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Protein synthesis studies increasingly focus on delineating the nature of conformational changes occurring as the ribosome exerts its catalytic functions. Here, we use FRET to examine such changes during single-turnover EF-G-dependent GTPase on vacant ribosomes and to elucidate the mechanism by which fusidic acid (FA) inhibits multiple-turnover EF-G.GTPase. Our measurements focus on the distance between the G' region of EF-G and the N-terminal region of L11 (L11-NTD), located within the GTPase activation center of the ribosome. We demonstrate that single-turnover ribosome-dependent EF-G GTPase proceeds according to a kinetic scheme in which rapid G' to L11-NTD movement requires prior GTP hydrolysis and, via branching pathways, either precedes P(i) release (major pathway) or occurs simultaneously with it (minor pathway). Such movement retards P(i) release, with the result that P(i) release is essentially rate-determining in single-turnover GTPase. This is the most significant difference between the EF-G.GTPase activities of vacant and translocating ribosomes [Savelsbergh, A., Katunin, V. I., Mohr, D., Peske, F., Rodnina, M. V., and Wintermeyer, W. (2003) Mol. Cell 11, 1517-1523], which are otherwise quite similar. Both the G' to L11-NTD movement and P(i) release are strongly inhibited by thiostrepton but not by FA. Contrary to the standard view that FA permits only a single round of GTP hydrolysis [Bodley, J. W., Zieve, F. J., and Lin, L. (1970) J. Biol. Chem. 245, 5662-5667], we find that FA functions rather as a slow inhibitor of EF-G.GTPase, permitting a number of GTPase turnovers prior to complete inhibition while inducing a closer approach of EF-G to the GAC than is seen during normal turnover.

Methodology was developed for specifically anchoring Escherichia coli 70S ribosomes onto a chemically modified, cysteine-reactive glass surface. Immobilized ribosomes maintain the capability of binding a polyuridylic acid (poly(U)) template, enabling investigation of mechanical properties of individual ribosome-poly(U) complexes using laser tweezers. Streptavidin-coated polystyrene microspheres bound specifically to the biotinylated 3' end of long (up to 10,000 bases) poly(U) strands. A novel optical method was built to control the position of the laser trap along the microscope optical axis at 2 nm resolution, facilitating measurement of the force-extension relationship for poly(U). Some immobilized ribosome-poly(U) complexes supported 100 pN of force applied at the 3' end of the mRNA. Binding of N-acetylated Phe-tRNA(Phe), an analog of the initiator fMet-tRNA(Met), enhanced the population of complexes that could withstand high forces. The persistence length of poly(U) RNA homopolymer, modeled as a worm-like chain, was found to be 0.79 +/- 0.05 nm and the backbone elasticity was 900 +/- 140 pN, similar to values for single-stranded DNA.

Here we examine the enantioselectivity of the allosteric and substrate binding sites of murine ribonucleotide reductase (mRR). l-ADP binds to the active site and l-ATP binds to both the s- and a-allosteric sites of mR1 with affinities that are only three- to 10-fold weaker than the values for the corresponding d-enantiomers. These results demonstrate the potential of l-nucleotides for interacting with and modulating the activity of mRR, a cancer chemotherapeutic and antiviral target. On the other hand, we detect no substrate activity for l-ADP and no inhibitory activity for N3-l-dUDP, demonstrating the greater stereochemical stringency at the active site with respect to catalytic activity.

Alpha(1)-antitrypsin (AT) is the most abundantly circulating human proteinase inhibitor in the serpin family. The polymerization of AT, leading to alpha(1)-antitrypsin deficiency, has been studied extensively in vitro by a variety of ensemble methods. Here we report the use of fluorescence correlation spectroscopy to gain further insight into this process. Measurements of the distributions of diffusion times of polymerizing AT, carried out at 45, 50, and 55 degrees C, clearly show the existence of a kinetic lag phase, during which short oligomers are formed, prior to the formation of heterogeneous mixtures of longer polymers, and suggest that long polymers, which appear to be metastable, are produced through the condensation of shorter oligomers.

Mammalian ribonucleotide reductase (mRR) is a chemotherapeutic target. The enzyme is composed of 2 subunits (mR1 and mR2) and is inhibited by Ac-FTLDADF (denoted P7), corresponding to the C-terminus of mR2, which competes with mR2 for binding to mR1. mRR has 2 physiologically important active forms, mR12mR22 and mR16(mR22)j (j = 1–3). Here we report on the mechanism of action of recently identified peptide derivatives having higher activities than P7 toward inhibition of one or both active forms. A significant feature of both P7 and these new inhibitors is that they are more potent vs. mR12mR22 than mR16(mR22)j. For some of these peptides, this is due in part to their preferential binding to the mR1 monomer. The possible application of these peptide derivatives in cancer chemotherapy is discussed. © 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2005

Mammalian ribonucleotide reductase (mRR) is a chemotherapeutic target. The enzyme is composed of two subunits (mR1 and mR2) and is inhibited by Ac-FTLDADF (denoted P7), corresponding to the C-terminus of mR2, which disrupts mRR quaternary structure by competing with mR2 for binding to mR1. The tripeptide FmocWFF acts similarly. Here we report on the use of small, focused libraries to identify Fmoc derivatives of tetra and hexapeptides having comparable or considerably higher activities than P7 toward inhibition of mRR.

Ribosome recycling factor (RRF) and elongation factor-G (EF-G) are jointly essential for recycling bacterial ribosomes following termination of protein synthesis. Here we present equilibrium and rapid kinetic measurements permitting formulation of a minimal kinetic scheme that accounts quantitatively for RRF and EF-G interaction on the Escherichia coli ribosome. RRF and EF-G (a) each form a binary complex on binding to a bare ribosome which undergoes isomerization to a more stable complex, (b) form mixed ternary complexes on the ribosome in which the affinity for each factor is considerably lower than its affinity for binding to a bare ribosome, and (c) each bind to two sites per ribosome, with EF-G having considerably higher second-site affinity than RRF. Addition of EF-G to the ribosome-RRF complex induces rapid RRF dissociation, at a rate compatible with the rate of ribosome recycling in vivo, but added RRF does not increase the lability of ribosome-bound EF-G. Added thiostrepton slows the initial binding of EF-G, and prevents both formation of the more stable EF-G complex and EF-G-induced RRF dissociation. These findings are relevant for the mechanism of post-termination complex disassembly.

