A rapid separation of bovine brain S-100a and S-100b proteins and related conformation studies

S-100 protein absorbs to the calmodulin antagonist W-7 coupled to epoxy-activated Sepharose 6B in the presence of Ca²⁺ and is eluted by ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid buffer. S-100a and S-100b were separated and isolated by Ca²⁺-dependent affinity chromatography on W-7 Sepharose. The Ca²⁺-induced conformational changes of S-100a and S-100b were examined using circular dichroism, ultraviolet difference spectra, and a fluorescence probe. Differences in Ca²⁺-dependent conformational changes between S-100a and S-100b became apparent. Circular dichroism studies revealed that both S-100a and S-100b undergo a conformational change upon binding of Ca²⁺ in the aromatic and far-uv range. In the presence or absence of Ca²⁺, the aromatic CD spectrum of S-100a differed completely from that of S-100b, possibly due to the single tryptophan residue of S-100a. Far-uv studies indicate that α-helical contents of both S-100a and S-100b decreased with addition of Ca²⁺. Ca²⁺-induced conformational changes of S-100a and S-100b were also detected by uv difference spectra. The spectrum of S-100a also differed from that of S-100b. Fluorescence studies using 2-p-toluidinylnaphthalene-6-sulfonate (TNS), a hydrophobic probe for protein, revealed a slight difference in conformational changes of these two components. The interaction of TNS and S-100b was observed with concentrations above 3 μm Ca²⁺; on the other hand, S-100a required concentrations above 8 μm. This finding was supported by the difference in the binding affinities of S-100a and S-100b to the W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide)-Sepharose column; both S-100a and S-100b bound the column in the presence of Ca²⁺ but S-100a was eluted prior to S-100b. These results suggest that S-100a and S-100b differ in their dependence on Ca²⁺ and that the affinity-chromatographic separation of S-100a from S-100b on the W-7-Sepharose column makes feasible a rapid purification of these two components.     

Conformation and dynamics of bovine brain S-100a protein determined by fluorescence spectroscopy.

We have used time-resolved laser fluorescence spectroscopy to investigate the intensity and anisotropy decays of the single tryptophan residue in bovine brain S-100a (alpha beta) protein. The steady-state and acrylamide quenching results indicated that the Trp 90 of the alpha-subunit was partially buried in a relatively nonpolar environment at pH 7.5. Both Ca2+ and pH 8.5 slightly enhanced the exposure of the residue to the solvent, but the residue remained partially buried in the calcium complex at both pH values. The best representation of the intensity decays was a linear combination of three exponential terms, regardless of solvent condition and temperature. The three lifetimes (tau i) were in the range of 0.4-5 ns and insensitive to emission wavelength, but their fractional amplitudes (alpha i) shifted in favor of the shortest component (alpha 1) when the decays were measured at the blue end of the emission spectrum. These results suggest that an excited-state interaction between the indole ring and the side chain of an adjacent residue may be responsible for the observed shortest lifetime. In the presence of Ca2+, the three lifetimes remained relatively unaltered, but the values of alpha 1 decreased by a factor of 2.3 at pH 7.2 and a factor of 1.8 at pH 8.2. This Ca(2+)-induced decrease may be attributed to disruption of the putative excited-state interaction resulting from reorientations of the alpha-helical segments flanking a Ca(2+)-binding loop (residues 62-73). At both pH 7.2 and 8.4, the anisotropy decays of the apoprotein followed a biexponential decay law.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-lived tryptophan fluorescence in phosphoglycerate mutase

