Significance of mutations on the structural perturbation of thymidylate synthase: implications for their involvement in subunit exchange.

Wild-type thymidylate synthase (WT-TS) from Escherichia coli and several of its mutants showed varying degrees of susceptibility to trypsin. While WT-TS was resistant to trypsin as were the mutants C146S, K48E, and R126K, others such as Y94A, Y94F, C146W, and R126E were digested but at different rates from one another. The peptides released from the mutants were identified by mass spectrometry and Edman sequence analysis. The known crystal structures for WT-TS, Y94F, and R126E, surprisingly, showed no structural differences that could explain the difference in their susceptibility to trypsin. One explanation is that the mutations could perturb the dynamic equilibrium of the dimeric state of the mutants as to increase their dissociation to monomers, which being less structured than the dimer, would be hydrolyzed more readily by trypsin. Earlier studies appear to support this proposal since conditions that promote subunit dissociation in solutions of R126E with other inactive mutants, such as dilution, low concentrations of urea, and elevated pH, greatly enhance the rate of restoration of TS activity. Analytic ultracentrifuge studies with various TSs in urea, or at pH 9.0, or that have been highly diluted are, for the most part, in agreement with this thesis, since these conditions are associated with an increase in dissociation to monomers, particularly with the mutant TSs. However, these studies do not rule out the possibility that conformation differences among the various TS dimers are responsible for the differences in susceptibility to trypsin, particularly at high concentrations of protein where the WT-TS and mutants are mainly dimers.

Three-dimensional structure of the R115E mutant of T4-bacteriophage 2'-deoxycytidylate deaminase.

2'-Deoxycytidylate deaminase (dCD) converts deoxycytidine 5'-monophosphate (dCMP) to deoxyuridine 5'-monophosphate and is a major supplier of the substrate for thymidylate synthase, an important enzyme in DNA synthesis and a major target for cancer chemotherapy. Wild-type dCD is allosterically regulated by the end products of its metabolic pathway, deoxycytidine 5'-triphosphate and deoxythymidine 5'-triphosphate, which act as an activator and an inhibitor, respectively. The first crystal structure of a dCD, in the form of the R115E mutant of the T4-bacteriophage enzyme complexed with the active site inhibitor pyrimidin-2-one deoxyribotide, has been determined at 2.2 A resolution. This mutant of dCD is active, even in the absence of the allosteric regulators. The molecular topology of dCD is related to that of cytidine deaminase (CDA) but with modifications for formation of the binding site for the phosphate group of dCMP. The enzyme has a zinc ion-based mechanism that is similar to that of CDA. A second zinc ion that is present in bacteriophage dCD, but absent in mammalian dCD and CDA, is important for the structural integrity of the enzyme and for the binding of the phosphate group of the substrate or inhibitor. Although the R115E mutant of dCD is a dimer in solution, it crystallizes as a hexamer, mimicking the natural state of the wild-type enzyme. Residues 112 and 115, which are known to be important for the binding of the allosteric regulators, are found in a pocket that is at the intersubunit interfaces in the hexamer but distant from the substrate-binding site. The substrate-binding site is composed of residues from a single protein molecule and is sequestered in a deep groove. This groove is located at the outer surface of the hexamer but ends at the subunit interface that also includes residue 115. It is proposed that the absence of subunit interactions at this interface in the dimeric R115E mutant renders the substrate-binding site accessible. In contrast, for the wild-type enzyme, binding of dCTP induces an allosteric effect that affects the subunit interactions and results in an increase in the accessibility of the binding site.

Label-free amplified bioaffinity detection using terahertz wave technology.

A new affinity biosensor based on pulsed terahertz (THz) wave technology has been used to monitor binding between biotin and avidin molecules. Amplified detection of avidin-biotin binding is obtained on supported membranes composed of biotin layers on quartz surface, which is modified with octadecanol. Agarose particles are conjugated with avidin and then applied to biotin, which is already bound to the octadecanol quartz surface, the biotin binds to the conjugate rapidly and causes an enhancement of the THz difference signal between biotin and biotin-avidin complexes by a factor greater than eight fold when compared to the same sample without agarose beads. The technique was able to detect less than 10.3 ng/cm2 avidin, thus, giving the THz system a detection capability of sub-thin solid films better than ellipsometry and reflectometry techniques. Further improvement is underway using highly refractive beads together with appropriate surface chemistry. This newly developed method is being saliently optimized for future application, including the detection of DNA hybridization and ligand-analyte affinity binding.

