Interactions between canine plasminogen and 8-anilino-1-naphthalene sulfonate: structural insights from a fluorescent probe.

Canine plasminogen (DPgn) binds the fluorescent molecule, 8-anilino-1-naphthalene sulfonate (ANS). Binding is favored by low pH as shown by steady-state fluorescence, native PAGE and isothermal titration calorimetry. Binding occurs at multiple sites. A strong site binds ANS with a Ka of 3.3 x 10(4) M(-1). Two weaker sites were observed with association constants of 6.2 x 10(3) M(-1) and 360 M(-1), respectively. ANS binds equally well to both the closed and opened conformations of DPgn at pH 3.0. At low pH there are changes in protein tertiary structure as monitored by sedimentation velocity analysis and circular dichroism. Binding sites for ANS were located in the proteolytic domain as well as the kringle domains. However, it is unlikely that binding occurs within the lysine binding sites of each respective kringle. Taken together, the data suggest that ANS may be binding to a pH induced molten globule state of DPgn.

Characterization of a complex formed between human plasminogen and recombinant sheep prion: pressure and thermal sensitivity of complex formation.

Scrapie is thought to be caused by one or more conformations of a proteinacious particle called a prion. The infectious form(s) is referred to as the scrapie form of the prion protein (PrPsc) whereas a benign form, the cellular conformer, is referred to as PrPC. The cellular conformation of the sheep prion protein formed a 1:1 complex with human plasminogen. The complex precipitated at 0 degrees C (Ksp = 17* 10(-12) M2). This precipitation reaction was sensitive to both temperature and pressure. When subjected to hydrostatic pressure the precipitate dissolved. At 25 degrees C the complex was soluble with a dissociation constant of about 10(-7) M as determined by isothermal titration calorimetry. Absorption, fluorescence and circular dichroism spectroscopy showed that neither protein, in the complex, underwent a detectable structural change so long as proteolytic inhibitors were present. In the absence of proteolytic inhibitors, plasminogen slowly cleaved the prion protein.

Water as it applies to the function of enzymes.

Escherichia coli and Saccharomyces cerevisiae can metabolize, grow, and divide over osmotic pressures ranging from 0.24 atm to about 100 atm [Record, T. M. et al. (1999). Trends Biochem. Sci. 23,143-148,190-194; Wood, J. M. (1999). Microbiol. Mol. Bio. Rev. 63, 230-262; Marachal, P. A., and Gervais, P. (1994). Appl. Microbiol. Biotechnol. 42, 617-622]. At the higher end of the range, they perform their functions with difficulty, but they can survive. Over the full span of pressures, the activity of water goes from 0.9998 to 0.93. Neither of the authors can survive at anything like these extremes; some of their enzymes and enzymatic complexes would "fall apart," would either cease to function or would denature. We would very much like to know just how the two microbes manage.

Reaction of canine plasminogen with 6-aminohexanoate: a thermodynamic study combining fluorescence, circular dichroism, and isothermal titration calorimetry.

The thermodynamics of the binding of 6-aminohexanoate (6-AH) to dog glu-plasminogen has been studied. Fluorescence titrations revealed four binding sites. Three yielded positive fluorescence changes on ligand binding; one yielded a negative fluorescence change. The fluorescence data gave no indication of cooperative interactions. Binding was studied using circular dichroism (CD). Near 295 nm there were small changes associated with binding ligand. These were magnified at 235 nm, a wavelength that is mainly associated with tryptophan bands. The dissociation constants obtained from the fluorescence were applied to the CD data and fit quite well. Below 220 nm, there were no significant differences between samples with or without 6-AH and, therefore, no substantial change in the secondary structure of the protein. Isothermal titration calorimetry was used in combination with the binding constants from fluorescence to study the enthalpy and entropy contributions to 6-AH binding. The enthalpies of association for the four sites are all negative. Their absolute values are small for the tight sites and large for the weakest. -TDeltaS is negative for the tight sites and positive for the weakest. The binding of 6-AH to plasminogen is entropically driven for the two tightest sites and enthalpically driven for the weakest site. The binding of 6-AH to lys-plasminogen has been studied and differs slightly from binding to glu-plasminogen. Most importantly, the binding of 6-AH for the weak site goes from enthalpy- to entropy-driven as is found with the other sites.

