Random Coil In Solution Research Articles

Imaging and tracking of single hyaluronan molecules diffusing in solution

Hyaluronan is an important soluble component of the extracellular matrix of many tissues with well known space-filling, lubricating and signaling functions. As such, hyaluronan can regulate cell adhesion, migration, differentiation and proliferation. Ultrastructural studies showed the existence of fibers and networks of hyaluronan molecules at surfaces, while bulk studies of hyaluronan in solution indicated that the polymer forms random coils. Here, we show that single hyaluronan molecules can be visualized and tracked in three-dimensional samples at room temperature in aqueous buffer. Using a wide-field fluorescence microscope equipped with laser excitation and an sensitive and fast EMCCD camera for fluorescence detection, single FITC-labeled hyaluronan molecules from rooster comb were detected in aqueous solutions. Freely moving hyaluronan-FITC could be tracked over up to 20 images acquired at a frame rate of 98 Hz. Analysis of the trajectories revealed Brownian motion of hyaluronan in tris-buffered saline with an average diffusion coefficient D=3.0+/-0.2 microm(2)/s. These observations confirm the concept that hyaluronan molecules form random coils in solution. The possibility of following the tracks of single hyaluronan molecules in solution facilitates the analysis of processes that lead to the formation of more organized forms of hyaluronan and its interactions with cells with very high spatial and temporal accuracy.

Spectroscopic and Functional Characterization of Human β-Synuclein

To elucidate potential biological roles of human beta-synuclein, we studied its conformational changes under various conditions. The structural and functional properties of beta-synuclein were characterized using biochemical and bio-physical methods including: a functional assay, mass spectrometry, size exclusion chromatography, circular dichroism (CD), and fluorescence spectroscopy. The results showed beta-synuclein has a high proportion of random coil in solution.

Muc5b and Muc5ac are the major oligomeric mucins in equine airway mucus

Horses frequently suffer from respiratory diseases, which, irrespective of etiology, are often associated with airway mucus accumulation. Studies on human airways have shown that the key structural components of the mucus layer are oligomeric mucins, which can undergo changes of expression and properties in disease. However, there is little information on these gel-forming glycoproteins in horse airways mucus. Therefore, the aims of this study were to isolate equine airways oligomeric mucins, characterize their macromolecular properties, and identify their gene products. To this end, pooled tracheal washes, collected from healthy horses and horses suffering from respiratory diseases, were solubilized with 6 M guanidinium chloride (GdmCl). The oligomeric mucins were purified by density gradient centrifugation followed by size exclusion chromatography. Biochemical and biophysical analyses showed the mucins were stiffened random coils in solution that were polydisperse in size (M(r) = 6-20 MDa, average M(r) = 14 MDa) and comprised of disulfide-linked subunits (average M(r) = 7 MDa). Agarose gel electrophoresis showed that the pooled mucus sample contained at least two populations of oligomeric mucins. Electrospray ionization tandem mass spectrometry of tryptic digests of the unfractionated mucin preparation showed that the oligomeric mucins Muc5b and Muc5ac were present. In summary, we have shown that equine airways mucus is a mixture of Muc5b and Muc5ac mucins that have a similar macromolecular organization to their human counterparts. This study will form the basis for future studies to analyze the contribution of these two mucins to equine airways pathology associated with mucus accumulation.

Structure and properties of regenerated Antheraea pernyi silk fibroin in aqueous solution

Antheraea pernyi silk fibroin fibers were dissolved by aqueous lithium thiocyanate to obtain regenerated A. pernyi silk fibroin solution. By means of circular dichroism, 13C NMR and Raman spectroscopy, the molecular conformation of regenerated A. pernyi silk fibroin in aqueous solution was investigated. The relationship of environmental factors and sol–gel transformation behavior of regenerated A. pernyi silk fibroin was also studied. The molecular conformations of regenerated A. pernyi silk fibroin mainly were α-helix and random coil in solution. There also existed a little β-sheet conformation. It was obviously different with Bombyx mori silk fibroin, whose molecular conformation in solution was only random coil but no α-helix existence. With the increase of temperature and solution concentration and with the decrease of solution pH value, the gelation velocity of regenerated A. pernyi silk fibroin solution increased. Especially, it showed that A. pernyi silk fibroin was more sensitive to temperature than B. mori silk fibroin during the sol–gel transformation. The velocity increased obviously when the temperature was above 30 °C. During the sol–gel transformation, the molecular conformation of regenerated A. pernyi silk fibroin changed from random coil to β-sheet structure. The results of these studies provided important insight into the preparation of new biomaterials by silk fibroin protein.

Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin

A 15-residue peptide dimer G15 derived from the cell lytic protein granulysin has been shown to exert potent activity against microbes, including E. coli, but not against human Jurkat cells [Z. Wang, E. Choice, A. Kaspar, D. Hanson, S. Okada, S.C. Lyu, A.M. Krensky, C. Clayberger, Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin. J. Immunol. 165 (2000) 1486–1490]. We investigated the target membrane selectivity of G15 using fluorescence, circular dichroism and 31P NMR methods. The ANS uptake assay shows that the extent of E. coli outer membrane disruption depends on G15 concentration. 31P NMR spectra obtained from E. coli total lipid bilayers incorporated with G15 show disruption of lipid bilayers. Fluorescence binding studies on the interaction of G15 with synthetic liposomes formed of E. coli lipids suggest a tight binding of the peptide at the membrane interface. The peptide also binds to negatively charged POPC/POPG (3:1) lipid vesicles but fails to insert deep into the membrane interior. These results are supported by the peptide-induced changes in the measured isotropic chemical shift and T1 values of POPG in 3:1 POPC:POPG multilamellar vesicles while neither a non-lamellar phase nor a fragmentation of bilayers was observed from NMR studies. The circular dichroism studies reveal that the peptide exists as a random coil in solution but folds into a less ordered conformation upon binding to POPC/POPG (3:1) vesicles. However, G15 does not bind to lipid vesicles made of POPC/POPG/Chl (9:1:1) mixture, mimicking tumor cell membrane. These results explain the susceptibility of E. coli and the resistance of human Jurkat cells to G15, and may have implications in designing membrane-selective therapeutic agents.

The Chaoulli challenge: getting a grip on waiting lists.

<h3>Abstract</h3> Functional amyloids are present in a wide variety of organisms ranging from bacteria to humans. Experience-dependent aggregation of the cytoplasmic polyadenylation element-binding (CPEB) prion-like protein to a translationally active state has emerged as a plausible biochemical substrate of long-lasting memories. CPEB aggregation is driven by prion-like domains (PLD) that are highly divergent in sequence across species. Here, we describe the amyloid-like features of the neuronal <i>Aplysia</i> CPEB (<i>Ap</i>CPEB) PLD <i>in vitro</i> using single-molecule and bulk biophysical methods and compare them with those previously reported for neuronal <i>Drosophila</i> CPEB, Orb2 PLD. The existence of transient oligomers and mature filaments suggests similarities in the late stages of the assembly pathway for both PLDs. However, while prior to aggregation the Orb2 PLD monomer remains as a random coil in solution, <i>Ap</i>CPEB PLD adopts a diversity of conformations comprising <i>α</i>-helical structures that evolve to coiled-coil species, suggesting structural differences at the beginning of their amyloid assembly pathways. Our results show how divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.

Laccase-catalyzed oxidation of naphthol in the presence of soluble polymers

The oxidation of 1-naphthol and 2-naphthol was studied in presence of recombinant laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) from Polyporus pinsitus (rPpL), Myceliophthora thermophila (rMtL), Coprinus cinereus (rCcL) and Rhizoctonia solani (rRsL). During 1-naphthol oxidation a violet colored insoluble product was formed. The product of 2-naphthol oxidation was a white insoluble precipitate. Almost two moles of 1-naphthol was oxidized by one mole of oxygen. Kinetic measurements of oxygen consumption showed that laccases have been inactivated during naphthols oxidation. Water soluble polymers and albumins changed oxidation profile. The polymers investigated were divided into three groups. The first group includes polymers which increase total oxygen consumption without changing an initial rate: polyvinyl alcohol, ficoll, a copolymer of acrylamide, N-vinylpyrrolidone and ethyl acrylate, hydroxyethyl cellulose, dextrans 500, 110 and 20, polyvinylpyrrolidone, polyethylene glycol and albumins. These polymers intermediately bound hydrophobic naphtholic radicals preventing inactivation of laccases. Cationic polymers such as diethylaminoethyl-dextran, poly- l-lysine and protasan that inhibited the initial oxidation rate, but did not change total oxygen consumption, were attributed to the second group. The inhibition was associated with complexation of positively charged polymers with negatively charged laccases since isoelectric points of rPpL and of rMtL were 3.5 and 4.2, respectively. The polymers such as alginic acid, polyacrylic acid and heparin were attributed to the third group. These polymers carried negative charges and formed random coil in solution due to electrostatic repulsion. They did not interact with laccases and did not combine intermediates. Therefore, they practically did not change total oxygen consumption and initial rate of naphthol oxidation.

NMR and molecular modeling of complex carbohydrates and carbohydrate-protein interactions. Applications to anti-bacteria vaccines.