Differential effects of alpha subunit Asparagine56 oligosaccharide structure on equine lutropin and follitropin hybrid conformation and receptor-binding activity.

The gonadotropins, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and chorionic gonadotropin (CG), are cysteine-knot growth-factor superfamily glycoproteins composed of a common alpha subunit noncovalently associated with a hormone-specific beta subunit. The cysteine-knot motifs in both subunits create two hairpin loops, designated L1 and L3, on one side of the knot, with the intervening long loop, L2, on the opposite side. As the average alpha-subunit loop 2 oligosaccharide mass increased from 1482 to 2327, LH and FSH receptor-binding affinities of the dual-specificity eLH declined significantly, while the decrease in FSH receptor-binding affinity for eFSH was not significant. In the present study, we characterized hormone-specific glycosylation of alphaL2 oligosaccharides in eLHalpha, eFSHalpha, and eCGalpha preparations. MALDI mass spectrometry revealed 28-57 structures, including high mannose, hybrid, bi-, and triantennary oligosaccharides. The same intact subunit preparations and their alphaL2 loop-deglycosylated derivatives were combined with either eLHbeta or eFSHbeta, and the circular dichroism (CD) spectrum for each preparation was determined. We predicted that hybrid hormone preparations obtained by combining intact eLHalpha, eFSHalpha, and eCGalpha preparations with eLHbeta might exhibit differences in conformation that would disappear when the alphaL2 oligosaccharide attached to alphaAsn(56) was removed by selective peptide-N-glycanase digestion (N(56)dg-alpha). CD data supported the first prediction; however, elimination of alphaL2 oligosaccharide actually increased the conformational differences. The intact alpha subunit:eFSHbeta hybrids had virtually identical CD spectra, as expected. However, the N(56)dg-alpha:eFSHbeta hybrid spectra differed from each other. Oligosaccharide removal altered the conformation of most hybrids, suggesting that alphaAsn(82) oligosaccharide (located in alphaL3) also influenced gonadotropin conformation.

Allophycocyanin and energy transfer.

Allophycocyanin is a biliprotein located in the core of the phycobilisome. The biliprotein is isolated and purified as a trimer (alpha3beta3), where a monomer is an alphabeta structure. Each alpha and beta subunit has a single noncyclic tetrapyrrole chromophore, called phycocyanobilin. The trimer of allophycocyanin has an unusual absorption maximum at 650 nm with a shoulder at 620 nm, while the monomer has an absorption maximum at 615 nm. Two explanations have been proposed for the 650-nm maximum. In one, this maximum is produced by the interaction of a particular local protein environment for three of the chromophores, causing them to red shift, while the other three chromophores are at a higher energy. Energy is transferred from the high- to the low-energy chromophores by Förster resonance energy transfer, the donor-acceptor model. In the second proposal, there is strong exciton coupling between two chromophores of the trimer that closely approach across the monomer-monomer interface. The strong interaction causes exciton splitting and a red shift in the absorption. There are three of these strongly coupled chromophore pairs, and energy is transferred between the two-exciton states of a pair by internal conversion. A variety of biophysical methods have been used to examine this question. Although evidence supporting both models has been produced, sophisticated ultra fast fluorescence results from a plethora of approaches now firmly point to the latter strong coupling hypothesis as being more likely. Between the different strongly coupled pairs, Förster resonance energy transfer should occur. For monomers of allophycocyanin, Förster resonance energy transfer occurs between the two chromophores.