Understanding the fluorescence changes of human plasminogen when it binds the ligand, 6-aminohexanoate: a synthesis.

This work attempts to explain several aspects of the response of plasminogen to 6-aminohexanoate (6-AH). These responses include the overall fluorescent changes that occur when plasminogen binds the ligand, the changes shown by the individual domains when they bind the ligand, and the changes in structure shown by the holoprotein when it binds 6-AH. The results have implications for understanding the physicochemical behavior of all kringle based proteins.

The effects of hydrostatic pressure on the conformation of plasminogen.

Plasminogen undergoes a large conformational change when it binds 6-aminohexanoate. Using ultraviolet absorption spectroscopy and native PAGE, we show that hydrostatic pressure brings about the same conformational change. The volume change for this conformational change is -33 mL.mol-1. Binding of ligand and hydrostatic pressure both cause the protein to open up to expose surfaces that had previously been buried in the interior.

The individual tyrosines of proteins: their spectra may or may not differ from those in water or other solvents.

The overall derivative spectrum of a protein is the sum of the individual derivative spectra just as the overall ultraviolet spectrum of a protein is the sum of its component parts. The RNase and DNA binding protein Sso7d has two tyrosines and one tryptophan. We used two mutant forms of the protein to show that the individual aromatics contribute derivative spectra that can be explained on the basis of their environments. We used mutant forms of iso-1-cytochrome c to estimate the contributions of the single tryptophan and three of the five tyrosines to the overall derivative spectrum. The tryptophan spectrum is not exceptional. The comparable tyrosine spectra are more complex. The derivative spectrum of individual tyrosines does not correspond to that expected on the basis of concentration. This is a reflection of two factors: (1) the extent to which mutations are sensed distally through the introduction and compression of packing defects; and (2) the extent to which electronic transitions of tyrosine are influenced by nearby atoms. This influence could take the form of tyrosine residing in an area where the dielectric coefficient is not uniform; it could also result from tyrosine bumping into neighboring atoms with lower frequency than it does in solution.

The water channel of cytochrome c oxidase: inferences from inhibitor studies.

Cytochrome c oxidase couples electron transfer to proton transfer from inside the mitochondrion to the cytosol. Protons pass through a channel; it is closed except when protons are pumped. Electron transfer is also coupled to a water cycle. Water moves into and out of the oxidase during electron transfer, presumably through a channel. The three processes are coupled because of the common dependence on electron transfer. If water and protons had to pass through the same channel for the proton to pass, it might be possible to block the pore by entraining small molecules in the flow. The data in this report indicate that there is a correlation between the ability of a compound to inhibit the oxidase and its size. Formamide and formaldehyde are potent inhibitors. Larger and smaller molecules are poor inhibitors. Formamide introduces an internal block in electron transfer. It is a slow-onset, reversible inhibitor, dependent on turnover to manifest its effects. Vesicular oxidase is less influenced by formamide than is soluble oxidase; formamide must pass a permeability barrier to act. The data are consistent with a proton channel with constrictions at both ends that open to yield a pore of approximately 4 A.

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 polarity of tyrosine 67 in yeast iso-1-cytochrome c monitored by second derivative spectroscopy.

