Anticancer versus antigrowth activities of three analogs of the growth-inhibitory peptide: relevance to physicochemical properties.

A 34-amino acid peptide has been synthesized based on an amino acid sequence from the third domain of native full-length alpha-fetoprotein, which has been shown to have both antigrowth and anticancer activities. This peptide, known as the growth-inhibitory peptide (GIP), has two cysteine residues and demonstrates reduced antigrowth activity after long-term storage, presumably due to disulfide bond formation. The disulfide bridge problem was addressed by replacing the two naturally occurring cysteines with either glycines, alanines, or serines (to produce the G-, A- and S-peptides, respectively). The non-hydrophobic G- and S-peptides were found to exist as dimers, while the more hydrophobic C- and A-peptides formed trimers in solution under certain conditions of pH and peptide concentration. The A-peptide was already known to display anticancer activity; however, the G- and S-serine analogs have not been studied in depth since they had demonstrated low antigrowth activities in rodent uterine assays. Using both in vivo and in vitro assays, the A-, G- and S-peptides were shown to exhibit various degrees of cancer growth suppression. An in vitro culture assay, using MCF-7 breast cancer cells, demonstrated that both the G- and S-peptides showed modest cancer growth suppression, while the A- analog showed strong inhibition at doses ranging from 10(-5) M to 10(-7) M. In contrast, an in vivo ascites tumor study of all four peptides showed them to have notable activity in the suppression of mouse mammary tumor growth. Overall, our data indicated that physicochemical properties, such as hydrophobicity, oligomeric state and secondary structure, contribute to the anticancer activity of both the active C- peptide and its analogs. In addition, the antigrowth rodent uterine assay was not always predictive of the anticancer potential of the peptide forms, suggesting a difference between the mechanism of peptide action in the antigrowth models and that in the anticancer assay. Notably, the antigrowth assay failed to predict the marked anticancer activity of the analogs against a mammary tumor, indicating that the growth bioassay cannot always be relied upon as a screening protocol.

Fluorescence polarization studies on four biliproteins and a bilin model for phycoerythrin 545.

Fluorescence (excitation) polarization spectroscopy in the wavelength region of the bilin chromophores was applied to phycoerythrocyanin (CV-phycocyanin), phycocyanins 645 and 612, and phycoerythrin 545. The cryptomonad biliproteins - phycoerythrin 545 and phycocyanins 612 and 645 - were studied as both protein dimers having an alpha(2)beta(2) polypeptide structure and as alphabeta monomers. The cyanobacterial phycoerythrocyanin (CV-phycocyanin) was a trimeric oligomer. The changes in polarization across the spectrum were attributed to transfers of energy between bilins. Cryptomonad biliproteins are isolated as dimers. The similarities between their steady-state fluorescence polarization spectra and those of the corresponding monomers suggested that the monomers' conformations were analogous to the dimers. This supports the use of monomers in the study of dimer bilin organization. The unusual polarization spectrum of phycoerythrin 545 was explained using a model for the topography of its bilins. Obtaining the emission spectra of phycoerythrin 545 at several temperatures and a deconvolution of the dimer circular dichroism spectrum also successfully tested the bilin model. Circular dichroism spectroscopy was used to determine which polarization changes are formed by Förster resonance energy transfers and which may be produced by internal conversions between high- and low-energy states of pairs of exciton-coupled bilins. Attempts were made to assign energy transfer events to the corresponding changes in fluorescence polarization for each of the four biliproteins.

Circular dichroism analysis of the glucan binding domain of Streptococcus mutans glucan binding protein-A.

The glucan binding domain (GBD) of the glucan binding protein-A (GBP-A) from the cariogenic bacterium Streptococcus mutans was studied using circular dichroism (CD) analysis, Chou-Fasman-Rose secondary structure prediction, and absorption and fluorescence spectroscopy. Our data show that the binding domain undergoes a conformational shift upon binding to the ligand dextran. The CD spectrum shows two positive bands at 280 nm and 230 nm which were assigned to aromatic residues. The 230-nm band was seen at 20 degrees C and 30 degrees C, lost intensity at 40 degrees C, and was eliminated at 45 degrees C coinciding with complete denaturation. The protein was stable at physiological pH, but precipitated at pH 5. A pH of 10 changed the secondary structure but had no effect on the 230-nm band. Analysis of the CD data in the far UV using the SELCON computer program revealed a high content of beta-sheets and a lack of alpha-helical structures. Secondary structure prediction based on the amino acid sequence of GBD agreed with the CD analysis. The fluorescence emission maximum at 339 nm suggested that the majority of the tryptophans were located in the interior of the protein. This maximum shifted to higher energy upon binding to the ligand dextran.

