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Post-SELEX modification of DNA aptamers is an established strategy to improve their affinity or inhibitory characteristics. In this study, we examined the possibility of increasing the recognition interface between the thrombin-binding aptamer HD1 (TBA) and thrombin by adding a chemically modified side chain to selected nucleotide residues. A panel of 22 TBA variants with N3-modified residues T3 and T12 was prepared by a two-step modification procedure. Aptamers were characterized by a combination of biophysical and biochemical methods. We identified mutants with enhanced affinity and improved anticoagulant activity. The crystal structures of thrombin complexes with three selected modified variants revealed that the modified pyrimidine base invariably allocates in proximity to thrombin residues Tyr76 and Ile82 due to the directing role of the unmodified TT loop. The modifications induced an increase in the contact areas between thrombin and the modified TBAs. Comparative analysis of the structural, biochemical, and biophysical data suggests that the non-equivalent binding modes of the mutants with thrombin in the T3- and T12-modified series account for the observed systematic differences in their affinity characteristics. In this study, we show that extending the recognition surface between the protein and modified aptamers is a promising approach that may improve characteristics of aptamer ligands., Graphical Abstract, Smirnov et al. describe a post-SELEX approach for the thrombin aptamer HD1 that consists in expanding the aptamer-protein interface through attachment of non-natural side chains at specific aptamer residues. The authors report synthesis and characterization of 22 aptamer mutants and present crystal structures of thrombin complexes with three selected variants.

Recently, mixed duplex/quadruplex oligonucleotides have attracted great interest for use as biomedical aptamers. In the case of anti-thrombin aptamers, the addition of duplex-forming sequences to a G-quadruplex module identical or very similar to the best-known G-quadruplex of the Thrombin Binding Aptamer (HD1) results in new or improved biological properties, such as higher activity or different recognition properties with respect to HD1. Remarkably, this bimodular fold was hypothesized, based on its sequence, for the only anti-thrombin aptamer in advanced clinical trial, NU172. Whereas cation modulation of G-quadruplex conformation and stability is well characterized, only few data from similar analysis on duplex/quadruplex oligonucleotides exist. Here we have performed a characterization of structure and stability of four different duplex/quadruplex anti-thrombin aptamers, including NU172, in the presence of different cations and in physiological-mimicking conditions in comparison to HD1, by means of spectroscopic techniques (UV and circular dichroism) and differential scanning calorimetry. Our data show a strong reciprocal influence of each domain on the stability of the other and in particular suggest a stabilizing effect of the duplex region in the presence of solutions mimicking the physiological conditions, strengthening the idea that bimodular aptamers present better therapeutic potentialities than those containing a single G-quadruplex domain.

Locked nucleic acids (LNAs) are formed by bicyclic ribonucleotides where the O2′ and C4′ atoms are linked through a methylene bridge and the sugar is blocked in a 3′-endoconformation. They represent a promising tool for therapeutic and diagnostic applications and are characterized by higher thermal stability and nuclease resistance with respect to their natural counterparts. However, structural descriptions of LNA-containing quadruplexes are rather limited, since few NMR models have been reported in the literature. Here, the first crystallographically derived model of an all-LNA-substituted quadruplex-forming sequence 5′-TGGGT-3′ is presented refined at 1.7 Å resolution. This high-resolution crystallographic analysis reveals a regular parallel G-quadruplex arrangement terminating in a well defined thymine tetrad at the 3′-end. The detailed picture of the hydration pattern reveals LNA-specific features in the solvent distribution. Interestingly, two closely packed quadruplexes are present in the asymmetric unit. They face one another with their 3′-ends giving rise to a compact higher-order structure. This new assembly suggests a possible way in which sequential quadruplexes can be disposed in the crowded cell environment. Furthermore, as the formation of ordered structures by molecular self-assembly is an effective strategy to obtain nanostructures, this study could open the way to the design of a new class of LNA-based building blocks for nanotechnology.

Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated with the swapping on the N-terminal. The first direct experimental evidence on the formation of a C-terminal swapped dimer (C-dimer) obtained from the monomeric derivative of BS-RNase, although under non-native conditions, is here reported. The X-ray model of this dimer reveals a quaternary structure different from that of the C-dimer of RNase A, due to the presence of three mutations in the hinge peptide 111–116. The mutations increase the hinge peptide flexibility and decrease the stability of the C-dimer against dissociation. The biological implications of the structural data are also discussed.

This work describes major features and specificities of glutathione (GSH) biosynthesis in the cold-adapted Pseudoalteromonas haloplanktis. Two steps of ATP hydrolysis, catalysed by γ-glutamyl-cysteine ligase (PhGshA) and glutathione synthetase (PhGshB), drive GSH formation; however, differently from other sources, this psychrophile contains two redundant PhGshAs. The biochemical properties of recombinant rPhGshB and rPhGshA II were previously reported; here we report the characterization of rPhGshA I, together with a structural comparison with rPhGshA II. The availability of the three enzymes involved in GSH biosynthesis allowed the reconstitution of two systems producing this thiol in P. haloplanktis. Both systems were active, although with different reaction rates; in particular, rPhGshB worked more efficiently with rPhGshA I compared to rPhGshA II, also because of the different expression of the corresponding genes. However, the lower KM of rPhGshA II for cysteine compared to rPhGshA I could permit an effective GSH production even under conditions of low cysteine content. Coupling of the kinetics of GSH production with that of ATP hydrolysis was realised for the first time and proved the rigorous stoichiometry of GSH biosynthesis, resulting in two moles of ATP hydrolysed per one mole of GSH formed. Furthermore, after dissecting the reaction rates for GSH biosynthesis, we demonstrated that synthesis of this thiol was rate-limited by the step catalysed by rPhGshB, at least in the cold-adapted source. Therefore, the GshA rate-limited step postulated in other sources could be revised or restricted to conditions of limiting substrate amounts.

Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human ?-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5'-5' polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin-mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin-aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs.

Eukaryotic protein carboxyl methyltransferase catalyzes a two-substrates reaction in which the methyl group of S-adenosylmethionine is transferred to the free carboxyl group of D-aspartyl and L-isoaspartyl-containing peptide or protein substrates. It has been previously shown that at least three binding sites are required for the interaction of adenosylmethionine with the enzyme and/or the protein substrate [Oliva A., Galletti P., Zappia V., Paik W. K. & Kim S. (1980) Eur. J. Biochem. 104, 595–602], while very little is know concerning the structural requirements of the protein substrate. In this study several synthetic tripeptides were selected in order to elucidate the structural requirements of the methyl-accepting substrates. The results obtained with this series of peptides suggested that: (1) three residues appear to be the minimal length, so far identified, required for a productive enzyme-substrate interaction, several dipeptides being ineffective as substrates [McFadden P. N. & Clarke S. (1986) J. Biol. Chem. 261, 11 503–11 511]; (2) the isoaspartyl residue is not recognized unless its α-amino group is involved in a carboamide bond; (3) an hydrogen atom on the amide linkage following the isoaspartyl residue is essential for both recognition and catalysis; (4) oligopeptides containing both D-aspartyl and D-isoaspartyl residues are not recognized by this methyltransferase. On the basis of these results, interaction sites between the peptide substrate and the enzyme molecule have been proposed. This paper also reports the first application of fast-atom-bombardment mass spectrometry to the detection of the products of the enzymatic methyl esterification reaction. By this soft ionization technique, the methylesterified peptides as well as the corresponding cyclic imides generated during the spontaneous demethylation process have been identified.