Phosphoglycerate mutase (PGM; EC 2.7.5.3) isolated from rat and rabbit muscle has been shown to possess an unusually long-lived fluorescence component when excited by ultraviolet light below 310 nm. On the basis of spectral and physical measurements, this 16.4 (+/- 0.2) ns fluorescence lifetime at room temperature is assigned to a tryptophan residue in an unusual environment. The emission profile of this long-lived tryptophan is red shifted from the other tryptophans of PGM by approximately 25 nm. PGM has been crystallized and sequenced from yeast where it has been shown to be a tetramer with 29K subunits. However, we have not been able to detect the existence of an unusually long-lived fluorescence component in the yeast isomer. The long fluorescence lifetime is lost upon denaturation of rabbit PGM and is partially restored upon introduction of the protein to a nondenaturing environment, suggesting the long lifetime is not the result of a covalent modification. The PGM molecule was studied by a number of techniques including time-resolved tryptophan fluorescence, quenching studies of tryptophan fluorescence, and enzyme activity studies. The long-lived fluorescence has been shown to be statistically quenched by Br-, I-, and Cu2+ in the submillimolar region while the acrylamide quenching shows the tryptophan is marginally accessible to solvent. Characterization of the long-lived fluorescence and its possible sources are discussed.     

Purification and characterization of adipose tissue S-100b protein

We have purified S-100 protein from bovine brain using Ca2+-dependent affinity chromatography on N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7)-Sepharose (Endo, T., Tanaka, T., Isobe, T., Kasai, H., Okuyama, T., and Hidaka, H. (1981) J. Biol. Chem. 256, 12485-12489). By essentially the same procedure, W-7-Sepharose binding protein has been purified to apparent homogeneity from bovine abdominal adipose tissue. Electrophoretically, the purified protein from adipose tissue co-migrated with brain S-100b protein both in the presence and absence of sodium dodecyl sulfate and the protein was indistinguishable from brain S-100b region in terms of amino acid composition, two-dimensional tryptic peptide mapping and reactivity with anti-brain S-100b serum. Immunohistochemical analysis confirmed the existence of S-100b protein in the adipose cell where the protein seems to be located in both the nucleus and cytoplasm. Thus, the results indicate that the adipose cells contain the protein possibly identical with brain S-100b protein. In addition, the contents of S-100b protein in various rat tissues were measured by enzyme immunoassay method using the anti-bovine brain S-100b serum. Significant amounts of S-100b protein were found not only in the adipose tissue but also in the peripheral tissue such as trachea and skin. These observations suggest that S-100b protein should no longer be considered as a protein specific to nervous tissues.     

An IgA-binding peptide derived from a streptococcal surface protein.

Surface proteins that bind to the Fc part of human IgA are expressed by many strains of Streptococcus pyogenes, a major human pathogen. Studies of these proteins have been complicated by their size and by their ability to bind human plasma proteins other than IgA. Here, we describe a synthetic 50-residue peptide, derived from streptococcal protein Sir22, that binds human IgA but not any of the other plasma proteins known to bind to Sir22. The peptide binds serum IgA and secretory IgA and binds IgA of both subclasses. Evidence is presented that the peptide folds correctly both in solution and when it is immobilized and that it readily renatures after denaturation. Together, these data indicate that the peptide corresponds to a protein domain that binds IgA with high specificity. This is the first report of an IgA-binding domain that retains its properties in isolated form.     

Tyrosine and tyrosinate fluorescence of S-100b. A time-resolved nanosecond fluorescence study. The effect of pH, Ca(II), and Zn(II)

The properties of the tyrosine and tyrosinate emissions from brain S-100b have been studied by nanosecond time-resolved fluorescence at emission wavelengths in the range 305 to 365 nm. The effect of pH on the fluorescence has been studied at pH 6.5, 7.5, and 8.5 for the Ca(II) apo and holo forms of the protein, and for the apo and holo forms in the presence and absence of Zn(II) at pH 7.5. The fluorescence decay is biexponential at pH 8.5 and triexponential at pH 6.5 and 7.5. The three components of the decay have wavelength and metal ion dependent lifetimes in the ranges 0.06 to 1.05 ns, 0.49 to 3.76 ns, and 3.60 to 14.5 ns. The observation of a long lifetime component at wavelengths characteristic of emission from tyrosinate suggests that in class A proteins this may be a useful diagnostic of the environment of tyrosine in their native structures. The time-resolved emission spectra provide evidence for efficient, subnanosecond protolysis of the excited state of the single tyrosine (Tyr17) under all conditions studied except in 6 M guanidium chloride in which the protein shows only emission from tyrosine (lambda em 305 nm), suggesting that the tyrosinate emission is a property of the tertiary structure of the native protein. The Zn(II)-dependence of the fluorescence is fully consistent with the earlier suggestion that Tyr17 is near the Zn(II) binding site and remote from the high affinity Ca(II) binding site.     