Studies on C-phycocyanin from Cyanidium caldarium, a eukaryote at the extremes of habitat.

C-Phycocyanin, a biliprotein, was purified from the red alga, Cyanidium caldarium. This alga grows at temperatures up to 57 degrees C, a very high temperature for a eukaryote, and at pH values down to 0.05. Using the chromophores on C-phycocyanin as naturally occurring reporter groups, the effects of temperature on the stability of the protein were studied by circular dichroism and absorption spectroscopy. The protein was unchanged from 10 to 50 degrees C, which indicates that higher temperatures are not required to cause the protein to be photosynthetically active. At 60 and 65 degrees C, which are above the temperatures at which the alga can survive, the protein undergoes irreversible denaturation. Gel-filtration column chromatography demonstrated that the irreversibility is caused by the dissociation of the trimeric protein to its constitutive polypeptides. Upon cooling, the alpha and beta polypeptides did not reassemble to the trimer. Unlike phycocyanins 645 and 612, the C-phycocyanin does not show a reversible conformational change at moderately high temperatures. At constant temperature, the C-phycocyanin was more stable than a mesophilic counterpart. It is designated a temperature-resistant protein.

Interrelationships among biological activity, disulfide bonds, secondary structure, and metal ion binding for a chemically synthesized 34-amino-acid peptide derived from alpha-fetoprotein.

A 34-amino-acid peptide has been chemically synthesized based on a sequence from human alpha-fetoprotein. The purified peptide is active in anti-growth assays when freshly prepared in pH 7.4 buffer at 0.20 g/l, but this peptide slowly becomes inactive. This functional change is proven by mass spectrometry to be triggered by the formation of an intrapeptide disulfide bond between the two cysteine residues on the peptide. Interpeptide cross-linking does not occur. The active and inactive forms of the peptide have almost identical secondary structures as shown by circular dichroism (CD). Zinc ions bind to the active peptide and completely prevents formation of the inactive form. Cobalt(II) ions also bind to the peptide, and the UV-Vis absorption spectrum of the cobalt-peptide complex shows that: (1) a near-UV sulfur-to-metal-ion charge-transfer band had a molar extinction coefficient consistent with two thiolate bonds to Co(II); (2) the lowest-energy visible d-d transition maximum at 659 nm, also, demonstrated that the two cysteine residues are ligands for the metal ion; (3) the d-d molar extinction coefficient showed that the metal ion-ligand complex was in a distorted tetrahedral symmetry. The peptide has two cysteines, and it is speculated that the other two metal ion ligands might be the two histidines. The Zn(II)- and Co(II)-peptide complexes had similar peptide conformations as indicated by their ultraviolet CD spectra, which differed very slightly from that of the free peptide. Surprisingly, the cobalt ions acted in the reverse of the zinc ions in that, instead of stabilizing anti-growth form of the peptide, they catalyzed its loss. Metal ion control of peptide function is a saliently interesting concept. Calcium ions, in the conditions studied, apparently do not bind to the peptide. Trifluoroethanol and temperature (60 degrees C) affected the secondary structure of the peptide, and the peptide was found capable of assuming various conformations in solution. This conformational flexibility may possibly be related to the biological activity of the peptide.

The basal transcription factors TBP and TFB from the mesophilic archaeon Methanosarcina mazeii: structure and conformational changes upon interaction with stress-gene promoters.