Bovine seminal ribonuclease (BS-RNase) is an intriguing homodimeric enzyme which exists as two conformational isomers, characterized by distinct catalytic and biological properties, referred to as M × M and M = M. Reduction of inter-chain disulfide bridges produces a stable monomeric derivative (M) which is still active. This paper reports the screening and optimization of crystallization conditions for growing single diffraction-quality crystals for the various BS-RNase forms. The crystallization trials were performed using both the vapor diffusion and microbatch methods. The M × M dimer was crystallized in the free form from polyethylene glycol (PEG) 4000 at pH 8.5 and as a complex with the substrate analog uridylyl(2′-5′)guanosine (UpG) from an unbuffered ammonium sulfate (AS) solution. These two crystal types diffract X-rays to 2.5 and 1.9 Å resolution, respectively. Two different crystal types were obtained both for the M = M dimer and for the monomeric derivative. (M = M)a crystals, grown from PEG 4000 (8% w/v) at pH 5.6, diffract X-rays to 4.0 Å. At higher PEG concentration (15% w/v) a different crystal type was obtained, (M = M)b, which showed a better diffraction limit (2.5 Å). For the monomer, type (M)a and (M)b crystals, diffracting X-rays to 2.5 Å resolution, were obtained from AS at pH 6.5 and from PEG 4000 at pH 8.5, respectively. A comparison with previously crystallized forms of the dimer M × M and its complexes with uridylyl(2′-5′)adenosine and 2′-deoxycytidylyl(3′-5′)-2′-deoxyadenosine is also presented. The three-dimensional structure analysis of (M × M) · UpG and (M = M)b is in progress. © 1999 Elsevier Science B.V. All rights reserved.

International audience; Bacteria react to adverse environmental stimuli by clustering into organized communities called biofilms. A remarkably sophisticated control system based on the dinucleotide 3'-5' cyclic diguanylic acid (c-di-GMP) is involved in deciding whether to form or abandon biofilms. The ability of c-di-GMP to form self-intercalated dimers is also thought to play a role in this complex regulation. A great advantage in the quest of elucidating the catalytic properties of the enzymes involved in c-di-GMP turnover (diguanylate cyclases and phosphodiesterases) would come from the availability of an experimental approach for in vitro quantification of c-di-GMP in real-time. Here, we show that c-di-GMP can be detected and quantified by circular dichroism (CD) spectroscopy in the low micromolar range. The method is based on the selective ability of manganese ions to induce formation of the intercalated dimer of the c-di-GMP dinucleotide in solution, which displays an intense sigmoidal CD spectrum in the near-ultraviolet region. This characteristic spectrum originates from the stacking interaction of the four mutually intercalated guanines, as it is absent in the other cyclic dinucleotide 3'-5' cyclic adenilic acid (c-di-AMP). Thus, near-ultraviolet CD can be used to effectively quantify in real-time the activity of diguanylate cyclases and phosphodiesterases in solution.

Thrombin plays a pivotal role in the coagulation cascade; therefore, it represents a primary target in the treatment of several blood diseases. The 15-mer DNA oligonucleotide, 5'-GGTTGGTGTGGTTGG-3', known as Thrombin Binding Aptamer (TBA), is a highly potent inhibitor of the enzyme. TBA folds as an antiparallel chair-like G-quadruplex structure, with two G-tetrads surrounded by two TT loops on one side and a TGT loop on the opposite side. Previous crystallographic studies have shown that TBA binds thrombin exosite I by its TT loops, T3T4 and T12T13. In order to get a better understanding of the thrombin-TBA interaction, we have undertaken a crystallographic characterization of the complexes between thrombin and two TBA mutants, TBA??T3 and TBA??T12, which lack the nucleobase of T3 and T12, respectively. The structural details of the two complexes show that exosite I is actually split into two regions, which contribute differently to TBA recognition. These results provide the basis for a more rational design of new aptamers with improved therapeutic action. This article is protected by copyright. All rights reserved.

The non-enzymatic deamidation of asparagine residues in proteins is a widely occurring reaction, both in vivo and in vitro. Although the importance of this process is commonly recognised, only little structural information is available on it. In order to evaluate the structural effects of this reaction in proteins, we have determined the crystal structure of a ribonuclease A derivative in which asparagine 67 has been replaced by an isoaspartyl residue, as a consequence of an in vitro deamidation reaction. The overall structure of the model, refined to a crystallographic R-factor of 0.159 at a resolution of 1.9 A, is very similar to that of the native protein, but considerable deviations are observed in the region delimited by the disulphide bridge 65-72. In particular, the insertion of an extra methylene group in the main chain at residue 67 breaks up the hydrogen bond network that makes this region rather rigid in ribonuclease A. On the basis of the structure observed, some of the slightly but significantly different properties of this deamidated derivative, with respect to the native enzyme, can be explained.