In order to characterize the conformational epitope of the group B meningococcal polysaccharide and of the type III group B Streptococcus capsular polysaccharide NMR measurements were done on a wide variety of native and modified polysaccharides and oligosaccharides. Since these saccharides are highly mobile and exist as random coils in solution, the analysis of the NMR data and molecular modeling was done to take into account this inherent flexibility. The conformational model of extended high-order helices being selected upon binding to a protein, although still hypothetical at this stage, has proven useful in explaining the serology for the conformational epitopes for polysaccharides of group B Neisseria meningitidis, group B Streptococcus type III and Streptococcus pneumoniae type 14.

Synthesis and preliminary characterisation of charged derivatives and hydrogels from scleroglucan

The synthesis of negatively and positively charged polyelectrolytes from scleroglucan is described. Polycarboxylates were synthesised through nucleophilic substitution with chloroacetic acid or through a selective 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO)-mediated oxidation of the primary alcohol groups. Amine groups were introduced through nucleophilic substitution with 2-chloroethylamine or 3-chloropropylamine. Reaction conditions were varied to obtain insight into the influence of variables on the degree of substitution. The conformational behaviour of the obtained polyelectrolytes was studied as a function of pH, temperature and solvent. For the products with a low degree of modification, evidence of an ordered conformation was found, whereas the polymers with a higher degree of modification behaved as random coils in solution. The negatively charged polymers were reticulated using the Ugi four-component condensation, obtaining negatively charged hydrogels. The positively charged polymers were reticulated using diethyl squarate (3,4-diethoxy-3-cyclobutene-1,2-dion, DES) to obtain positively charged hydrogels.

Membrane binding and pore formation of the antibacterial peptide PGLa: thermodynamic and mechanistic aspects.

The antibacterial peptide PGLa exerts its activity by permeabilizing bacterial membranes whereas eukaryotic membranes are not affected. To provide insight into the selectivity and the permeabilization mechanism, the binding of PGLa to neutral and negatively charged model membranes was studied with high-sensitivity isothermal titration calorimetry (ITC), circular dichroism (CD), and solid-state deuterium nuclear magnetic resonance ((2)H NMR). The binding of PGLa to negatively charged phosphatidylcholine (PC)/phosphatidylglycerol (PG) (3:1) vesicles was by a factor of approximately 50 larger than that to neutral PC vesicles. The negatively charged membrane accumulates the cationic peptide at the lipid-water interface, thus facilitating the binding to the membrane. However, if bulk concentrations are replaced by surface concentrations, very similar binding constants are obtained for neutral and charged membranes (K approximately 800-1500 M(-)(1)). Membrane selectivity is thus caused almost exclusively by electrostatic attraction to the membrane surface and not by hydrophobic insertion. Membrane insertion is driven by an exothermic enthalpy (DeltaH approximately -11 to -15 kcal/mol) but opposed by entropy. An important contribution to the binding process is the membrane-induced random coil --> alpha-helix transition of PGLa. The peptide is random coil in solution but adopts an approximately 80% alpha-helical conformation when bound to the membrane. Helix formation is an exothermic process, contributing approximately 70% to the binding enthalpy and approximately 30% to the free energy of binding. The (2)H NMR measurements with selectively deuterated lipids revealed small structural changes in the lipid headgroups and in the hydrocarbon interior upon peptide binding which were continuous over the whole concentration range. In contrast, isothermal titration calorimetry of PGLa solutions with PC/PG(3:1) vesicles gave rise to two processes: (i) an exothermic binding of PGLa to the membrane followed by (ii) a slower endothermic process. The latter is only detected at peptide-to-lipid ratios >17 mmol/mol and is paralleled by the induction of membrane leakiness. Dye efflux measurements are consistent with the critical limit derived from ITC measurements. The endothermic process is assigned to peptide pore formation and/or lipid perturbation. The enthalpy of pore formation is 9.7 kcal/mol of peptide. If the same excess enthalpy is assigned to the lipid phase, the lipid perturbation enthalpy is 180 cal/mol of lipid. The functional synergism between PGLa and magainin 2 amide could also be followed by ITC and dye release experiments and is traced back to an enhanced pore formation activity of a peptide mixture.