Class I ribonucleotide reductases (RRs), which are well-recognized targets for cancer chemotherapeutic and antiviral agents, are composed of two different subunits, R1 and R2, and are inhibited by oligopeptides corresponding to the C-terminus of R2, which compete with R2 for binding to R1. These peptides specifically inhibit the RRs from which they are derived, and closely homologous RRs, but do not inhibit less homologous RRs. Here we review results obtained for oligopeptide inhibition of RRs from several sources, including related x-ray, NMR, and modeling results. The most extensive studies have been performed on herpes simplex virus-RR (HSV-RR) and mammalian-RR (mRR). A common model fits the data obtained for both enzymes, in which the C-terminal residue of the oligopeptide (Leu for HSV-RR, Phe for mRR) binds with high specificity to a narrow and deep hydrophobic subsite, and two or more hydrophobic groups at the N-terminal portion of the peptide bind to a broad and shallow second hydrophobic subsite. The studies have led to the development of highly potent and specific inhibitors of HSV-RR and promising inhibitors of mRR, and indicate possible directions for the development of inhibitors of bacterial and fungal RRs.

Intracellular RNA molecules adopt a wide variety of 3D structures, many of which have been studied using high-resolution structural techniques such as X-ray crystallography and NMR. However, while canonical structural features for a class of RNA molecules are assumed based on such studies, structural variations within a class are extremely difficult to detect, as only a limited set of structures are available. For example, the number of unique transfer-RNA (tRNA) molecules in eukaryotes can be ∼100, not including variant products of post-transcriptional modification. It has been hypothesized that tRNA accommodation in the ribosome during translation is modulated by tRNA stiffness, which in turn affects the translation kinetics. Despite this, no experimental studies that probe tRNA stiffness are available. In this work, we electrophoretically drive individual tRNA molecules through ∼3nm diameter pores in order to study their deformation kinetics during translocation. Surprisingly, we find that all six tRNAs studied here exhibit unique translocation kinetics, which we believe correspond to variations in their mechanical properties. Implementation of machine learning algorithms reveal a multidimensional set of parameters that can be used to ascertain the identity of a tRNA from a single ionic current pulse with 70-90% accuracy. Our results pave the way for a deeper understanding of RNA tertiary structure and its dependence on post-transcriptional modifications.

Soluble inorganic pyrophosphatase (PPase), which converts inorganic pyrophosphate (PP(i)) into usable phosphate, is almost universally present as a central enzyme of phosphorus metabolism and uses divalent metal ion as a necessary cofactor. PPase from Saccharomyces cerevisiae (Y-PPase) is the best studied with respect to both structure and mechanism. Here we report the first combined use of stopped flow and quenched flow techniques to study the PPase reaction in both the forward (PP(i) hydrolysis) and back (PP(i) synthesis) directions. The results of these studies permit direct comparison of different divalent metal-ion effects (Mg(2+), Mn(2+), Co(2+)) on microscopic rate constants at pH 7.0. For the Mn-enzyme, on which all of the high-resolution X-ray studies have been conducted, they demonstrate that the rate-determining step changes as a function of pH, from hydrolysis of enzyme-bound PP(i) at low pH to release of the more tightly bound P(i) at high pH. They also provide evidence for two kinetically important forms of the product complex EM(4)(P(i))(2), supporting an earlier suggestion based on crystallographic evidence, and allow informed speculation as to the identities of acidic and basic groups essential for optimal PPase catalytic activity.

Photolabile oligonucleotides (PHONTs) bind to rRNA sequences to which they are complementary and, on photolysis, incorporate into neighboring ribosomal components. Here we report on photocrosslinking results obtained with PHONTs targeting 23S rRNA nucleotides 1882-1892, in the long lateral arm of the 50S subunit (PHONT 1892), and 1085-1093, in the L11 binding domain (PHONT 1093). Photolysis of the PHONT 1892.50S and PHONT 1093.50S complexes leads to formation of 'long-range' crosslinks from C1892 to U1094/A1095 and G1950, and from G1093 to U1712/1716 and U1926, that are clearly incompatible with published crystal structures of 50S subunits. These results provide strong evidence that within the 50S subunit (a) the L11 binding domain can extend in an arm-like fashion, accessing large areas of the ribosome, and (b) the lateral arm can bend about the noncanonical helix at its center. Such motions may have functional relevance in identifying regions that undergo major conformational change as the ribosome moves through its catalytic cycle.

Prostate-specific antigen (PSA) and its SDS-stable complex with the serine proteinase inhibitor (serpin) alpha(1)-antichymotrypsin (ACT), which is the dominant form of PSA in serum, are in widespread use as markers for the diagnosis of prostate cancer, and there is increasing evidence for the involvement of PSA proteinase activity itself in the development of prostate and other cancers. However, both the formation and degradation of the PSA-ACT complex, denoted PSA*ACT* to indicate substantial changes in the structure of both proteins on complex formation, have been incompletely studied. Here we determine rate and equilibrium constants for the steps involved in PSA*ACT* formation and demonstrate that (a) the effects of added NaCl, polyamines, and Zn(2+) on this process parallel their effects on PSA catalytic activity [Hsieh, M.-C., and Cooperman, B. S. (2000) Biochim. Biophys. Acta 1481, 75-87], (b) the effect of added NaCl in dramatically increasing the rate of ACT inhibition of PSA correlates with salt-induced changes in PSA conformation, and (c) the PSA*ACT* complex is subject to proteolysis by human neutrophil elastase. Possible clinical implications of these findings are considered.

The chapter describes an approach to form defined photo-cross-links from targeted RNA sites within the ribosome, with an emphasis on those that are important functionally. In this approach, radioactive, photolabile derivatives of oligonucleotides (PHONTs) having sequences complementary to rRNA sequences are bound to their targeted sequences in intact ribosomal subunits and, on photolysis, form cross-links with neighboring ribosomal components. The PHONT approach offers several advantages. First, it allows targeting of sequences of particular functional or structural significance throughout the ribosome structure. Second, the cross-links formed provide a defined upper-limit distance for the separation of the linked components within the ribosome, given by the length of the tether. The chapter first describes the PHONT approach in general before presenting the results of recent applications of the approach to the study of the peptidyltransferase center (PTC). It identifies four principal elements in PHONT design: first, the backbone structure; second, the placement of the photolabile group within the oligonucleotide sequence; third, the length and flexibility of the tether linking the photolabile group with the oligonucleotide backbone; and fourth, the introduction of radioactivity. As is evident in the descriptions, PHONT design continues to evolve. Currently the YAMMP approach is applied to develop a three-dimensional model of the PTC based on cross-linking results, which provide the clearest set of constraints for model construction.