Transcription of archaeal non-stress genes involves the basal factors TBP and TFB, homologs of the eucaryal TATA-binding protein and transcription factor IIB, respectively. No comparable information exists for the archaeal molecular-chaperone, stress genes hsp70(dnaK), hsp40(dnaJ), and grpE. These do not occur in some archaeal species, but are present in others possibly due to lateral transfer from bacteria, which provides a unique opportunity to study regulation of stress-inducible bacterial genes in organisms with eukaryotic-like transcription machinery. Among the Archaea with the genes, those from the mesophilic methanogen Methanosarcina mazeii are the only ones whose basal (constitutive) and stress-induced transcription patterns have been determined. To continue this work, tbp and tfb were cloned from M. mazeii, sequenced, and the encoded recombinant proteins characterized in solution, separately and in complex with each other and with DNA. M. mazeii TBP ranks among the shortest within Archaea and, contrary to other archaeal TBPs, it lacks tryptophan or an acidic tail at the C terminus and has a basic N-terminal third. M. mazeii TFB is similar in length to archaeal and eucaryal homologs and all have a zinc finger and HTH motifs. Phylogenetically, the archaeal and eucaryal proteins form separate clusters and the M. mazeii molecules are closer to the homologs from Archaeoglobus fulgidus than to any other. Antigenically, M. mazeii TBP and TFB are close to archaeal homologs within each factor family, but the two families are unrelated. The purified recombinant factors were functionally active in a cell-free in vitro transcription system, and were interchangeable with the homologs from Methanococcus thermolithotrophicus. The M. mazeii factors have a similar secondary structure by circular dichroism (CD). The CD spectra changed upon binding to the promoters of the stress genes grpE, dnaK, and dnaJ, with the changes being distinctive for each promoter; in contrast, no effect was produced by the promoter of a non-stress-gene. Factor(s)-DNA modeling predicted that modifications of H bonds are caused by TBP binding, and that these modifications are distinctive for each promoter. It also showed which amino acid residues would contact an extended TATA box with a B recognition element, and evolutionary conservation of the TBP-TFB-DNA complex orientation between two archaeal organisms with widely different optimal temperature for growth (37 and 100 degrees C).

The binding domain structure of retinoblastoma-binding proteins.

The retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu-X-Cys-X-Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb-binding protein to another. In this report, we present a diagnostic computer pattern for a high-affinity Rb-binding domain featuring the three conserved residues as well as the conserved physico-chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico-chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding.

Cyanobacterial phycobilisomes

Cyanobacterial phycobilisomes harvest light and cause energy migration usually toward photosystem II reaction centers. Energy transfer from phycobilisomes directly to photosystem I may occur under certain light conditions. The phycobilisomes are highly organized complexes of various biliproteins and linker polypeptides. Phycobilisomes are composed of rods and a core. The biliproteins have their bilins (chromophores) arranged to produce rapid and directional energy migration through the phycobilisomes and to chlorophyll a in the thylakoid membrane. The modulation of the energy levels of the four chemically different bilins by a variety of influences produces more efficient light harvesting and energy migration. Acclimation of cyanobacterial phycobilisomes to growth light by complementary chromatic adaptation is a complex process that changes the ratio of phycocyanin to phycoerythrin in rods of certain phycobilisomes to improve light harvesting in changing habitats. The linkers govern the assembly of the biliproteins into phycobilisomes, and, even if colorless, in certain cases they have been shown to improve the energy migration process. The Lcm polypeptide has several functions, including the linker function of determining the organization of the phycobilisome cores. Details of how linkers perform their tasks are still topics of interest. The transfer of excitation energy from bilin to bilin is considered, particularly for monomers and trimers of C-phycocyanin, phycoerythrocyanin, and allophycocyanin. Phycobilisomes are one of the ways cyanobacteria thrive in varying and sometimes extreme habitats. Various biliprotein properties perhaps not related to photosynthesis are considered: the photoreversibility of phycoviolobilin, biophysical studies, and biliproteins in evolution. Copyright 1998 Academic Press.

R-phycoerythrins having two conformations for the same aggregate.

The visible circular dichroism (CD) spectrum of an R-phycoerythrin (Porphyra tenera) is composed of several positive bands. The protein in aqueous buffer very slowly exhibits changes in the CD spectrum of its chromophores, a band at 489 nm undergoes an increase in intensity and a red shift. When the band reached a 493 nm maximum, the spectrum became very stable. The aggregation state of the protein did not change during this spectral conversion. The chromophore CD spectrum was also obtained in the presence of a low concentration of urea or sodium thiocyanate, and the identical change in the CD was noted, but the change was much faster. The visible absorption and CD in the far UV spectra were unaffected by urea. Unchanged visible absorption and protein secondary structure (61% alpha helix) contradicted by comparatively salient alterations in the visible CD spectra suggested very subtle structural changes are influencing some of the chromophores. For a second R-phycoerythrin (Gastroclonium coulteri), the CD of the chromophores had a negative band on the blue edge of the spectrum. This is the first negative CD band observed for any R-phycoerythrin. Treatment of this protein with low concentrations of urea produced a change in the visible CD with the negative band being completely converted to a positive band. Fluorescence studies showed that the treatment by urea did not affect energy migration. Deconvolution of the CD spectra were used to monitor the chromophores. The results demonstrated that the same aggregate of each R-phycoerythrin could exist in two conformations, and this is a novel finding for any red algal or cyanobacterial biliprotein. The two forms of each protein would differ in tertiary structure, but retain the same secondary structures.