3D domain swapping is the process by which two or more protein molecules exchange part of their structure to form intertwined dimers or higher oligomers. Bovine pancreatic ribonuclease (RNase A) is able to swap the N-terminal α-helix (residues 1–13) and/or the C-terminal β-strand (residues 116–124), thus forming a variety of oligomers, including two different dimers. Cis - trans isomerization of the Asn113-Pro114 peptide group was observed when the protein formed the C-terminal swapped dimer. To study the effect of the substitution of Pro114 on the swapping process of RNase A, we have prepared and characterized the P114A monomeric and dimeric variants of the enzyme. In contrast with previous reports, the crystal structure and NMR data on the monomer reveals a mixed cis - trans conformation for the Asn113-Ala114 peptide group, whereas the X-ray structure of the C-terminal swapped dimer of the variant is very close to that of the corresponding dimer of RNase A. The mutation at the C-terminus affects the capability of the N-terminal α-helix to swap and the stability of both dimeric forms. The present results underscore the importance of the hydration shell in determining the cross-talk between the chain termini in the swapping process of RNase A.

Superoxide dismutases are enzymes that protect biological systems against oxidative damage caused by superoxide radicals. In this paper, a detailed characterization is presented on the stability of SmSOD, the dimeric cambialistic superoxide dismutase from the dental pathogenic microorganism Streptococcus mutans, towards temperature and guanidine hydrochloride. Thermal and chemical denaturations were investigated by means of circular dichroism, fourth-derivative UV spectroscopy and fluorescence measurements. Data indicate that SmSOD is endowed with a significant thermostability and that both its thermal and guanidine hydrochloride-induced unfolding processes occur through a three-state model, characterized by a catalytically active dimeric intermediate species. To our knowledge, SmSOD is the smallest known dimeric protein that populates a well-structured active dimeric rather than a monomeric intermediate during unfolding processes.

The Root effect describes the drastic drop of oxygen affinity and loss of cooperativity at acidic pH expressed in the hemoglobins (Hb) of certain fish. The comparison between the deoxy structures of the Root effect Hb from the Antarctic fish Trematomus bernacchii (HbTb) at different pHs (pH = 6.2 and pH = 8.4) shows that the most significant differences are localized at the CDα region, where a salt bridge between Asp48 and His55 breaks during the low-to-high pH transition. In order to shed light on the relationship between pH, CDα loop structure and dynamics, and oxygen access to the active site in the alpha chain of HbTb, different computer simulation techniques were performed. Our results highlight the importance of the protonation of His55 in regulating oxygen access, underscoring its pivotal role in the structural and functional properties of HbTb. These data provide further support to the hypothesis that this residue might contribute to the release of Root protons in HbTb and underline the fact that an efficient transport of molecular oxygen in Hbs relies on a subtle balance of tertiary structure and protein conformational flexibility. © 2011 IUBMB IUBMB Life, 63(3): 175–182, 2011

Superoxide dismutases (SODs) are metalloenzymes catalysing the dismutation of superoxide anion radicals into molecular oxygen and hydrogen peroxide. Here, we present the crystal structure of a cold-adapted Fe-SOD from the Antarctic eubacterium Pseudoalteromonas haloplanktis (PhSOD), and that of its complex with sodium azide. The structures were compared with those of the corresponding homologues having a high sequence identity with PhSOD, such as the mesophilic SOD from Escherichia coli (EcSOD) or Pseudomonas ovalis, and the psychrophilic SOD from Aliivibrio salmonicida (AsSOD). These enzymes shared a large structural similarity, such as a conserved tertiary structure and arrangement of the two monomers, an almost identical total number of inter- and intramolecular hydrogen bonds and salt bridges. However, the two cold-adapted SODs showed an increased flexibility of the active site residues with respect to their mesophilic homologues. Structural information was combined with a characterisation of the chemical and thermal stability performed by CD and fluorescence measurements. Despite of its psychrophilic origin, the denaturation temperature of PhSOD was comparable with that of the mesophilic EcSOD, whereas AsSOD showed a lower denaturation temperature. On the contrary, the values of the denaturant concentration at the transition midpoint were in line with the psychrophilic/ mesophilic origin of the proteins. These data provide additional support to the hypothesis that cold-adapted enzymes achieve efficient catalysis at low temperature, by increasing the flexibility of their active site; moreover, our results underline how fine structural modifications can alter enzyme flexibility and/or stability without compromising the overall structure of typical rigid enzymes, such as SODs.