There is a critical need for techniques that directly monitor protein synthesis within cells isolated from normal and diseased tissue. Fibrotic disease, for which there is no drug treatment, is characterized by the overexpression of collagens. Here, we use a bioinformatics approach to identify a pair of glycine and proline isoacceptor tRNAs as being specific for the decoding of collagen mRNAs, leading to development of a FRET-based approach, dicodon monitoring of protein synthesis (DiCoMPS), that directly monitors the synthesis of collagen. DiCoMPS aimed at detecting collagen synthesis will be helpful in identifying novel anti-fibrotic compounds in cells derived from patients with fibrosis of any etiology, and, suitably adapted, should be widely applicable in monitoring the synthesis of other proteins in cells.

Reduction of NDPs by murine ribonucleotide reductase (mRR) requires catalytic (mR1) and free radical-containing (mR2) subunits and is regulated by nucleoside triphosphate allosteric effectors. Here we present a new, comprehensive, and quantitative model for allosteric control of mRR enzymatic activity based on molecular mass, ligand binding, and enzyme activity studies. In this model, nucleotide binding to the specificity site (s-site) drives formation of an active R1(2)R2(2) dimer, ATP or dATP binding to the adenine-specific site (a-site) results in formation of an inactive tetramer, and ATP binding to the newly described hexamerization site (h-site) drives formation of active R1(6)R2(6) hexamer. In contrast, an earlier phenomenological model [Thelander, L., and Reichard, P. (1979) Annu. Rev. Biochem. 67, 71-98] (the "RT" model) ignores aggregation state changes and mistakenly rationalizes ATP activation versus dATP inhibition as reflecting different functional consequences of ATP versus dATP binding to the a-site. Our results suggest that the R1(6)R2(6) heterohexamer is the major active form of the enzyme in mammalian cells, and that the ATP concentration is the primary modulator of enzyme activity, coupling the rate of DNA biosynthesis with the energetic state of the cell. Using the crystal structure of the Escherichia coliR1 hexamer as a model for the mR1 hexamer, a scheme is presented that rationalizes the slow isomerization of the tetramer form and suggests an explanation for the low enzymatic activity of tetramers complexed with R2. The similar specific activities of R1(2)R2(2) and R1(6)R2(6) are inconsistent with a proposed model for R2(2) docking with R1(2) [Uhlin, U., and Eklund, H. (1994) Nature 370, 533-539], and an alternative is suggested.

The wealth of kinetic and structural information makes inorganic pyrophosphatases (PPases) a good model system to study the details of enzymatic phosphoryl transfer. The enzyme accelerates metal-complexed phosphoryl transfer 10 10 -fold: but how? Our structures of the yeast PPase product complex at 1.15 Å and fluoride-inhibited complex at 1.9 Å visualize the active site in three different states: substrate-bound, immediate product bound, and relaxed product bound. These span the steps around chemical catalysis and provide strong evidence that a water molecule (O nu ) directly attacks PPi with a p K a vastly lowered by coordination to two metal ions and D117. They also suggest that a low-barrier hydrogen bond (LBHB) forms between D117 and O nu , in part because of steric crowding by W100 and N116. Direct visualization of the double bonds on the phosphates appears possible. The flexible side chains at the top of the active site absorb the motion involved in the reaction, which may help accelerate catalysis. Relaxation of the product allows a new nucleophile to be generated and creates symmetry in the elementary catalytic steps on the enzyme. We are thus moving closer to understanding phosphoryl transfer in PPases at the quantum mechanical level. Ultra-high resolution structures can thus tease out overlapping complexes and so are as relevant to discussion of enzyme mechanism as structures produced by time-resolved crystallography.

The pattern of yeast pyrophosphatase (Y-PPase) inhibition by fluoride suggests that it replaces active site Mg(2+)-bound nucleophilic water, for which two different locations were proposed previously. To localize the bound fluoride, we investigate here the effects of mutating Tyr(93) and five dicarboxylic amino acid residues forming two metal binding sites in Y-PPase on its inhibition by fluoride and its five catalytic functions (steady-state PP(i) hydrolysis and synthesis, formation of enzyme-bound PP(i) at equilibrium, phosphate-water oxygen exchange, and Mg(2+) binding). D117E substitution had the largest effect on fluoride binding and made the P-O bond cleavage step rate-limiting in the catalytic cycle, consistent with the mechanism in which the nucleophile is coordinated by two metal ions and Asp(117). The effects of the mutations on PP(i) hydrolysis (as characterized by the catalytic constant and the net rate constant for P-O bond cleavage) were in general larger than on PP(i) synthesis (as characterized by the net rate constant for PP(i) release from active site). The effects of fluoride on the Y-PPase variants confirmed that PPase catalysis involves two enzyme.PP(i) intermediates, which bind fluoride with greatly different rates (Baykov, A. A., Fabrichniy, I. P., Pohjanjoki, P., Zyryanov, A. B., and Lahti, R. (2000) Biochemistry 39, 11939-11947). A mechanism for the structural changes underlying the interconversion of the enzyme.PP(i) intermediates is proposed.

Ribosomal proteins L2, L3 and L4, together with the 23S RNA, are the main candidates for catalyzing peptide bond formation on the 50S subunit. That L2 is evolutionarily highly conserved led us to perform a thorough functional analysis with reconstituted 50S particles either lacking L2 or harboring a mutated L2. L2 does not play a dominant role in the assembly of the 50S subunit or in the fixation of the 3'-ends of the tRNAs at the peptidyl-transferase center. However, it is absolutely required for the association of 30S and 50S subunits and is strongly involved in tRNA binding to both A and P sites, possibly at the elbow region of the tRNAs. Furthermore, while the conserved histidyl residue 229 is extremely important for peptidyl-transferase activity, it is apparently not involved in other measured functions. None of the other mutagenized amino acids (H14, D83, S177, D228, H231) showed this strong and exclusive participation in peptide bond formation. These results are used to examine critically the proposed direct involvement of His229 in catalysis of peptide synthesis.