The average tyrosine polarity of 10 yeast iso-1-cytochrome c proteins and two horse heart cytochrome c proteins was assayed by second derivative spectroscopy. Yeast iso-1-cytochrome c contains five tyrosines, one of which (tyrosine 67) is in the heme pocket. The wild-type protein and the Y67F, N52V Y67F, and N521 Y67F proteins were used to differentiate events that were occurring in or near the heme pocket from those occurring closer to the protein's surface. The wild-type protein shows a substantial change in the second derivative spectrum as the protein goes from oxidized to reduced; mutants lacking tyrosine 67 do not show this change. This indicates that it is primarily the spectrum of tyrosine 67 that changes as the protein cycles between the oxidized and reduced state. One thing that contributes to the overall polarity of the heme pocket is a water molecule hydrogen bonded to several of the nearby residues. The wild-type protein has one water molecule in the heme pocket but this can be increased or decreased by introducing mutations into the protein. N52A has two water molecules and N52I has no water molecule in the pocket. The three proteins allowed us to assess the contribution of water to the inferred heme crevice polarity. The number of water molecules in the crevice correlates with the perceived polarity of the pocket when one takes account of the fact that the second water molecule in the crevice of the N52A mutant takes the position and hydrogen bonding pattern of the amide it replaces.

The effects of sodium perchlorate on rabbit muscle enolase--Spectral characterization of the monomer.

Incubation of rabbit beta beta enolase in NaClO4 (< or = O.3 M) results in a loss of enzymatic activity and striking changes in the second-derivative ultraviolet spectrum of enolase. HPLC gel filtration shows that dissociation of the dimeric enzyme is occurring. We have used molecular modelling, fluorescence and circular dichroic spectroscopy to examine the structural differences between the monomeric and dimeric forms of this protein. In the dimer, the tyrosine residues are in a non-polar environment; upon dissociation, two of them that were at the dimer interface become exposed. This results in large changes in the second-derivative spectrum. Both the tryptophan fluorescence emission spectrum and the aromatic region of the CD spectrum indicate that there are also changes in the environment of other aromatic residues. No perturbations in the peptide bond region of the CD spectrum are observed. We propose that the major structural effect of NaClO4 is to increase the flexibility of the loops connecting the helices and strands of the alpha/beta barrel of enolase. These loops, which contain about half of the aromatic residues, contain some of the residues of the active site and other residues involved in subunit contacts. Increased flexibility of the loops could disrupt both subunit interactions and the structure of the active site.

Second derivative spectroscopy of enolase at high hydrostatic pressure: an approach to the study of macromolecular interactions.

Second derivative spectroscopy in the ultraviolet region of proteins has been used to study the polarity of the regions surrounding tyrosine residues. We show here that it can also be a tool to study the degree to which proteins associate and that it can be effectively combined with hydrostatic pressure in order to evaluate equilibrium dissociation constants and reaction volumes. Hydrostatic pressure causes yeast enolase to dissociate. Clear changes in the second derivative spectra of enolase were observed as pressure was increased. At enolase concentrations of about 20 microM, the midpoint of the transition is about 1800 bar. All aspects of the transition are reversible up to 2700 bar. It is likely that the transition observed is the result of enolase dimers dissociating into monomers. The second derivative spectra indicate that one or more tyrosine residues is in an unusually polar environment in the dimer, an environment that is less polar in the monomer. Three tyrosines (6, 11, 130) are near the dimer interface. Tyrosines 6 and 11 are pointing into the water-filled crevice between the subunits and are close to several immobilized waters. All three are close to a network of intersubunit salt bridges and hydrogen bonds. We believe that the average tyrosine polarity in the dimer reflects the exposure of these tyrosines to immobilized water and the fixed dipole of the salt bridge. The water in the crevice between the subunits should be more mobile in the monomer; the salt bridge does not exist in the monomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations of iso-1-cytochrome c at positions 13 and 90. Separate effects on physical and functional properties.