A synthetic peptide derived from p34cdc2 is a specific and efficient substrate of src-family tyrosine kinases.

A peptide derived from p34cdc2, cdc2(6-20)NH2 with the amino acid sequence of KVEKIGEGTYGVVYK-amide, was found to be a specific and efficient substrate for a pp60c-src-related protein tyrosine kinase from bovine spleen (STK). Glu-12 and Thr-14 were identified to be substrate specificity determinants in this peptide (Cheng, H.-C., Litwin, C. M. E., Hwang, D. M., and Wang, J. H. (1991) J. Biol. Chem. 266, 17919-17925). In this study, we demonstrated the presence of cdc2(6-20)NH2 peptide tyrosine kinase activity in the membrane fractions of bovine brain, spleen, thymus, lung, liver, and kidney. Hydroxylapatite column chromatography of thymus membrane extract revealed four protein tyrosine kinases, TK-I, TK-II, TK-III, and TK-IV, with different relative activities toward cdc2(6-20)NH2 and a general tyrosine kinase substrate, poly(Glu/Tyr). Only TK-I and TK-II showed significant activity toward cdc2(6-20)NH2, they were suggested as belonging to the src-family by virtue of their cross-reactivity with an antibody against a synthetic peptide corresponding to a conserved sequence of src-family kinases. Further immunological characterization using antibodies specific to individual src-related protein tyrosine kinases suggested that TK-I, TK-II, and STK are bovine homologs of p56lck, p55fyn, and p56lyn, respectively. Substrate specificity and kinetic characterization of src-family tyrosine kinases including human platelet pp60c-src, bovine p56lyn, p56lck, and p55fyn, as well as several non-src-related tyrosine kinases including epidermal growth factor receptor, p43v-abl, TK-III, and TK-IV showed that all the src-family tyrosine kinases but none of the other kinases displayed efficient cdc2(6-20)NH2 phosphorylation. In all cases, the high efficiency of cdc2(6-20)NH2 peptide phosphorylation could be markedly attenuated when Glu-12 and Thr-14 of the peptide were substituted, respectively, by valine and serine.     

S-100-like immunoreactivity in a planarian

Summary The presence of an S-100-like immunoreactivity was investigated in the planarian Dugesia gonocephala. By microcomplement fixation assay, measurable amounts of S-100-like immunoreactive material (0.11g/mg soluble protein) were detected in planarian high-speed supernatants. The index of immunological dissimilarity between ox S-100 and planarian S-100-like immunoreactive material was higher than that previously calculated between ox S-100 and all the vertebrates tested. By the immunohistochemical PAP method, S-100-like immunoreactivity was only detectable in the cilia of the epidermal cells. Although the biological meaning of S-100-like immunoreactivity in planarian remains to be clarified, the present data introduce new perspectives into the investigation of S-100.     