Mammalian ribonucleotide reductase (mRR), a potential target for cancer intervention, is composed of two subunits, mR1 and mR2, whose association is critical for enzyme activity. In this article we describe the structural features of the mRR-inhibitor Ac-F-c[ELAK]-DF (Peptide 3) while bound to the mR1 subunit as determined by transferred NOEs. Peptide 3 is a cyclic analogue of the N-acetylated form of the heptapeptide C-terminus of the mR2 subunit (Ac-FTLDADF), which is the link between the two subunits and previously shown to be the minimal sequence inhibitor mRR by competing with mR2 for binding to mR1. Structural refinement employing an ensemble-based, full-relaxation matrix approach resulted in two structures varying in the conformations of F(1) and the cyclic lactam side chains of E(2) and K(5). The remainder of the molecule, both backbone and side chains, is extremely well-defined, with an RMSD of 0.54 A. The structural features of this conformationally constrained analogue provide unique insight into the requirements for binding to mR1, critical for further inhibitor development.

The 23S rRNA nucleotides 2604-12 and 2448-58 fall within the central loop of domain V, which forms a major part of the peptidyl transferase center of the ribosome. We report the synthesis of radioactive, photolabile 2'-O-methyloligoRNAs, PHONTs 1 and 2, complementary to these nucleotides and their exploitation in identifying 50S ribosomal subunit components neighboring their target sites. Photolysis of the 50S complex with PHONT 1, complementary to nts 2604-12, leads to target site-specific photoincorporation into protein L2 and 23S rRNA nucleotides A886, Alpha1918, A1919, G1922-C1924, U2563, U2586, and C2601. Photolysis of the 50S complex with PHONT 2, complementary to nts 2448-58, leads to target site-specific probe photoincorporation into proteins L2, L3, one or more of proteins L17, L18, L21, and of proteins L9, L15, L16, and 23S rRNA nucleotides C2456 and psi2457. Chemical footprinting studies show that 2'-O-methyloligoRNA binding causes little distortion of the peptidyl transferase center but do provide suggestive evidence for the location of flexible regions within 23S rRNA. The significance of these results for the structure of the peptidyl transferase center is considered.

We report the synthesis of a radioactive, photolabile 2'-O-methyloligoRNA probe, 2258-53/52(SAz)-48, PHONT1, and its exploitation in identifying 23S rRNA nucleotides neighboring the so-called 'P-loop'. The probe is complementary to nt 2248-2258 in Escherichia coli 50S subunits. PHONT1 contains a p-azidophenacyl group attached to a phosphorothioate bridge between the nucleotides complementary to the positions 2252-2253, such that the photogenerated nitrene is maximally 17-19 A from 23S RNA nucleotides G2252 and G2253. PHONT1 binds to the 50S subunit, and photoincorporates within or immediately adjacent to its target site, as well as into several nucleotides falling between G2357 and A2430. The significance of these results for the structure of the peptidyl transferase center is considered. The PHONT approach is generally applicable to studies of complex RNA-containing molecules.

[formula: see text] A series of lactam-bridged peptide inhibitors (2-6) of mammalian ribonucleotide reductase (mRR) has been designed and synthesized on the basis of the heptapeptide N-AcFTLDADF (1), corresponding to the C-terminus of the R2 subunit of mRR. Inhibition studies revealed a direct relation between ring size and activity, with peptide 5 being 2.5 times more potent than peptide 1.

Based on the primary structure, soluble inorganic pyrophosphatases can be divided into two families which exhibit no sequence similarity to each other. Family I, comprising most of the known pyrophosphatase sequences, can be further divided into prokaryotic, plant and animal/fungal pyrophosphatases. Interestingly, plant pyrophosphatases bear a closer similarity to prokaryotic than to animal/fungal pyrophosphatases. Only 17 residues are conserved in all 37 pyrophosphatases of family I and remarkably, 15 of these residues are located at the active site. Subunit interface residues are conserved in animal/fungal but not in prokaryotic pyrophosphatases.

Serpins form enzymatically inactive covalent complexes (designated E*I*) with their target proteinases, corresponding most likely to the acyl enzyme that resembles the normal intermediate in substrate turnover. Formation of E*I* involves large changes in the conformation of the reactive center loop (residues P17 to P9′) and of the serpin molecule in general. The “hinge” region of the reactive center loop, including residues P10–P14, shows facile movement in and out of β-sheet A, and this movement appears to be crucial in determining whether E*I* is formed (the inhibitor pathway) or whether I is rapidly hydrolyzed to I* (the substrate pathway). Here, we report stopped-flow and rapid quench studies investigating the pH dependence of the conversion of the α1-antichymotrypsin·α-chymotrypsin encounter complex, E·I, to E*I*. These studies utilize fluorescent derivatives of cysteine variants of α1-antichymotrypsin at the P11 and P13 residues. Our results demonstrate three identifiable intermediates, EIa, EIb, and EIc, between E·I and E*I* and permit informed speculation regarding the nature of these intermediates. Partitioning between inhibitor and substrate pathways occurs late in the process of E*I* formation, most likely from a species occurring between EIc and E*I*.

Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg2+-binding affinity for two of the three sites present in E-PPase (3.5- to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20- to 40-fold). By contrast, Mg2+-binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer. These data indicate that E-PPase requires in total only three Mg2+ ions per active site for best performance, rather than four, as previously believed. As measured by equilibrium dialysis, Mg2+ binds to 2.5 sites per monomer, supporting the notion that one of the tightly binding sites is located at the trimer–trimer interface. Mg2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg2+ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site.

Nucleotides 518-533 form a loop in ribosomal 30 S subunits that is almost universally conserved. Both biochemical and genetic evidence clearly implicate the 530 loop in ribosomal function, with respect both to the accuracy control mechanism and to tRNA binding. Here, building on earlier work, we identify proteins and nucleotides (or limited sequences) site-specifically photolabeled by radioactive photolabile oligoDNA probes targeted toward the 530 loop of 30 S subunits. The probes we employ are complementary to 16 S rRNA nucleotides 517-527, and have aryl azides attached to nucleotides complementary to nucleotides 518, 522, and 525-527, positioning the photogenerated nitrene a maximum of 19-26 A from the complemented rRNA base. The crosslinks obtained are used as constraints to revise an earlier model of 30 S structure, using the YAMMP molecular modeling package, and to place the 530 loop region within that structure.