Spectroscopic study of the interaction of aluminum ions with human transferrin.

Transferrin is the plasma protein responsible for transporting Fe3+ from the absorption to the utilization site. Interactions of apo- and holo-transferrin with Al3+ were studied by circular dichroism (CD), UV-visible, and fluorescence spectrometry. Binding of Al3+ to both metal-ion binding sites of apo-transferrin was confirmed by fluorescence studies. No interaction of Al3+ with holo-transferrin was observed, indicating that Al3+ cannot displace Fe3+ under the experimental conditions employed. An increase in tryptophan fluorescence (lambda max at 330 nm) by excitation at either 280 or 295 nm was observed after Al3+ interaction with apo-transferrin. There was no shift in wavelength of the fluorescence band of apo-transferrin after interaction with Al3+, but the intensity did increase. Since excitation at 295 nm is specific for tryptophan residues, tryptophan but not tyrosine must be responsible for the change in fluorescence intensity. Decreased fluorescence is the result of Fe3+ binding to apo-transferrin. The CD spectrum of apo-transferrin was slightly affected in the far UV by Al3+ binding, but a salient change was noted in the near UV at approximately 288 nm where tyrosine and tryptophan absorb. It is concluded that a small conformational change in the protein was induced by Al3+ binding to apo-transferrin.

Fluorescence studies on R-phycoerythrin and C-phycoerythrin.

Biliproteins are photosynthetic light-harvesting proteins, which transfer excitons with high efficiencies over relatively long distances until they arrive at a photosynthetic reaction center. Purified R-phycoerythrin (isolated from a red alga) and C-phycoerythrin (isolated from a cyanobacterium), each of which contains several chromophores, were studied by a combination of fluorescence emission, fluorescence excitation polarization, and absorption methods. The polarization spectra of both these biliproteins showed that there was a minimum of two spectrally distinct sensitizing chromophores, which, after absorbing photons, transfer excitons to the lowest-energy (fluorescing) chromophores. Some of these spectroscopic data were used to deconvolute the absorption spectra into the spectra of the two sensitizing and one fluorescing chromophores. It was shown that the higher-energy sensitizing chromophore could readily transfer its excitation energy to the fluorescing chromophore using the lower-energy sensitizing chromophore as an intermediary. However, there was sufficient spectral overlap between the higher-energy sensitizing chromophore and the fluorescing chromophore so that direct transfer between them could not be ruled out.

Stability of allophycocyanin's quaternary structure.

The dissociation of allophycocyanin trimers to monomers was examined under a variety of conditions. For alkyl ureas and alcohols the dissociation increased as the straight-chain alkyls increased in length. The effect of branching chains was smaller. Tetrapropylammonium chloride was found to be a very effective agent for trimer dissociation when compared to ureas and alcohols with similar or longer alkyl chains. An explanation for these observations is that the hydrocarbons have an affinity for nonpolar regions in the contact areas between monomers in a trimeric structure. A comparison among several inorganic salts demonstrated that the chaotropic salts (NaSCN greater than NaClO4 much greater than NaNO3 greater than NaBr) fostered increased trimer dissociation, while nonchaotropes (KF, (NH4)2SO4, K phosphate, and NaCl) produced no measurable amounts of monomer. Allophycocyanin dissolved in D2O was much more stable against dissociation than when dissolved in H2O. All the above observations were consistent with hydrophobic forces being the dominant source of trimer stabilization. The equilibrium constant for the dissociation of trimers to monomers was calculated to be about 6 X 10(-16) mol2 liter-2. Calculations were made of the apparent total number of amino acids (40) in the two contact regions on each monomer. An absorption change analogous but not necessarily identical to a conversion of allophycocyanin II to III was noted when (NH4)2SO4 was present. When allophycocyanin's nonexchangeable hydrogens were replaced by deuteriums, it was more readily dissociated to monomers.

Effect of aromatic molecules on the aggregation of C-phycocyanin. Quantum chemical calculations on phycocyanobilin and phycoerythrobilin.

The energies of the highest occupied and lowest empty molecular orbitals were calculated for the chromophore groups of the proteins phycocyanin and phycoerythrin. These tetrapyrrole groups on the algal proteins are shown to provide them with the potential of ating as efficient electron donors and acceptors. In addition, the pi electron charges and bond orders were also computed.