S-100b protein regulates aggregation and fusion of cardiolipin vesicles

We have recently shown that S-100b protein interacts with the polar surface of cardiolipin vesicles [6]. This interaction produces changes in the secondary structure of S-100b as well as changes in the structural organization of cardiolipin vesicles. We report here on the effects of S-100b on cardiolipin vesicles as investigated by turbidity, terbium-dipicolinate fluorescence and freeze-fracture. Experiments were carried out in the absence and in the presence of Ca2+. In the absence of Ca2+ (0.1 mM EDTA), S-100b favors the aggregation and fusion of vesicles to some extent. Under these conditions, electron microscope analyses reveal the presence of fused vesicles along with particles similar to those observed in protein reconstituted systems or to lipid particles observed during fusional processes. In the presence of Ca2+, S-100b counteracts the Ca2(+)-dependent tendency of vesicles to aggregate and fuse. Under these conditions, bilayer phases along with hexagonal phases can be observed by electron microscopy. The latter effects of S-100b are not due to chelation of Ca2+ because of the relative concentrations of S-100b and Ca2+ under our experimental conditions and since much larger concentrations of EDTA are required to produce the S-100b effects. We propose that the dimeric nature of S-100b plays a major role in these events. In the absence of Ca2+, the S-100b molecules probably cross-link adjacent vesicles, one subunit contacting one vesicle and the other subunit contacting another vesicle through electrostatic bonds. In the presence of Ca2+, due to the large changes occurring in the conformation of the protein (which loses about 52% of its alpha-helical content), S-100b associates strongly with the polar surface of individual vesicles, thus generating some kind of physical barrier to aggregation and fusion of vesicles.     

Non-alcoholic Steatohepatitis Induces a Decrease in the Levels of S-100b in the Rat Brain

Using an experimental model of non-alcoholic (alimentary-induced) steatohepatitis in rats, we found that this pathological condition created by consumption of a special diet for 16 weeks results in a drop in the brain mass (by 22%, on average) and also in decreases in the size and morphological modifications of astrocytes and the level of a calcium-binding protein, S-100b. The latter shifts were greater in the hindbrain, including the cerebellum (34.8%), than in the hemispheres, including the thalamus, hypothalamus, hippocampus, and cortex (18.0%).     

Identification of S-100 proteins and S-100-binding proteins in a detergent-resistant EDTA/KCl-extractable fraction from bovine brain membranes

The Triton X-100-resistant residue of brain membranes contains appreciable amounts of S-100 proteins. This fraction of S-100 can be solubilized by high concentrations of EDTA plus or minus high concentrations of KCl. Whereas KCl (0.6 M) extracts the detergent-resistant S-100, NaCl (1 M) does not. Endogenous Ca2+ is required and is sufficient for S-100 to remain associated with the detergent-resistant residue. However, 0.6 M KCl extracts a further fraction of Triton X-100-resistant S-100. In contrast, the Triton X-100-extractable fraction of S-100 resists the action of EDTA. These data suggest that Ca2+ regulates the extent of association of S-100 with Triton X-100-resistant components in brain membranes, whereas the association of S-100 with the lipid bilayer of brain membranes and/or with some intrinsic membrane proteins is less Ca2+-regulated. Several S-100-binding proteins are identified in the detergent-resistant residue of brain membranes by an overlay procedure.     

Ca2+ and Zn2+-binding properties of nitrated S-100b protein from bovine brain.

The single tyrosine residue in S-100b protein was nitrated by treatment with tetranitromethane in 0.1 M-Tris/HCl buffer, pH 8.0, containing 2 mM-EDTA. The nitrated protein did not differ significantly in secondary structure from its native unmodified counterpart, as revealed by far-u.v. c.d. measurements. The effect of Ca2+ on the modified protein was different from that on the native protein, e.g. addition of Ca2+ resulted in a loss of helical content from 55 to 47% with the native protein whereas Ca2+ had no significant effect on the gross conformation of the nitrated derivative. Near-u.v. c.d. studies also indicated a very minimal effect on the tyrosine residue and this was also reflected in the u.v.-absorption difference spectrum. Polyacrylamide-gel electrophoresis in the absence of SDS showed the nitrated S-100b to move faster in the presence of EDTA compared with the calcium-bound state, suggesting that the modified protein does bind Ca2+ although it does not undergo a major conformational change in response to Ca2+ addition. In contradistinction, Zn2+ binding was not influenced by nitration, as demonstrated by aromatic c.d. and u.v.-difference spectroscopy. It is clear from this study that the single tyrosine residue in S-100b is critical to sense the Ca2+-induced conformational changes in the protein.     