We demonstrate the functional activity of single ribosomal complexes, opening the way for detailed studies of the trajectories of protein synthesis. Our approach employs a single-molecule detection system, capable of picoseconds to minutes resolution, to observe a growing peptide labeled at its N terminus with the fluorophore tetramethylrhodamine (TMR). Single complexes of mRNA-programmed ribosomes with TMR-Met-tRNA f Met or TMR-Met-Phe-tRNA Phe are immobilized on mica and observed by fluorescence. Immobilized ribosome·mRNA·TMR-Met-tRNA f Met complexes form peptide bonds with puromycin. Single-molecule detection reveals dynamics on the scale of seconds at the ribosomal peptidyl transferase center.

Serpins, serine proteinase inhibitors, form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases, that only slowly release I*, in which the P1-P1′ linkage is cleaved. Recently we presented evidence that the serpin antichymotrypsin (ACT, I) reacts with the serine proteinase chymotrypsin (Chtr, E) to form an E*I* complex via a three-step mechanism, E + I ⇌ E ·I ⇌ EI′ ⇌ E*I* in which EI′, which retains the P1-P1′ linkage, is formed in a partly or largely rate-determining step, depending on temperature (O’Malley, K. H, Nair, S. A., Rubin, H., and Cooperman, B. S. (1997) J. Biol. Chem. 272, 5354–5359). Here we extend these studies through the introduction of a new assay for the formation of the postcomplex fragment, corresponding to ACT residues 359 (the P1′ residue) to 398 (the C terminus), coupled with rapid quench flow kinetic analysis. We show that the E·I encounter complex of wild type-rACT and Chtr forms both E*I* and postcomplex fragment with the same rate constant, so that both species arise fromEI′ conversion to E*I*. These results support our earlier conclusion that the P1-P1′ linkage is preserved inEI′ and imply that E*I* corresponds to a covalent adduct of E and I, either acyl enzyme or the tetrahedral intermediate formed by water attack on acyl enzyme. Furthermore, we show that the A347R (P12) variant of rACT, which is a substrate rather than an inhibitor of Chtr, has a rate constant for postcomplex fragment formation from the E·I complex very similar to that observed for WT-rACT, implying that EI′ is the common intermediate from which partitioning to inhibitor and substrate pathways occurs. These results are used to elaborate a proposed scheme for ACT interaction with Chtr that is considered in the light of relevant results from studies of other serpin-serine proteinase pairs.

Soluble inorganic pyrophosphatase (PPase) is one of the better understood phosphoryl-transfer enzymes and is distinctive in having four divalent metal ions at the active site. Here we determine pH profiles for wild-type Saccharomyces cerevisiae PPase (Y-PPase) and for 14 of its active site variants and consider the effects of active site mutation on the pH-independent parameters and acid dissociation constants that characterize these profiles against the framework of the proposed structure of the activated complex. The results obtained (a) support the current mechanistic model in which a hydroxide ion, stabilized by binding to two metal ions at the active site and by an extended system of hydrogen bonds within the active site, is the nucleophile that attacks enzyme-bound inorganic pyrophosphate and (b) provide evidence that the acid group that is necessary for maximal activity is a water molecule coordinated to a third metal ion, as shown by the general rise in the pKa of this group that is a consequence of almost all of the mutations. We further compare the present results to those previously observed for the corresponding mutations in Escherichia coli PPase [E-PPase; Salminen et al. (1995) Biochemistry 34, 782-791]. Such comparison provides a measure of the extent to which different portions of the active site are conserved. We find that some corresponding mutations have different effects on catalytic function, demonstrating that even in the context of very similar active sites, interactions of the mutated site with less well conserved portions of the enzyme, in this case outside the active site, can lead to different outcomes. On the other hand, one region of the active site is highly conserved, suggesting that it may represent a common feature of phosphoryl-transfer enzymes or a vestige of a primitive ur-PPase active site.

We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50 S ribosomal subunit components neighboring its target site, nucleotides 803-811 in 23 S rRNA. Photolysis of the complex formed between the probe and 50 S subunits leads to site-specific probe photoincorporation into proteins L15, L17, and L20, labeled to greater extents, and L13 and L21, labeled to lesser extents. Portions of each of these proteins thus lie within 23 A of nucleotide U803. These results lead us to conclude that nucleotides 803-811 fall on the side of the L13-L17-L20-L21 protein cluster [Walleczek et al. (1988) EMBO J. 7, 3571-3576] that points from the back of the 50 S particle toward the peptidyl transferase center within the 50 S subunit. Such placement is consistent with the observation that an oligoDNA probe directed to nucleotides 803-811 decreases P-site binding of tRNA [Hill et al. (1990) Biochim. Biophys. Acta 1050, 45-50].

Escherichia coli inorganic pyrophosphatase is a tight hexamer of identical subunits. Replacement of both His136 and His140 by Gln in the subunit interface results in an enzyme which is trimeric up to 26 mg/mL enzyme concentration in the presence of Mg2+, allowing direct measurements of Mg2+ binding to trimer by equilibrium dialysis. The results of such measurements, together with the results of activity measurements as a function of [Mg2+] and pH, indicate that Mg2+ binds more weakly to one of the three sites per monomer than it does to the equivalent site in the hexamer, suggesting this site to be located in the trimer:trimer interface. The otherwise unobtainable hexameric variant enzyme readily forms in the presence of magnesium phosphate, the product of the pyrophosphatase reaction, but rapidly dissociates on dilution into medium lacking magnesium phosphate or pyrophosphate. The kcat values are similar for the variant trimer and hexamer, but Km values are 3 orders of magnitude lower for the hexamer. Thus, while stabilizing hexamer, the two His residues, per se, are not absolutely required for active-site structure rearrangement upon hexamer formation. The reciprocal effect of hexamerization and product binding to the active site is explained by destabilization of alpha-helix A, contributing both to the active site and the subunit interface.

We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50S ribosomal subunit components neighboring the alpha-sarcin loop. The probe is complementary to 23S rRNA nt 2653-2674. Photolysis of the complex formed between the probe and 50S subunits leads to site-specific probe photoincorporation into proteins L2, the most highly labeled protein, L1, L15, L16 and L27, labeled to intermediate extents, and L5, L9, L17 and L24, each labeled to a minor extent. Portions of each of these proteins thus lie within 23 A of nt U2653. These results lead us to conclude that the alpha-sarcin loop is located at the base of the L1 projection within the 50S subunit. Such placement, near the peptidyl transferase center, provides a rationale for the extreme sensitivity of ribosomal function to cleavage of the alpha-sarcin loop.