The free-energy profile and the classical kinetics of the heme carbomonoxide binding-unbinding reaction have been derived by means of a theoretical method for the separated chains of human (HbA) and Trematomus newnesi major component (HbTn) hemoglobin. The results reveal that the alpha- and beta-chains of HbA have similar values of kinetic constants for the dissociation of the Fe-CO state, in agreement with experimental data. Comparisons of the present findings with the data obtained for the alpha- and beta-chains of HbTn and with theoretical and experimental results previously collected on myoglobin provide a detailed picture of this important biochemical reaction in globins. The sequence and structural differences among the globins are not reflected in meaningful variations in the rate of CO dissociation. These data support the conclusion that the differences observed for the reaction with CO of globins, if any, involve the rate of ligand migration to the solvent, rather than the Fe-CO complex formation/rupture. Furthermore, our results agree with the recent discovery that globin family proteins exhibit common dynamics, thus confirming the observation that the dynamic properties of proteins are strongly related to their overall architecture.

Recent studies have demonstrated that hemoglobins isolated from Antarctic fish undergo peculiar oxidation processes. Here we show, by combining crystallographic and spectroscopic data, that the oxidation pathway of Trematomus bernacchii hemoglobin (HbTb) is distinct from that observed for the major component of Trematomus newnesi (Hb1Tn), despite the high sequence identity of the two proteins and structural similarity of their ferrous and fully oxidized states. Resonance Raman analysis of HbTb autoxidation upon air-exposure reveals the absence of the oxidized pentacoordinated state that was observed for Hb1Tn. The HbTb oxidation pathway is characterized by two ferric species: an aquo hexacoordinated high spin state and a bis-histidyl hexacoordinated low spin form, which appear in the early stages of the oxidation process. The high resolution structure of an intermediate along the oxidation pathway has been determined at 1.4 A resolution. The analysis of the electron density of the heme pocket shows, for both the α and the β iron, the coexistence of multiple binding states. In this partially oxidized form, HbTb exhibits significant deviations from the canonical R state both at the local and global level. The analysis of these modifications highlights the structural correlation between key functional regions of the protein. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 1117–1125, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

The properties of hemoproteins strictly depend on the type and orientation of axial ligands. Here, the orientations of axially coordinated His in bis-His complexes and the heme geometry in protein data bank have been analyzed. The effect of the bis-histidyl formation on the heme cavity of Antarctic fish hemoglobins has been also evaluated. The results show that protein matrix exerts a major effect on the conformation of axially ligated histidines: the imidazoles in bis-His complexes occupy a preferred relative orientation in globins and in model systems, whereas they adopt a variety of relative orientations in other hemoproteins. The bis-histidyl adducts affect the heme geometry inducing larger distortions from planarity with respect to other ligands. These deviations are larger in bis-His multiheme cytochromes than in globins. In Antarctic fish hemoglobins the bis-histidyl adduct adopts preferentially a distorted coordination and the formation of the bis-His complex induces a slight but significant modification in the shape, area and volume of the heme cavity.