Purification and characterization of a pp60c-src-related tyrosine kinase that effectively phosphorylates a synthetic peptide derived from p34cdc2.

A protein tyrosine kinase has been purified from the particulate fraction of bovine spleen to a specific activity of 0.217 mumol/min/mg at 100 microM ATP and 3 mM [Val5] angiotensin II. Both the angiotensin phosphorylation activity and immunoreactivity towards an antibody preparation raised against a synthetic peptide containing the autophosphorylation site of pp60c-src, Cys-src(403-421), were monitored during the purification. The purified sample displayed three closely spaced protein bands with molecular weights of 50-55 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All bands could be phosphorylated exclusively on tyrosine residues under autophosphorylation conditions. All reacted on immunoblots with an antibody raised against a synthetic peptide corresponding to the consensus autophosphorylation site of members of the pp60c-src family of tyrosine kinases. Tryptic phosphopeptide maps of the three proteins were essentially indistinguishable. The results suggest that the purified enzyme preparation contained mainly three closely related pp60c-src-family protein tyrosine kinases or a pp60src-family protein tyrosine kinase modified posttranslationally to give three closely spaced protein bands on sodium dodecyl sulfate gel. Neither of these proteins appears to be pp60c-src or p56lck. The spleen protein tyrosine kinase was found to phosphorylate a p34cdc2 kinase peptide, Cys-cdc2(8-20), which contained the regulatory tyrosine residue Tyr-15 about 20 times better than [Val5]angiotensin II or Cys-src(403-421) peptide at a peptide substrate concentration of 1 mM. In contrast, epidermal growth factor receptor kinase partially purified from A431 cells did not show preference for Cys-cdc2(8-20) as its substrate. Although Cys-cdc2(8-20) contained two tyrosine residues, only the tyrosine corresponding to Tyr-15 in p34cdc2 was phosphorylated by the spleen tyrosine kinase. The observation suggests that the primary structure surrounding Tyr-15 of p34cdc2 contains substrate structural determinants specific for the spleen tyrosine kinase.     

Interaction of S-100b protein with cardiolipin vesicles as monitored by electron spin resonance, pyrene fluorescence and circular dichroism

The interaction of S-100b protein with cardiolipin (CL) vesicles has been studied by electron spin resonance, pyrene fluorescence, and circular dichroism. Electron spin resonance and pyrene fluorescence data indicate that S-100b binds to the polar surface of vesicles Ca2+-independently. In the presence of Ca2+, S-100b potentiates the Ca2+-induced clustering of the polar headgroups of CL molecules and causes a further reduction in the Ca2+-dependent decrease in the lateral mobility of the pyrene inserted into the lipid bilayer, which points to an effect of the protein on the hydrophobic core of the lipid bilayer through a larger perturbation of its polar surface. Circular dichroism analyses indicate that CL vesicles cause a decrease in the alpha-helical content of S-100b, analogous to that produced by Ca2+ and that the effects of CL vesicles and of Ca2+ on the secondary structure of the protein are supra-additive. By this technique, we found that the affinity of Ca2+ for S-100b increases substantially in the presence of CL vesicles, even in the presence of physiologic concentrations of KCl, suggesting that once S-100b had interacted with CL vesicles it assumes a new conformation in which its Ca2+-binding properties are greatly enhanced. These results are discussed in relation to binding of S-100b proteins to natural membranes, and to a possible involvement of S-100b in the regulation of membrane structural organization.     