Tyrosine 55 and lysine 104 are evolutionarily conserved residues that form a hydrogen bond in the active site of Escherichia coli inorganic pyrophosphatase (E-PPase). Here we used site-directed mutagenesis to examine their roles in structure stabilization and catalysis. Though these residues are not part of the subunit interface, Y55F and K104R (but not K104I) substitutions markedly destabilize the hexameric structure, allowing dissociation into active trimers on dilution. A K104I variant is nearly inactive while Y55F and K104R variants exhibit appreciable activity and require greater concentrations of Mg2+ and higher pH for maximal activity. The effects on activity are explained by (a) increased pK(a)s for the catalytically essential base and acid at the active site, (b) decreases in the rate constant for substrate (dimagnesium pyrophosphate) binding to enzyme-Mg2 complex vs enzyme-Mg3 complex, and (c) parallel decreases in the catalytic constant for the resulting enzyme-Mg2-substrate and enzyme-Mg3-substrate complexes. The results are consistent with the major structural roles of Tyr55 and Lys104 in the active site. The microscopic rate constant for PPi hydrolysis on either the Y55F or K104R variants increases, by a factor of 3-4 in the pH range 7.2-8.0, supporting the hypothesis that this reaction step depends on an essential base within the enzyme active site.

Escherichia coli ribosomal protein L23 was derivatized with [3H]2,4-dinitrofluorobenzene both at the N terminus and at internal lysines. Dinitrophenyl-L23 (DNP-L23) was taken up into 50 S subunits from a reconstitution mixture containing rRNA and total 50 S protein depleted in L23. Unmodified L23 competed with DNP-L23 for uptake, indicating that each protein form bound in an identical or similar position within the subunit. Modified L23, incorporated at a level of 0.7 or 0.4 DNP groups per 50 S, was localized by electron microscopy of subunits complexed with antibodies to dinitrophenol. Antibodies were seen at two major sites with almost equal frequency. One site is beside the central protuberance, in a region previously identified as the peptidyltransferase center. The second location is at the base of the subunit, in the area of the exit site from which the growing peptide leaves the ribosome. Models derived from image reconstruction show hollows or canyons in the subunit and a tunnel that links the transferase and exit sites. Our results indicate that L23 is at the subunit interior, with separate elements of the protein at the subunit surface at or near both ends of this tunnel.

Serine proteinase inhibitors (serpins) form enzymatically inactive, 1:1 complexes (denoted E*I*) with their target proteinases that release free enzyme and cleaved inhibitor only very slowly. The mechanism of E*I* formation is incompletely understood and continues to be a source of controversy. Kinetic evidence exists that formation of E*I* proceeds via a Michaelis complex (E.I) and so involves at least two steps. In this paper, we determine the rate of E*I* formation from alpha-chymotrypsin and alpha1-antichymotrypsin using two approaches: first, by stopped-flow spectrofluorometric monitoring of the fluorescent change resulting from reaction of alpha-chymotrypsin with a fluorescent derivative of alpha1-antichymotrypsin (derivatized at position P7 of the reactive center loop); and second, by a rapid mixing/quench approach and SDS-polyacrylamide gel electrophoresis analysis. In some cases, serpins are both substrates and inhibitors of the same enzyme. Our results indicate the presence of an intermediate between E.I and E*I* and suggest that the partitioning step between inhibitor and substrate pathways precedes P1-P1' cleavage.

The translation machinery is the engine of life. Extracting the cytoplasmic milieu from a cell affords a lysate capable of producing proteins in concentrations reaching to tens of micromolar. Such lysates, derivable from a variety of cells, allow the facile addition and subtraction of components that are directly or indirectly related to the translation machinery and/or the over-expressed protein. The flexible nature of such cell-free expression systems, when coupled with high throughput monitoring, can be especially suitable for protein engineering studies, allowing one to bypass multiple steps typically required using conventional in vivo protein expression.

The current report represents a further advance- ment of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Forster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the back- ground transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2- Ibaraki and measured, through DiCoMPS, the syn- thesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fl- tRNA Ile UAU -generated FRET signals were specific- ally enhanced in infected cells, increased in the course of infection and were diminished on siRNA- mediated knockdown of NS3. Our results establish an experimental approach for the single-cell meas- urement of the levels of synthesis of a specific viral protein.

Pauses regulate the rhythm of ribosomal protein synthesis. Mutations disrupting even minor pauses can give rise to improperly formed proteins and human disease. Such minor pauses are difficult to characterize by ensemble methods, but can be readily examined by single-molecule (sm) approaches. Here we use smFRET to carry out real-time monitoring of the expression of a full-length protein, the green fluorescent protein variant Emerald GFP. We demonstrate significant correlations between measured elongation rates and codon and isoacceptor tRNA usage, and provide a quantitative estimate of the effect on elongation rate of replacing a codon recognizing an abundant tRNA with a synonymous codon cognate to a rarer tRNA. Our results suggest that tRNA selection plays an important general role in modulating the rates and rhythms of protein synthesis, potentially influencing simultaneous co-translational processes such as folding and chemical modification.

The structures of Escherichia coli soluble inorganic pyrophosphatase (E-PPase) and Thermus thermophilus soluble inorganic pyrophosphatase (T-PPase) have been compared to find the basis for the superior thermostability of T-PPase. Both enzymes are D3 hexamers and crystallize in the same space group with very similar cell dimensions. Two rather small changes occur in the T-PPase monomer: a systematic removal of Ser residues and insertion of Arg residues, but only in the C-terminal part of the protein, and more long-range ion pairs from the C-terminal helix to the rest of the molecule. Apart from the first five residues, the three-dimensional structures of E-PPase and T-PPase monomers are very similar. The one striking difference, however, is in the oligomeric interactions. In comparison with an E-PPase monomer, each T-PPase monomer is skewed by about 1 A in the xy plane, is 0.3 A closer to the center of the hexamer in the z direction, and is rotated by approximately 7 degrees about its center of gravity. Consequently, there are a number of additional hydrogen bond and ionic interactions, many of which form an interlocking network that covers all of the oligomeric surfaces. The change can also be seen in local distortions of three small loops involved in the oligomeric interfaces. The complex rigid-body motion has the effect that the hexamer is more tightly packed in T-PPase: the amount of surface area buried upon oligomerization increases by 16%. The change is sufficiently large to account for all of the increased thermostability of T-PPase over E-PPase and further supports the idea that bacterial PPases, most active as hexamers or tetramers, achieve a large measure of their stabilization through oligomerization. Rigid-body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic.

