle. We believe this to be the first volumetric cycle to have been measured for the conformational transitions of a protein. The results are discussed in terms of hydration contributions from deprotonation of the protein, other hydration effects, and the formation and/or enlargement of packing defects in the protein's tertiary structure during the steps of folding.

Determination of Km and kcat for signal peptidase I using a full length secretory precursor, pro-OmpA-nuclease A.

An effective method for the determination of the activity of signal peptidase I (SPase I) of Escherichia coli is established using the hybrid protein pro-OmpA-nuclease A as substrate. Pro-OmpA-nuclease A, a hybrid secretory precursor was purified to homogeneity under denaturing conditions. When this protein was refolded, it could be quantitatively processed by purified SPase I. The Km of signal peptidase I was 0.0165 mM. The kcat was 8.73 s-1. The Km is 50 to 100 times lower than that obtained with peptide substrates indicating that SPase I has a significantly greater affinity for the protein substrate. The turnover number, kcat, is two to four orders of magnitude greater as well. Thus, the specificity constant, kcat/Km is six orders of magnitude greater with pro-OmpA-nuclease A than with peptide substrates. This is the first determination of kinetics of SPase I with a protein substrate.

Slow-folding kinetics of ribonuclease-A by volume change and circular dichroism: evidence for two independent reactions.

The slow refolding of guanidine-HCl-denatured ribonuclease-A was studied by volume change and by kinetic CD at 222 and 276 nm. Dilatometric measurements revealed that on refolding there is a fast volume change of +232 mL/mol of protein. This is followed by a very slow nonexponential change that takes about 25 min to reach equilibrium. By adding varying amounts of (NH4)2SO4, the slow volume change curve was resolved into 2 concurrent reactions. The faster of the 2 slow events entails a negative volume change of -64 mL/mol of protein and appears to arise from proline isomerization. The slower process, attended by a positive change of +53 mL/mol of protein, has properties consistent with the "XY" reaction of Lin and Brands (1983, Biochemistry 22:563-573). This reaction is so named because the conformational nature of neither its initial (Y) nor its final state (X) is known; the transition is characterized solely by its absorbance and fluorescence kinetics. These are the first direct physical measures attributable to the "XY" process. The early formation of a compact structure in the event responsible for the rapid +232-mL/mol volume change, however, is consistent with the sequential model of folding (Cook KH, Schmid FX, Baldwin RL, 1979, Proc Natl Acad Sci USA 76:6157-6161; Kim PS, Baldwin RL, 1980, Biochemistry 19:6124-6129). The usefulness of volume change measurements as a method of detecting structural rearrangements was confirmed by finding agreement between time constants obtained from parallel volume change and kinetic CD experiments. The measured volume changes arise from both changes in hydration and changes in the packing of atoms in the interior of the protein.

Limited proteolysis of myelin basic protein in a system mimetic of the myelin interlamellar aqueous space.

We have investigated the early steps of myelin basic protein (MBP) degradation in a membrane mimetic system (reverse micelles), resembling the interlamellar aqueous spaces where the protein is located in the myelin sheath. MBP, unfolded in buffer, refolds on incorporation into the micelles, resulting in reduced accessibility to three proteolytic enzymes, trypsin, cathepsin D, and Staphylococcus aureus V8 protease, in comparison with aqueous solution. Eleven cleavage sites seen in buffer are removed from proteolytic attack in micellar solution. These sites delineate a protected protein domain displaying a potential beta-sheet structure capable of interacting with the myelin membrane. An additional site not seen in buffer is attacked in the micelles. Experiments with a structure inducer, 15% 1-propanol in buffer, reveal that the refolding pattern of MBP in reverse micelles is specific to the membrane biomimetic system and is not produced by organic solvent per se. Micellar digestions of MBP generate long peptides, two of which, isolated after tryptic digestion, have been found to be immunodominant in multiple sclerosis patients. The findings suggest the structure induced in MBP by the micelles resembles that leading to production of the self-peptides recognized by T cells during proteolytic breakdown of MBP in autoimmune demyelinating diseases.

Glycation of lysozyme in a restricted water environment.

Fast atom bombardment mass spectrometry (FAB) was used to determine the glycation sites of lysozyme in a restricted water environment. A 30-day incubation at 25 degrees C, and 65% relative humidity (R.H.) resulted in glycation at lysine-1 while a much shorter (3-day) incubation at 50 degrees C and 65% R.H. resulted in diglycation at lysine-1 as well as glycation at lysine-13 and lysine-33.

"Hydration memory" of lysozyme: a misinterpretation.

The effects of sugars and similar additives on the catalytic activity of lysozyme have been attributed (Laretta-Garde et al., Biochim. Biophys. Res. Commun. 155: 816-822 (1988] to a "hydration memory" of the protein for solution conditions to which it was previously exposed. By measuring catalytic activity versus substrate concentration, tryptophan fluorescence and near ultraviolet circular dichroism all in the presence and absence of 50% (w/v) sucrose, we show that the effects can be explained, instead, on the basis of well known properties of proteins.

Dioxins and dibenzofurans in blood and adipose tissue of Agent Orange-exposed Vietnam veterans and matched controls.