Circular dichroism and fluorescence of a tyrosine side-chain residue monitors the concentration-dependent equilibrium between U-shaped and coiled-coil conformations of a peptide derived from the catalytic core of HIV-1 integrase

The peptide denoted K159 (30 residues) derives from the catalytic core (CC) sequence of HIV-1 integrase (IN, residues 147-175). In the crystal structure of CC, the corresponding segment belongs to the alpha4 helix (residues 148-168, including residues Glu 152, Lys 156 and Lys 159, crucial for enzyme activity and DNA recognition), a loop (residues 169-171) and a part of the alpha5 helix (171-175), involved in enzyme dimerization. We used the fluorescence and the circular dichroism (CD) properties in the near-UV of the aromatic side chain of a tyrosine residue added at the C-terminal end of K159 in order to analyze the behavior of the concentrated and diluted peptide in aqueous trifluoroethanol (TFE), in an attempt to connect the information obtainable at high (NMR), medium (CD) and low (fluorescence) concentrations of the peptide. Altogether, the C-terminal tyrosine residue provided indirect information on the global conformation of K159 and on the local orientation and environment of the residue. The propensity of TFE to stabilize alpha-helical conformations in peptides was confirmed in CD and fluorescence experiments at relatively high (20-160 microM) and low (2-16 microM) concentrations, respectively. At relatively high concentration, stabilization of the peptide into alpha-helical conformation favored its auto-association likely in parallel coiled-coil dimers, as pointed out in our previous work [Eur. J. Biochem. 253 (1998) 236]. This was further confirmed by ANS (1-anilinonaphtalene-8-sulfonic acid) analysis and fluorescence temperature coefficient measurement. With diluted K159, a Stern-Volmer analysis with positively and negatively charged quenchers indicated that, when the intermolecular interactions were absent, the tyrosine was in a positively charged environment, as if the peptide folded into a U-shaped conformation similar to that present in the crystal structure of the enzyme.     

Conformational stability of a thrombin-binding peptide derived from the hirudin C-terminus.

The COOH-terminal region of hirudin represents an independent functional domain that binds to an anion-binding exosite of thrombin and inhibits the interaction of thrombin with fibrinogen and regulatory proteins in blood coagulation. The thrombin-bound structure of the peptide fragment, hirudin 55-65, has been determined by use of transferred NOE spectroscopy [Ni, F., Konishi, Y., & Scheraga, H. A. (1990) Biochemistry 29, 4479-4489]. The stability of the thrombin-bound conformation has been characterized further by a combined NMR and theoretical analysis of the conformational ensemble accessible by the hirudin peptide. Medium- and long-range NOE's were found for the free hirudin peptide in aqueous solution and in a mixture of dimethyl sulfoxide and water at both ambient (25 degrees C) and low (0 degrees C) temperatures, suggesting that ordered conformations are highly populated in solution. The global folding of these conformations is similar to that in the thrombin-bound state, as indicated by NOE's involving the side-chain protons of residues Phe(56), Ile(59), Pro(60), Tyr(63), and Leu(64). Residues Glu(61), Glu(62), Tyr(63), and Leu(64) all contain approximately 50% of helical conformations calculated from the ratio of the sequential dNN and d alpha N NOE's. Among the helical ensemble, active 3(10)-helical conformations were found by an analysis of the medium-range [(i,i+2) and (i,i+3)] NOE's involving the last six residues of the peptide. An analysis of the side-chain rotamers revealed that, upon binding to thrombin, there may be a rotation around the alpha CH-beta CH bond of Ile(59) such that Ile(59) adopts a gauche- (chi 1 = +60) conformation in contrast to the highly populated trans (chi 1 = -60) found for Ile(59) in the free peptide. However, the thrombin-bound conformation of the hirudin peptide is still an intrinsically stable conformer, and the preferred conformational ensemble of the peptide contains a large population of the active conformation. The apparent preference for a gauche- (chi 1 = +60) side-chain conformation of Ile(59) in the bound state may be explained by the existence of a positively charged arginine residue among the hydrophobic residues in the thrombin exosite.     