Residues at positions 13 (lysine or arginine) and 90 (glutamate or aspartate) of eukaryotic cytochromes c have been conserved during evolution; Cys102, however, is found only in yeast cytochrome c. The positively charged residue at position 13 and the negatively charged residue at position 90 are close together in those cytochromes c for which three-dimensional structures are available. We have replaced the amino acids at these two positions by cysteine in Saccharomyces cerevisiae iso-1-cytochrome c; in an earlier study, Cys102 was replaced by threonine without negatively influencing the physical or enzymic properties of the protein. The mutated proteins [R13C, C102T]cytochrome c (iso-1-cytochrome c containing Arg13-->Cys and Cys102-->Thr mutations), [D90C, C102T]cytochrome c (iso-1-cytochrome c containing Asp90-->Cys and Cys102-->Thr mutations) and [R13C, D90C, C102T]cytochrome c (iso-1-cytochrome c containing Arg13-->Cys, Asp90-->Cys, and Cys102-->Thr mutations) are functional in vivo. Free sulfhydryl titration shows that the doubly mutated forms each contain one sulfhydryl group while the triple mutant contains two sulfhydryl groups. The stability of mutant [R13C, C102T]cytochrome c resembles that of [C102T] cytochrome c, whereas the stability of [D90C, C102T]cytochrome c resembles the stability of [R13C, D90C, C102T]cytochrome c. The activity of cytochrome-c oxidase using cytochrome c was monitored polarographically. Compared to the wild-type or [C102T]cytochrome c, which shows two kinetic phases with cytochrome-c oxidase, [D90C, C102T]cytochrome c has much the same profile; [R13C, C102T]cytochrome c and [R13C, D90C, C102T]cytochrome c exhibit one kinetic phase with decreased activity. Electron-transfer activity of the mutant cytochromes c is inhibited by Hg2+. The inhibition is highest for the triple mutant, less for [R13C, C102T]cytochrome c, even less for [D90C, C102T]cytochrome c and insignificant for the wild type. It would appear as though the stability of the triple mutant follows the changes that result from the Asp90-->Cys mutation while the activity changes follow those of the Arg13-->Cys mutation.

The role of water in the dissociation of enolase, a dimeric enzyme.

Exposure of enolase to hydrostatic pressure results in a reversible inactivation of the enzyme; increasing the osmotic pressure, by adding glycerol, glucose, or sucrose to the solutions stabilizes the enzyme against the effects of hydrostatic pressure. The effects of both hydrostatic and osmotic pressure on the rate of inactivation have been determined. As hydrostatic pressure increases, the rate of inactivation increases. As osmotic pressure increases, the rate of inactivation decreases. We have interpreted these results using the following model: hydrostatic pressure causes the active, dimeric enzyme to dissociate into inactive monomers; during the dissociation, the subunit interfaces become hydrated. As osmotic pressure increases, hydration becomes more difficult and dissociation is reduced. The combined effects of hydrostatic and osmotic pressure suggest that much of this hydration occurs during formation of the transition state.

Responses of two protein-protein complexes to solvent stress: does water play a role at the interface?

We have analyzed the stability of the cytochrome c-cytochrome b5 and cytochrome c-cytochrome c oxidase complexes as a function of solvent stress. High concentrations of glycerol were used to displace the two equilibria. Glycerol promotes complex formation between cytochrome c and cytochrome b5 but inhibits that between cytochrome c and cytochrome c oxidase. The results with cytochrome b5 and cytochrome c were expected; the association of this complex is largely entropy driven. Our interpretation is that the cytochrome c-cytochrome b5 complex excludes water. The results with the cytochrome c oxidase and cytochrome c couple were not expected. We interpret them to mean that either glycerol is binding to the oxidase, thereby displacing the cytochrome c, or that water is required at this protein-protein interface. A requirement for substantial quantities of water at the interface of some protein complexes is logical but has been reported only once.

Cytochrome c and cytochrome c oxidase interactions: the effects of ionic strength and hydrostatic pressure studied with site-specific modifications of cytochrome c.