Alpha-fetoprotein growth inhibitory peptides: potential leads for cancer therapeutics.

alpha-Fetoprotein (AFP), known largely as a growth-promoting agent, also possesses a growth inhibitory motif recently identified as an occult epitopic segment of the molecule. This segment, a 34-amino acid stretch termed the growth inhibitory peptide (GIP), has been chemically synthesized, purified, and characterized. The purified 34-mer exhibits complex aggregation behaviors; initially, trimeric oligomers were formed that possess growth inhibitory activity in rodent uterine bioassays. These rodent growth assays have served as a prelude to the anticancer studies that followed. In solution, the trimers convert slowly to dimers containing intrapeptide disulfide bonds; such dimers are inactive in the antigrowth assays. Cysteine-to-alanine analogues of the GIP retain the antigrowth properties, while similar cysteine-to-glycine and cysteine-to-serine analogues demonstrate little, if any, growth regulatory activity. Chemical modifications of the cysteine residues also have little influence on the antigrowth activity of the GIP. Fragments of the 34-mer possess variable growth activities of their own, with an octamer from near the carboxyl terminus displaying estrogen-dependent antigrowth activity similar to that of the 34-mer. It was further observed that the GIP can bind both Zn(2+) and Co(2+); the Co(2+) peptide complex was shown to have a distorted tetrahedral symmetry, involving coordination of two cysteine and two histidine residues. The Zn(2+)-GIP complex had antigrowth activity and did not form the intrapeptide disulfide bond characteristic of the free GIP in aqueous solution. The GIP was tested in vitro for anticancer activity and was found to suppress the growth in 38 of 60 human cancer cell lines, representing nine different cancer types. In vivo studies of the GIP, certain analogues, and its fragments revealed anticancer activities in both isograft and xenograft animal tumor transplants. Furthermore, the 2C --> 2A replacement analogue was active against a breast tumor in vivo and in vitro and a prostate cancer in vitro. Thus, it is proposed that the GIP, its analogues, and its fragment peptides can potentially serve as lead compounds for cancer therapeutics.

Allophycocyanin: trimers, monomers, subunits, and homodimers.

Allophycocyanin is a photosynthetic light-harvesting pigment-protein complex located in the phycobilisomes of cyanobacteria and red algae. Using dynamic light scattering and circular dichroism, solutions of purified allophycocyanin were shown to consist of homogeneous trimers (alpha3beta3) with a nonspherical shape over a very wide range of protein concentrations at pH 6.0 and 20 degrees C. Deconvolutions of the visible circular dichroism spectrum of the trimer were carried out for the first determination of the individual spectra of all six-component chromophores. The chromophores were shown to be in different microenvironments that helped determine the spectrum of the trimer. Monomers (alpha beta) that were formed in either the presence of 0.50M NaSCN or at 45 degrees C were shown to be completely reversible to trimers. However, subunits (alpha and beta) that were formed in either the presence of 8M urea or at 60 degrees C, using spectroscopy and gel-filtration column chromatography, were observed to only partially reconstitute trimers. Homodimers (alpha2 and/or beta2) formed during the regeneration of trimers. The homodimer, which was detected for the first time when both subunits were present, was shown to be in equilibrium with its subunits. Unlike the trimer situation, subunits were found to fully reconstitute monomers in the presence of 0.50M NaSCN. These results suggest a route to trimer assembly from subunits with monomers serving as intermediaries and the homodimers forming in a nonproductive step that did not interfere with the overall assembly scheme.

Label-free bioaffinity detection using terahertz technology.

We report the first use of differential terahertz time-domain spectroscopy for bioaffinity sensing. Binding is observed by measuring the transmission of a thin layer of biotin bound to the sensor protein avidin. We demonstrate the THz wave transmission of a sub-micron-thick film and sensitivity to 0.1 microg cm(-2) of biotin. These results point the way for a host of biosensor applications using T-rays, or pulsed far-infrared (FIR) radiation.

Anti-prostate cancer and anti-breast cancer activities of two peptides derived from alpha-fetoprotein.

A chemically synthesized 34-amino-acid peptide and a select analog have been studied to determine their activities against the growth of prostate and breast cancer tumors. It was of interest to determine if the peptide has anti-prostate cancer activity. Previously, the peptide was shown to inhibit the growth of breast cancer tumor cells. The peptide inhibited the growth of both breast and prostate tumors. A novel experimental design for the peptide was in a study in which a time-release pellet was used to give daily peptide doses to mice that were subjected to a breast cancer tumor. The peptide was effective in inhibiting the growth of tumors in the mice. The 2 C-->2 A analog peptide, in which the two cysteines were replaced by alanines, was also active in inhibition of the growth of prostate and breast cancer cell lines and in an in vivo assay against breast cancer. A scrambled amino acid sequence of the peptide was used as a control in these tumor studies, and it had virtually no anti-cancer activity.