Rubiscolin, a delta selective opioid peptide derived from plant Rubisco.

We found that the sequences YPLDL and YPLDLF in the large subunit of spinach D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) met the structure YP-aliphatic amino acid which might have opioid activity. We then synthesized these peptides to test their opioid activity. The IC(50) of these peptides in mouse vas deferens assay were 51.0 microM and 24.4 microM, respectively, and those in delta receptor binding assay using [(3)H]deltorphin II as radioligand were 2.09 microM and 0.93 microM, respectively. Both peptides were selective for delta receptor. We named them rubiscolin-5 and -6, respectively. Rubiscolin-5 and -6 have antinociceptive activity in mice after i.c.v. or oral administration. The enzymatic conditions to release rubiscolin were investigated using both spinach Rubisco and synthetic fragment peptides. This is the first example of bioactive peptides derived from plant Rubisco.     

Studies on a growth-inhibitory peptide derived from alpha-fetoprotein and some analogs.

A 34-amino acid synthetic peptide was derived from the third domain of human alpha-fetoprotein, and the peptide was shown to inhibit estrogen-stimulated growth. Under certain conditions, however, the peptide lost growth-inhibitory activity. A biophysical study of the peptide was undertaken with a goal of obtaining completely reliable preparations. The peptide was studied using gel-filtration column chromatography as a function of peptide concentration and age of solution, and was found to exhibit complex aggregation behaviors. During the early period (0-3 h) after dissolving lyophilized peptide into pH 7.4 buffer, solutions were composed mostly of trimers. At higher peptide concentrations (> or = 3.0 g/L), the trimers aggregated extensively to a large aggregate (minimum size approximately 102 peptides). At 5.0-8.0 g/L, these large aggregates increased in size (up to approximately 146 peptides) until trimers were largely exhausted from solution. During the later times (>3 h) after sample preparation, the trimeric oligomer of the peptide dissociated slowly to form dimers for samples at 0.10-3.0 g/L. After their build-up, a very small number of dimers associated to form hexamers. Disulfide bonds stabilized the dimers as indicated by the conversion of dimers to trimers upon the addition of a reducing agent, and the failure of dimers to form in the presence of reducing agent. Reducing agent did not affect trimer or large aggregate formation. Trimers were found to be active in an assay monitoring inhibition of estrogen-stimulated growth, whereas dimers and large aggregates were inactive. The two cysteines in the peptide were modified to either S-methylcysteine or S-(2-aminoethyl)cysteine, and both derivatives showed significant growth-inhibition activity. A serine analog in which both cysteines were replaced had very different aggregation behavior than the cysteine peptide and lacked its growth inhibitory ability. Peptide aggregation is critically important in establishing the ability of the peptide to inhibit growth and have anticancer activity, but the state of its two cysteines is of little influence.     

Structure and dynamics of motilin. Time-resolved fluorescence of peptide hormone with single tyrosine residue.

Time-resolved fluorescence and CD spectroscopy were used to characterize the structure and dynamics of the peptide hormone motilin with a single tyrosine residue among its 22 amino acids. CD spectroscopy showed that secondary structure is independent of concentration in the range 1 x 10(-5)-2.6 x 10(-4) M, and of the presence of DOPC lipid vesicles, but is strongly induced by addition of hexafluoroisopropanol. The fluorescence studies with tyrosine as the intrinsic fluorophore, performed at the MAX synchrotron laboratory at Lund, showed that three fluorescence lifetimes (0.4 ns, 1.7 ns and 3.6 ns at 20 degrees C) and two rotational correlation times (0.4 ns and 5 ns at 20 degrees C) were needed to account for the data. The different decay times are interpreted as representing ground-state rotamers interconverting slowly on the ns time scale. The rotational correlation times are ascribed to local angular motion of the tyrosyl ring, and global motion of the whole peptide, respectively.