Bovine seminal ribonuclease exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer. The molecular envelope and the exposed surface of the two isomers are practically indistinguishable and their diversity is almost completely buried in the interior of the protein. Surprisingly, the cytotoxic and antitumor activity of the enzyme is a peculiar property of the swapped dimer. This buried diversity comes into light in the reducing environment of the cytosol, where the unswapped dimer dissociates into monomers, whereas the swapped one generates a metastable dimeric form (NCD-BS) with a quaternary assembly that allows the molecule to escape the protein inhibitor of ribonucleases. The stability of this quaternary shape was mainly attributed to the combined presence of Pro19 and Leu28. We have prepared and fully characterized by X-ray diffraction the double mutant P19A/L28Q (PALQ) of the seminal enzyme. While the swapped and unswapped forms of the mutant have structures very similar to that of the corresponding wild-type forms, the non-covalent form (NCD-PALQ) adopts an opened quaternary structure, different from that of NCD-BS. Moreover, model building clearly indicates that NCD-PALQ can be easily sequestered by the protein inhibitor. In agreement with these results, cytotoxic assays have revealed that PALQ has limited activity, whereas the single mutants P19A and L28Q display cytotoxic activity against malignant cells almost as large as the wild-type enzyme. The significant increase in the antitumor activity, brought about by the substitution of just two residues in going from the double mutant to the wild-type enzyme, suggests a new strategy to improve this important biological property by strengthening the interface that stabilizes the quaternary structure of NCD-BS.

A specialized class of RNases shows a high cytotoxicity toward tumor cell lines, which is critically dependent on their ability to reach the cytosol and to evade the action of the ribonuclease inhibitor (RI). The cytotoxicity and antitumor activity of bovine seminal ribonuclease (BSRNase), which exists in the native state as an equilibrium mixture of a swapped and an unswapped dimer, are peculiar properties of the swapped form. A dimeric variant (HHP2-RNase) of human pancreatic RNase, in which the enzyme has been engineered to reproduce the sequence of BSRNase helix-II (Gln28--Leu, Arg31--Cys, Arg32--Cys, and Asn34--Lys) and to eliminate a negative charge on the surface (Glu111--Gly), is also extremely cytotoxic. Surprisingly, this activity is associated also to the unswapped form of the protein. The crystal structure reveals that on this molecule the hinge regions, which are highly disordered in the unswapped form of BSRNase, adopt a very well-defined conformation in both subunits. The results suggest that the two hinge peptides and the two Leu28 side chains may provide an anchorage to a transient noncovalent dimer, which maintains Cys31 and Cys32 of the two subunits in proximity, thus stabilizing a quaternary structure, similar to that found for the noncovalent swapped dimer of BSRNase, that allows the molecule to escape RI and/or to enhance the formation of the interchain disulfides.

Bovine seminal ribonuclease (BS-RNase) is a unique member of the pancreatic-like ribonuclease superfamily. This enzyme exists as two conformational isomers with distinctive biological properties. The structure of the major isomer is characterized by the swapping of the N-terminal segment (M×M BS-RNase). In this article, the crystal structures of the ligand-free M×M BS-RNase and its complex with 2′-deoxycitidylyl(3′,5′)-2′-deoxyadenosine derived from isomorphous crystals have been refined. Interestingly, the comparison between this novel ligand-free form and the previously published sulfate-bound structure reveals significant differences. In particular, the ligand-free M×M BS-RNase is closer to the structure of M×M BS-RNase productive complexes than to the sulfate-bound form. These results reveal that M×M BS-RNase presents a remarkable flexibility, despite the structural constraints of the interchain disulfide bridges and the swapping of the N-terminal helices. These findings have important implications to the ligand binding mechanism of M×M BS-RNase. Indeed, a population shift rather than a substrate-induced conformational transition may occur in the M×M BS-RNase ligand binding process. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004