Vietnam veterans who were heavily exposed to Agent Orange exceeded matched control subjects in both blood and adipose tissue levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but not in the levels of the 12 other 2,3,7,8-substituted dioxins and dibenzofurans that were detected. Since only TCDD among these compounds was present in Agent Orange but all are present in the population of the industrialized world, it is likely that the elevated TCDD levels arose from wartime exposure. The high correlation (r = +.89) of blood with adipose tissue level suggests that there may be a mobile equilibrium between them and that blood measurement could replace adipose tissue measurement of TCDD levels, making the collection of human data less invasive.

Comparison of left and right ventricular end-systolic pressure-volume relations in congestive heart failure.

A hemodynamic-radionuclide study was performed to compare the relations between end-systolic pressure and volume in the left and right ventricles in 10 patients with biventricular failure, and to correlate the end-systolic pressure-volume slope with baseline variables of systolic function. During nitroprusside or nitroglycerin infusion, or a combination of both, linear relations were found between end-systolic pressure and volume for both ventricles. In 9 of 10 patients, the end-systolic pressure-volume slope was greater for the left ventricle (mean +/- SD 1.12 +/- 0.36 mm Hg X m2/ml) than for the right ventricle (0.46 +/- 0.27 mm Hg X m2/ml) (p less than 0.001). In all 10 patients, the volume-axis intercept of the pressure-volume relation was greater for the left ventricle (82 +/- 66 ml/m2) than for the right ventricle (2 +/- 30 ml/m2) (p less than 0.005). Right ventricular pressure-volume slope correlated weakly with baseline right ventricular ejection fraction (r = 0.69, p less than 0.05), strongly with the baseline right ventricular end-systolic pressure-volume ratio (r = 0.89) and inversely with baseline right ventricular end-systolic volume (r = -0.86). In conclusion, 1) in patients with severe biventricular failure, changes in systolic pressure influence end-systolic volume more strongly in the right than in the left ventricle. 2) For the right ventricle, the slope of the end-systolic pressure-volume relation is directly related to rest indexes of systolic function. 3) The greater the end-systolic volume at rest, the greater the predicted improvement in right ventricular emptying for any vasodilator-induced reduction in pulmonary artery end-systolic pressure.

Equilibrium (gated) radionuclide ejection fraction measurement in the pressure or volume overloaded right ventricle. Comparison of three methods.

Although equilibrium radionuclide angiographic measurement of right ventricular ejection fraction (RVEF) has been validated in patients with coronary artery disease, the accuracy of this technique has not been demonstrated in patients with other cardiac diseases which may result in RV pressure and/or volume overload. The accuracy of three methods of equilibrium radionuclide analysis for measuring RVEF was compared in several subgroups of patients with a variety of cardiac diseases, including congenital and valvular heart disease, cor pulmonale, and cardiomyopathy. It was concluded that RVEF may be accurately derived by equilibrium radionuclide ventriculography in patients with a wide variety of cardiac diseases, including those resulting in RV volume and/or pressure overload. Accuracy varies depending on analysis method and the nature of the hemodynamic derangement.

Vasodilator effect on right ventricular function in congestive heart failure and pulmonary hypertension: end-systolic pressure--volume relation.

The right ventricular (RV) end-systolic pressure-volume relation during vasodilator administration was studied in 10 patients with pulmonary arterial (PA) hypertension, most of whom had biventricular failure. RV end-systolic volumes were estimated from equilibrium radionuclide RV counts and forward cardiac output. Simultaneous radionuclide and hemodynamic values were measured at rest and during nitroglycerin and nitroprusside infusion. Vasodilator administration resulted in decreases in PA mean and systolic pressures in all 10 patients, with an average decrease in end-systolic pressure (p less than 0.001) from 48 +/- 11 to 38 +/- 9 and 35 +/- 10 mm Hg during nitroglycerin and nitroprusside administration, respectively. In each patient, a direct linear relation was observed between the vasodilator-induced decrease in PA end-systolic pressure and in RV end-systolic volume. Average RV end-systolic volume decreased (p less than 0.001), from 130 +/- 69 ml/m2 at baseline to 108 +/- 62 and 102 +/- 55 ml/m2 during nitroglycerin and nitroprusside infusion, respectively. The slope of the RV end-systolic pressure-volume relation was directly related to RV ejection fraction. Thus, the vasodilator-induced decrease in PA systolic pressure is accompanied by a linear decrease in RV end-systolic volume, with a slope which is dependent on RV systolic function. This linear relation is analogous to the left ventricular end-systolic pressure-volume relation.

Work in progress: the effect of heat on bleomycin cytotoxicity in vitro and on the accumulation of 57Co-bleomycin in heat-treated rat tumors.

The cytotoxic effects of the sequence and timing in combined hyperthermia and bleomycin treatment were tested in vitro using V79 Chinese hamster cells. The order of treatment was important; heat treatment followed by the administration of bleomycin yielded greater cytotoxicity than when the opposite order was used. To determine whether heat-treated tumors have an altered uptake of bleomycin, rat rhabdomyosarcoma (BA 1112) tumors were heated locally with RF current (43 degrees C, 90 min.), injected with 57Co-bleomycin, and imaged on a radioisotope camera. Results of tumor-to-background (T/B) ratio analysis indicate that (a) local hyperthermia (43 degrees C) does not appear to alter tumor uptake patterns of 57Co-bleomycin; and (b) intravenous and intraperitoneal injections produce similar T/B uptake ratios, typically between 2 and 3 at 120 minutes postinjection. In the BA 1112/WAG/Rij tumor system, local hyperthermia treatment does not seem to interfere with the subsequent accumulation of bleomycin in the tumor.