Glutamic acid 20 is an evolutionarily conserved residue found within the active site of the inorganic pyrophosphatase of Escherichia coli (E-PPase). Here we determine the effect of E20D substitution on the quaternary structure and catalytic properties of E-PPase. In contrast to wild-type enzyme, which is hexameric under a variety of conditions, E20D-PPase can be dissociated by dilution into nearly inactive trimers, as shown by electrophoresis of cross-linked enzyme, analytical ultracentrifugation, and measurement of catalytic activity as a function of enzyme concentration. Hexamer stability is increased in the presence of both substrate and Mg2+, is maximal at pH 6.5, and falls off sharply as the pH is lowered or raised from this value. Measured at saturating substrate, 20 mM Mg2+ and pH 7.2, E20D substitution (a) decreases activity towards inorganic pyrophosphate (PPi) hydrolysis and oxygen exchange between water and inorganic phosphate (P1), (b) increases the rate of net PPi synthesis, and (c) decreases the amount of enzyme-bound PPi in equilibrium with Pi in solution. Measurements of PPi hydrolysis rate as a function of both Mg2+ concentration and pH for the E20D variant show that its decreased activity is largely accounted for on the basis of an increased pKa of the catalytically essential base at the active site, and the need for a Mg2+ stoichiometry of 5 in the enzyme-substrate complex, similar to what is seen for the D97E variant. By contrast, wild-type PPase catalysis over a wide range of Mg2+ concentration and pH is dominated by an enzyme-substrate complex having a total of four Mg2+ ions. These results are consistent with a supporting role for Glu20 in PPase catalysis and demostrate that even conservative mutation at the active site can perturb the quaternary structure of the enzyme.

Both human neutrophil elastase (HNE) and free chymotrypsin (Chtr) proteolyze Chtr within the complex that Chtr forms with antichymotrypsin (ACT). As free Chtr is stable both to self-digestion and to digestion by HNE, these results are indicative of a stability and/or conformational change in Chtr that accompanies complex formation. As determined by both N-terminal sequence analysis and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS), the major initial sites of HNE cleavage of complexed Chtr are between gamma-chain residues A158/S159 and V188/S189. Significantly, this latter site is at the base of the S1 site that recognizes the P1 position of the serpin. A slower cleavage in the beta-chain between T139/G140 is also found. In addition, rACT is cleaved between residues V22/D23. The gamma-chain of complexed Chtr is also cleaved by free Chtr, but at different sites: L162/L163 and W172/G173. beta-Chain cleavages were also found between residues Q81/K82 and F114/S115. Cleavages similar to those described above were also found when Chtr was complexed with the L358F-rACT variant, but not for Chtr complexed with either of the smaller inhibitors bovine pancreatic trypsin inhibitor or turkey ovomucoid third domain, nor for the covalent adduct of Chtr with N-p-tosylphenylalanyl chloromethyl ketone. We conclude that the structural change in Chtr making it a proteinase substrate is coupled with the large conformational change in ACT following complex formation. Complexed Chtr is much less reactive toward proteolytic digestion in the presence of high salt than in its absence, in accord with the high-salt induced release of active enzyme from the Chtr.rACT complex and the suggestion that electrostatic interactions mediate the coupling of structural change between rACT and Chtr within the Chtr.rACT complex. Potential physiological consequences of this work are explored.

Steady-state rates of PPi hydrolysis by Escherichia coli inorganic pyrophosphatase (E-PPase) were measured as a function of magnesium pyrophosphatase (substrate) and free Mg2+ ion (activator) in the pH range 6.0-10.0. Computer fitting of hydrolysis data in combination with direct measures of Mg2+ binding to enzyme has resulted in a model that quantitatively accounts for our results. The major features of this model are the following: (a) E-PPase catalysis proceeds both with three and with four (and possibly with five) Mg2+ ions per active site; (b) catalysis requires both an essential base and an essential acid, and the pKas of these groups are modulated by the stoichiometry of bound Mg2+; and (c) the four-metal route predominates for concentrations of free Mg2+>0.2mM. The model straightforwardly accounts for the apparent linkage between increased pKa of an essential base and activity requirements for higher Mg2+ concentration observed for several active site variants. Microscopic rate constants for overall catalysis of PPi-Pi equilibration were determined at pH 6.5-9.3 by combined analysis of enzyme-bound PPi formation and rates of PPi hydrolysis, PPi synthesis, and Pi-H2O oxygen exchange. The catalytic activity of E-PPase at saturating substrate increases toward PPi hydrolysis and decreases toward PPi synthesis and Pi-H2O oxygen exchange with increasing pH. These changes are mainly due to an increased rate of dissociation of the second released Pi and a decreased rate of enzyme-bound PPi synthesis from enzyme-bound Pi, respectively, as the pH is raised .

It has recently been suggested that peptidyl transferase activity is primarily a property of ribosomal RNA and that ribosomal proteins may act only as scaffolding. On the other hand, evidence from both photoaffinity labeling studies and reconstitution studies suggest that protein L2 may be functionally important for peptidyl transferase. In the work reported here, we reconstitute 50S subunits in which the H229Q variant of L2 replaces L2, with all other ribosomal components remaining unchanged, and determine the catalytic and structural properties of the reconstituted subunits. We observe that mutation of the highly conserved His 229 to Gin results in a complete loss of peptidyl transferase activity in the reconstituted 50S subunit. This is strong evidence for the direct involvement of L2 in ribosomal peptidyl transferase activity. Control experiments show that, though lacking peptidyl transferase activity, 50S subunits reconstituted with H229Q-L2 appear to be identical with 50S subunits reconstituted with wild-type L2 with respect to protein composition and 70S formation in the presence of added 30S subunits. Furthermore, as shown by chemical footprinting analysis, H229Q-L2 appears to bind 23S RNA in the same manner as wild-type L2. Thus, the effect of H229 mutation appears to be confined to an effect on peptidyl transferase activity, providing the most direct evidence for protein involvement in this function to date.Key words: protein L2, site-specific mutagenesis, peptidyl transferase, reconstitution, histidine.