Seven cytochromes c, in which individual lysines have been modified to the propylthiobimane derivatives, have been prepared. These derivatives were also converted to the porphyrin cytochromes c by treatment with HF. The properties of both types of modified proteins were studied in their reactions with cytochrome c oxidase. The results show that lysines 25, 27, 60, 72, and 87 do not contribute a full charge to the binding interaction with the oxidase. These five residues, with the exception of the lysine-60 derivative, on the front surface of the protein and contain the solvent-accessible edge of the heme prosthetic group. By contrast, lysines 8 and 13 at the top of the front surface do contribute a full charge to the binding interaction with the oxidase. The removal of the positive charge on any one lysine weakens the binding to cytochrome c oxidase by at least 1 kcal (1 cal = 4.1868 J). The presence of bimane at lysines 13 and 87 clearly forces the separation of the cytochrome c and oxidase, but this does not occur with the other complexes. The bimane-modified lysine-13 protein, and to a lesser extent that modified at lysine 8, show the interesting effect of enhanced complex formation with cytochrome c oxidase when subjected to pressure, possibly because of entrapment of water at the newly created interface of the complex. Our observations indicate that the two proteins of the cytochrome c - cytochrome oxidase complex have preferred, but not obligatory, spatial orientations and that interaction occurs without either protein losing significant portions of its hydration shell.

Purification of sn-glycerol-3-phosphate dehydrogenase from Trypanosoma brucei brucei.

A protein has been purified from the membranes of bloodstream forms of Trypanosoma brucei brucei. The purified material contained a single polypeptide chain of molecular mass 67 kilodaltons as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; under "native" conditions it migrated through a Sephacryl S-300 column with a similar molecular mass. The purified protein catalysed electron transfer from sn-glycerol 3-phosphate to oxygen with the subsequent formation of water. Electron transfer by the purified enzyme to O2 was dependent on the presence of low concentrations of the mediator phenazine methosulfate. This protein is clearly the major membrane-bound sn-glycerol-3-phosphate dehydrogenase, but it also has some characteristics suggestive of the trypanosome alternative oxidase activities.

A nontraditional role for water in the cytochrome c oxidase reaction.

The passage of electrons through cytochrome c oxidase is directly related to the activity of water. Reducing the activity in a system containing reductant, oxygen, and cytochrome oxidase blocks electron transfer between reduced cytochrome a and oxidized cytochrome a3. The extent of the block is directly related to the osmotic pressure of the system, implying that the protein shell of the oxidase acts as a semipermeable membrane that excludes osmotic perturbants but not water. It appears that approximately 10 water molecules must enter and leave the oxidase in order for internal electron transfer to occur.

The formation of cytochrome P-450 from cytochrome P-420 is promoted by spermine.

This paper is concerned with camphor-bound bacterial cytochrome P-450 and processes that alter its spin-state equilibrium and influence its transition to the nonactive form, cytochrome P-420, as well as its renaturation to the native camphor-bound cytochrome P-450. Spermine, a polycation carrying a charge of 4 +, and potassium, a monovalent cation, were shown to differently cause an increase of high-spin content of camphor-bound cytochrome P-450. The spermine-induced spin transition saturates around 75% of the high spin; a further addition of KCl to the spermine-containing sample shifted the spin state to 95% of the high spin. The volume change of these spin transitions as measured by the use of high pressure indicated an excess of -40 mL/mol for the sample containing potassium as compared to that containing spermine. These results suggest that the proposed privileged site for potassium has not been occupied by spermine and that pressure forces both the camphor and the potassium ion from its sites, allowing solvent movement into the protein as well as ordering of solvent by the excluded camphor and potassium. Cytochrome P-420 was produced from cytochrome P-450 by hydrostatic pressure in the presence of potassium, spermine, and cysteine. Potassium cation shows a bigger effect on the stability of cytochrome P-450 than spermine or cysteine, as revealed by a higher value of the pressure of half-inactivation, P1/2, and a bigger inactivation volume change. However, potassium cation did not promote renaturation of cytochrome P-420 to cytochrome P-450 while the presence of spermine did.(ABSTRACT TRUNCATED AT 250 WORDS)