Bovine seminal ribonuclease, a homodimeric enzyme joined covalently by two interchain disulphide bonds, is an equilibrium mixture of two conformational isomers, MxM and M=M. The major form, MxM, whose crystal structure has been previously determined at 1.9 A resolution, presents the swapping of the N-terminal segments (residues 1-15) and composite active sites formed by residues of different chains. The three-dimensional domain swapping does not occur in the M=M form. The different fold of each N-terminal tail is directed by the hinge loop (residue 16-22) connecting the swapping domain to the body of the protein. Reduction and alkylation of interchain disulphide bridges produce a monomeric derivative and a noncovalent swapped dimer, which are both active. The free and nucleotide-bound forms of the monomer have been crystallized at an alkaline pH and refined at 1.45 and 1.65 A resolution, respectively. In both cases, the N-terminal fragment is folded on the main body of the protein to produce an intact active site and a chain architecture very similar to that of bovine pancreatic ribonuclease. In this new fold of the seminal chain, the hinge loop is disordered. Despite the difference between the tertiary structure of the monomer and that of the chains in the MxM form, the active sites of the two enzymes are virtually indistinguishable. Furthermore, the structure of the liganded enzyme represents the first example of a ribonuclease complex studied at an alkaline pH and provides new information on the binding of a nucleotide when the catalytic histidines are deprotonated.

An NAD + -dependent alcohol dehydrogenase from the extreme thermophilic archaebacterium Sulfolobus solfataricus has been crystallized in the holo-enzyme and apo-enzyme forms. Crystals of the holo-enzyme grow from 2-methyl-2,4-pentanediol at pH 8·4 with the addition of NADH and at pH 7·0 with the addition of NADH and dimethyl sulphoxide. Crystals of the apo-enzyme grow at pH 6·3 from a mixture of polyethylene glycol 4000 and propan-2-ol. The holo-enzyme crystallizes in C 2 with a dimer in the asymmetric unit, however the crystals are twinned and unsuitable for data collection. The apo-enzyme crystallizes in I 4 1 22 ( a = 126·82 A, b = 118·95 A) with a monomer in the asymmetric unit, and the single crystals diffract to 2·8 A.

Bovine seminal ribonuclease (BS-RNase) is a unique member of the pancreatic-like ribonuclease superfamily. The native enzyme is a mixture of two dimeric forms with distinct structural features. The most abundant form is characterized by the swapping of N-terminal fragments. In this paper, the crystal structure of the complex between the swapping dimer and uridylyl(2',5')adenosine is reported at 2.06 A resolution. The refined model has a crystallographic R-factor of 0.184 and good stereochemistry. The quality of the electron density maps enables the structure of both the inhibitor and active site residues to be unambiguously determined. The overall architecture of the active site is similar to that of RNase A. The dinucleotide adopts an extended conformation with the pyrimidine and purine base interacting with Thr45 and Asn71, respectively. Several residues (Gln11, His12, Lys41, His119, and Phe120) bind the oxygens of the phosphate group. The structural similarity of the active sites of BS-RNase and RNase A includes some specific water molecules believed to be relevant to catalytic activity. Upon binding of the dinucleotide, small but significant modifications of the tertiary and quaternary structure of the protein are observed. The ensuing correlation of these modifications with the catalytic activity of the enzyme is discussed.

Two complexes of bovine seminal ribonuclease with dinucleotides, uridylyl(2′-5′)adenosine (UpA) and 2′-deoxycytidylyl(3′-5′)-2′-deoxyadenosine (d(CpA)), were crystallized under unusual conditions involving a liquid-liquid phase separation. This phenomenon was induced by adding small aliquots of organic cosolutes to highly concentrated ammonium sulfate solutions. The liquid-liquid interface acts as a source of nucleation centers for growth of large crystals. Among the cosolutes tested in these salt-mediated crystallizations, 2-methyl-2,4-pentanediol was found to be the most efficient.

The cyclization of Ac–Gly–Asn–Gly–Gly–NHMe to the succinimide derivative has been studied in the pH range 5.5–10.4 at 37° and µ= 1 mol dm–3. Kinetic evidence indicates that the reaction is a multistep process with a change in the rate-determining step at pH 6.5–7.0.The suggested mechanism involves the pre-equilibrium deprotonation of the NH group next to the Asn residue, followed by nucleophilic attack of the nitrogen atom on the carbonyl carbon of the Asn side chain giving a cyclic tetrahedral intermediate. At acidic pH the cyclization step is rate determining, whereas, the removal of the leaving group by apparent general-base catalysis is the rate-determining step at neutral and basic pH. The literature data on the deamidation rate are discussed in light of the proposed mechanism.