Biliproteins and phycobilisomes from cyanobacteria and red algae at the extremes of habitat.

This review considers the properties of biliproteins from cyanobacteria and red algae that grow in extreme habitats. Three situations are presented: cyanobacteria that grow at high temperatures; a red alga that grows in acidic conditions at high temperature; and an Antarctic red alga that grows in the cold in dim light conditions. In particular, the properties of their biliproteins are compared to those from organisms from more usual environments. C-phycocyanins from two cyanobacteria able to grow at high temperatures are found to differ in their stabilities when compared to C-phycocyanin from mesophilic algae. They differ in opposite ways, however. One is more stable to dissociation than the mesophilic protein, and the other is more easily dissociated at low temperatures. The thermophilic proteins resist thermal denaturation much better than the mesophilic proteins. The most thermophilic cyanobacterium has a C-phycocyanin with a unique blue-shifted absorption maximum which does not appear to be part of the adaptation of the cyanobacterium to high temperature. The C-phycocyanin from the high-temperature red alga is able to resist dissociation better than mesophilic C-phycocyanins. Electron micrographs show the phycobilisomes of these algae. The Antarctic alga grows under ice at some distance down the water column. Its R-phycoerythrin has a novel absorption spectrum that gives the alga an improved ability to harvest blue light. This may enhance its survival in its light-deprived habitat.

Phycocyanin 645. The chromophore assay of phycocyanin 645 from the cryptomonad protozoa Chroomonas species.

Phycocyanin 645 was isolated and purified from the cryptomonad Chroomonas species. Its chromophore content was obtained from absorption spectra in acidic 8.0 M urea for both whole protein and the separated subunits. The principal method used to separate the alpha and beta subunits was gel filtration through a Sephacryl S-200 column in acidic urea. The subunits were shown to be completely separated during this procedure by sodium dodecyl sulfate-gel electrophoresis. Spectra were analyzed by three component Beer's law equations. The whole protein was found to consist of four phycocyanobilins (Amax at 662 nm), two cryptoviolins (Amax at 590 nm), and two unnamed bilins with an Amax at 697 nm. The separated subunits were analyzed, and the beta subunit was shown to have two phycocyanobilins for each cryptoviolin and alpha was composed of the 697-nm bilin exclusively. A comparison of the total amounts of alpha and beta from the Sephacryl columns showed that the molar ratios of phycocyanobilin on beta to the 697-nm bilin on alpha was 2:1, and the ratio of cryptoviolin on beta to 697-nm bilin on alpha was 1:1. We therefore propose that, assuming a symmetrical distribution, each beta subunit on the alpha 2 beta 2 protein has two phycocyanobilins and one cryptoviolin and each alpha subunit has one 697-nm bilin. This chromophore distribution differs from one previously reported in which the subunits were separated on a BioRex 70 cation exchange resin in 12% formic acid via a 4-10 M urea gradient.

Spectroscopic properties of tetrapyrroles on denatured biliproteins.

Four biliproteins (phycoerythrin 545, phycocyanin 612, phycocyanin 645, and C-phycocyanin) were denatured by a high concentration of urea and then studied by absorption spectroscopy. Low pH and high protein concentrations conserved the tetrapyrroles' color, and mercaptoethanol and dithiothreitol promoted bleaching. One of these tetrapyrroles, cryptoviolin, appeared not to be hypochromic in the presence of depleting phycocyanobilin, but its absorbance did decay when phycocyanobilin is absent. The product from the treatment of phycocyanobilin with mercaptoethanol or dithiothreitol overlapped spectrally with cryptoviolin and gave the false appearance of maintaining a constant cryptoviolin concentration. Failure to note this effect could result in erroneous cryptoviolin/phycocyanibilin ratios.

Chromophore interactions in allophycocyanin.

Allophycocyanin, which is normally isolated as a trimer (alpha 3 beta 3), has now been successfully dissociated into a monomer (alpha beta) with strikingly different spectroscopic properties. In particular, upon dissociation the characteristic 650-nm absorption and 661-nm fluorescence emission bands of the trimer are completely lost and its fluorescence polarization properties are sharply altered. The spectroscopic characteristics of allophycocyanin monomers are much closer to those of C-phycocyanin than to trimeric allophycocyanin. A model for trimeric allophyocyanin is presented in which the appearance of the 650-nm absorption band is induced by a particular kind of chromophore-chromophore interaction. Similar results are found for both allophycocyanin II and III.