We report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe and its exploitation in identifying 50S ribosomal subunit components neighboring its target site in 23S rRNA. The probe is complementary to 23S rRNA nucleotides 2475-2483, a single-stranded sequence (the 2475 loop) near the peptidyltransferase center of Escherichia coli ribosomes. On photolysis in the presence of 50S subunits, it site-specifically incorporates into proteins L1, L13, L16, L32, and L33 and into 23S rRNA nucleotides G2470, A2471, and G2472. These results provide clear evidence that C2475 in 23S rRNA is within 21 A (the distance between C2475 and the photogenerated nitrene) of proteins L1, L13, L16, L32, and L33. The implications of these results for the evolving model of the internal structure of the 50S subunit are considered.

Aspartic acid 97 in the inorganic pyrophosphatase of Escherichia coli (E-PPase) has been identified as an evolutionarily conserved residue forming part of the active site [Cooperman et al. (1992) Trends Biochem. Sci. 17, 262-266]. Here we determine the effect of D97E substitution on several kinetic and thermodynamic properties of E-PPase, including rate and equilibrium constants for enzyme-catalyzed PPi.Pi equilibration at pH 7.2 and 8.0, Mg2+ affinity in the presence and absence of substrate, and the Mg2+ and pH dependence of kcat and Km. We find the major effects of D97E substitution are to (a) decrease markedly the pH-independence rates of both PPi hydrolysis and, especially, PPi resynthesis on the enzyme, (b) selectively destabilize both the EMg4PPi complex and the transition state between this complex and the EMg2(MgPi)2 complex, (c) raise the pKa of the basic group "essential" for PPi hydrolysis and for productive PPi binding by 1.5 and > 2.2 log units, respectively, (d) distort a site to which Mg2+ binds in the absence of substrate such that occupancy of the site by Mg2+ no longer confers enzymatic activity, and (e) decrease the affinity of one of the two Mg2+ ions that binds to enzyme in the presence of substrate. That this multiplicity of effects arises from a single Asp to Glu substitution suggests, in the absence of any evidence for a generalized structural change, a tightly integrated active site in which the perturbation induced by conservative substitution at a single location can have widespread functional effects.

Using site-directed mutagenesis, we have completed replacing all 17 putative active site residues of Escherichia coli inorganic pyrophosphatase (PPase). We report here the production of 11 new variant proteins and their initial characterization, including thermostability, hydrophobicity, oligomeric structure, and specific activity at pH 8. Studies of the pH-rate profiles of 12 variants containing substitutions for potentially essential residues showed that the effect of the mutation was always to increase the pKa of a basic group essential for both substrate binding and catalysis by 1-3 pH units. The D70E variant had the lowest activity at all pHs; the K29R, R43K, and K142R variants also had low kcat/Km values. The principal effect seen in the other variant proteins was higher and sharper pH optima; their pH-independent kcat and kcat/Km values changed at most by a factor of 8. Our results suggest that the most likely candidate for the essential basic group affected by all mutations in the active site is a hydroxide ion stabilized by coordination to the essential Mg2+ ions. Analyzing our results using the structure recently obtained for E. coli PPase [Kankare et al. (1994) Protein Eng. 7, 823-830] led us to identify a group of residues, centered around Asp70 and including Tyr55, Asp65, Asp67, Asp102, and Lys104, that we believe binds the magnesium ions that are critical for the activity, possibly by stabilizing the essential hydroxide. Others, including Lys29, Arg43, and Lys142, are more spread out and more positively charged. They appear to be involved in binding substrate and product. Tyr55 is also a key part of the hydrophobic core of E. coli PPase; when it or residues that interact with it are conservatively mutated, there are changes in the overall structure of the enzyme as assayed by thermostability, hydrophobicity, or oligomeric structure.

Prostate-Specific Antigen (PSA), a serine protease, is believed to regulate the actions of extracellular matrix proteins such as fibronectin and Insulin-like Growth Factor-Binding Proteins (IGFBPs), possible factors involved in tumor progression and metastasis. Two phosphonate ester derivatives Cbz-(4-AmPhGly)p(OPh)2 (Inh I) and Cbz- (4-AmPhe)p(OPh)2 (Inh II), first mechanism-based inhibitors for PSA were synthesized using Oleksyszyn oxidation reaction, and their inhibitory activities on cleaving fibronectin (both human and bovine) and IGFBPs were investigated. To elucidate the role of PSA in the development and progression of prostate cancer, the regulation of the PSA activity on these two physiological substrates was clearly illustrated by modulating the concentrations of inhibitors, i.e. cleavages of both fibronectin and IGFBPs by PSA are inhibited by structure specific designed diphenyl phosphonate esters, consistent with the putative degradation. The inhibition data of two synthetic phosphonate ester derivatives, as lead compounds, will facilitate the development of more specific inhibitors of PSA activity, likely to modulate the physiological control of the IGF in human prostatic cell growth.

The three-dimensional structure of an uncleaved serpin, a variant of human antichymotrypsin engineered to be an inhibitor of human neutrophil elastase, has been determined by X-ray crystallographic methods and is currently being refined at 2.5 A resolution. It contains an intact reactive loop in a distorted helical conformation. A comparison of the current model with that of its cleaved counterpart suggests that the conformational 'stress' of the serpin in its uncleaved and uncomplexed state may not be confined solely to the reactive loop or beta-sheet A. It is intriguing that strand s4A is not pre-inserted into beta-sheet A of the native serpin, and this has profound implications for the mechanism of serpin function.

In this work we report the synthesis of a radioactive, photolabile oligodeoxyribonucleotide probe complementary to 16S rRNA nucleotides 1397-1405 and its exploitation in identifying 30S ribosomal subunit components neighboring its target site in 16S rRNA. Nucleotides 1397-1405 lie within a single-stranded sequence that has been linked to the decoding region of Escherichia coli ribosomes. On photolysis in the presence of activated 30S subunits, the photolabile oligodeoxyribonucleotide probe site-specifically incorporates into proteins S1, S7, S18, and S21 (identified by SDS-PAGE, RP-HPLC, and antibody affinity chromatography) and into three separate 16S rRNA regions, specifically, nucleotides A-1396, G-1405-A-1408, and A-1492 and A-1493. These results provide clear evidence that G-1405 in 16S rRNA is within 24 A (the distance between G-1405 and the photogenerated nitrene) of proteins S1, S7, S18, and S21 and each of the other nucleotides mentioned above, consistent with other studies of 30S internal structure. Although the probe binds to inactive 30S subunits about as well as to activated 30S subunits, photolysis of the inactive 30S.probe complex leads to a very different pattern of protein labeling, providing strong evidence, at the protein level, that the inactive to activated transition is accompanied by conformational change in the 1400 region of 16S rRNA.