At equilibrium in acidic solution the succinimide derivative, formed by cyclization reaction of the Asp side chain, predominates; at a pH close to the apparent pKa of the Asp residue this reaction is relatively fast with a t1/2 of a few days.

L-~t-Aspartiumylglycylglycine fl-benzyl methyl ester trifluoroacetate, C16H22N30+.C2F302, M r =465.39, orthorhombic, P2,212 ~, a=5.425 (2), b = 12.159 (1), c= 32.925 (8))k, V= 2171 (1)A 3, Z = 4, D x = 1.42 Mg m -a, /(Cu K~t) = 1.5418 A, Iz(Cu Ka) = 1.1 mm -1, F(000) = 968, room tem- perature, final R -- 0.071 for 1550 observed reflections. The title compound consists of the triftuoroacetate anion and the peptide with the amino terminus protonated. The conformation of the peptide linkages is trans and the main-chain conformation of the tripeptide is not fully extended. The packing is determined by extensive hydrogen bonding with every available donor atom participating. Introduction. Peptides containing a t-blocked aspartyl residue easily cyclize forming a substituted succinimide (Bodansky & Kwei, 1978). From a structural point of view the formation of this cycle requires that the dihedral angle ~, of the aspartyl residue (N~Ci-- C/----Ni+x) assumes a value close to -120 °, which falls in a disallowed region of the Ramachandran map for an L residue. It has been suggested that the deamidation of an asparaginyl side chain in proteins may occur through the formation of the cyclic imide as intermedi- ate (Aswad, 1984). As a part of our ongoing research into structural properties of t-blocked aspartyl peptides and aminosuccinyl peptides (Capasso, Mattia, Maz- zarella & Zagari, 1984a,b; Capasso, Mazzarella, Sica & Zagari, 1984), we have synthesized and studied, by X-ray analysis, the tripeptide L-Asp(fl-Bzl)-Gly-Gly- OMe.Tfa.

C21H21N3O5, Mr = 395.42, orthorhombic, P2(1)2(1)2(1), a = 5.935 (2), b = 13.037 (1), c = 25.992 (2) A, V = 2011 (1) A3, Z = 4, D chi = 1.31 Mg m-3, lambda(Cu K alpha) = 1.5418 A, mu = 0.74 mm-1, F(000) = 832, room temperature, final R = 0.046 for 1343 observed reflections. The title compound adopts a type II' beta-turn conformation in the solid state, stabilized by a 4----1 intramolecular hydrogen bond between the CO of the protecting benzyloxycarbonyl group and the NH2 of the terminal amide group. These results indicate that the beta-turn conformation, already found in the solid state for peptides embodying the sequence Asu-Gly or Asu-Ala, is highly preferred, even when the residue next to the aminosuccinyl moiety has a bulkier side chain. In the crystal, rows of hydrogen-bonded molecules are held together by van der Waals forces between hydrophobic phenyl groups.

Circular dichroism spectra of di-, tri-, and tetrapeptides containing an aminosuccinyl residue (Asu) have been studied in a variety of solvents covering a wide range of polarity and hydrogen bonding ability. The CD spectral features in the longer wavelength region are determined by the chiral contribution of the Asu moiety and can be used to study the conformational preferences of these compounds in solution. In Asu tripeptides the positive band near 260 nm, which appears in non proton acceptor solvents, is correlated to an intramolecularly hydrogen bonded type II′β-bend conformation, which previous studies have shown to be the preferred conformation in these solvents. This band can be ascribed to the asymmetrical perturbation of the succinimide chromophore induced by the carbonyl group which precedes the imide ring and is fixed at a short distance from the plane of the ring by the intramolecular hydrogen bond. The presence of this band in the CD spectra of Asu tetrapeptides indicates that these compounds also assume, in non polar solvents, the same β-bend conformation. This interpretation is confirmed by i.r. and tH-n.m.r. spectra, which give evidence of the presence in non polar solvents of an intramolecular hydrogen bond between the carbonyl group of the first residue and the NH group of the fourth.