Thermodynamics of the α-helix-coil transition of amphipathic peptides in a membrane environment: implications for the peptide-membrane binding equilibrium

Amphipathic α-helices are the membrane binding motif in many proteins. The corresponding peptides are often random coil in solution but are folded into an α-helix upon interaction with the membrane. The energetics of this ubiquitous folding process are still a matter of conjecture. Here, we present a new method to quantitatively analyze the thermodynamics of peptide folding at the membrane interface. We have systematically varied the helix content of a given amphipathic peptide when bound to the membrane and have correlated the thermodynamic binding parameters determined by isothermal titration calorimetry with the α-helix content obtained by circular dichroism spectroscopy. The peptides investigated were the antibiotic magainin 2 amide and three analogs in which two adjacent amino acid residues were substituted by their d-enantiomers. The thermodynamic parameters controlling the α-helix formation were found to be linearly related to the helicity of the membrane-bound peptides. Helix formation at the membrane surface is characterized by an enthalpy change of ΔHhelix≈−0.7 kcal/mol per residue, an entropy change of ΔShelix≈−1.9 cal/molK residue and a free energy change of ΔGhelix=−0.14 kcal/mol residue. Helix formation is a strong driving force of peptide insertion into the membrane and accounts for about 50 % of the free energy of binding. An increase in temperature entails an unfolding of the membrane-bound helix. The temperature dependence can be described with the Zimm-Bragg theory and the enthalpy of unfolding agrees with that deduced from isothermal titration calorimetry.

Electrostatic and hydrophobic contributions to the folding mechanism of apocytochrome c driven by the interaction with lipid.

In aqueous solution, while cytochrome c is a stably folded protein with a tightly packed structure at the secondary and tertiary levels, its heme-free precursor, apocytochrome c, shows all features of a structureless random coil. However, upon interaction with phospholipid vesicles or lysophospholipid micelles, apocytochrome c undergoes a conformational transition from its random coil in solution to an alpha-helical structure on association with lipid. The driving forces of this lipid-induced folding process of apocytochrome c were investigated for the interaction with various phospholipids and lysophospholipids. Binding of apocytochrome c to negatively charged phospholipid vesicles induced a partially folded state with approximately 85% of the alpha-helical structure of cytochrome c in solution. In contrast, in the presence of zwitterionic phospholipid vesicles, apocytochrome c remains a random coil, suggesting that negatively charged phospholipid headgroups play an important role in the mechanism of lipid-induced folding of apocytochrome c. However, negatively charged lysophospholipid micelles induce a higher content of alpha-helical structure than equivalent negatively charged diacylphospholipids in bilayers, reaching 100% of the alpha-helix content of cytochrome c in solution. Furthermore, micelles of lysolipids with the same zwitterionic headgroup of phospholipid bilayer vesicles induce approximately 60% of the alpha-helix content of cytochrome c in solution. On the basis of these results, we propose a mechanism for the folding of apocytochrome c induced by the interaction with lipid, which accounts for both electrostatic and hydrophobic contributions. Electrostatic lipid-protein interactions appear to direct the polypeptide to the micelle or vesicle surface and to induce an early partially folded state on the membrane surface. Hydrophobic interactions between nonpolar residues in the protein and the hydrophobic core of the lipid bilayer stabilize and extend the secondary structure upon membrane insertion.

Structural Determinants Present in the C-terminal Binding Protein Binding Site of Adenovirus Early Region 1A Proteins

The C-terminal binding protein (CtBP) has previously been shown to bind to a highly conserved six-amino acid motif very close to the C terminus of adenovirus early region 1A (Ad E1A) proteins. We have developed an enzyme-linked immunosorbent assay that has facilitated the screening of synthetic peptides identical or similar to the binding site on Ad E1A for their ability to bind CtBP and thus inhibit its interaction with Ad12 E1A. It has been shown that amino acids both C-terminal and N-terminal to the original proposed binding site contribute to the interaction of peptides with CtBP. Single amino acid substitutions across the binding site appreciably alter the Kd of the peptide for CtBP, indicative of a marked reduction in the affinity of the peptide for CtBP. The solution structures of synthetic peptides equivalent to the C termini of both Ad5 and Ad12 E1A and two substituted forms of these have been determined by proton NMR spectroscopy. Both the Ad12 and Ad5 peptides dissolved in trifluoroethanol/water mixtures were found to adopt regular secondary structural conformations seen as a series of beta-turns. An Ad12 peptide bearing a substitution that resulted in only very weak binding to CtBP (Ad12 L258G) was found to be random coil in solution. However, a second mutant (Ad12 V256K), which bound to CtBP rather more strongly (although not as well as the wild type), adopted a conformation similar to that of the wild type. We conclude that secondary structure (beta-turns) and an appropriate series of amino acid side chains are necessary for recognition by CtBP.

Solution conformation of an immunogenic peptide from HRV2: comparison with the conformation found in a complex with a Fab fragment of an anti-HRV2 neutralizing antibody.

The conformation of a [15]-peptide (H-VKAETRLNPDLQPTE-NH2) from VP2 of rhinovirus HRV2 complexed with a Fab fragment was previously shown by X-ray crystallographic studies to be similar to the one found in the corresponding region of HRV1A. Antibodies raised against this peptide bind to and neutralize HRV2. In order to identify structural features preserved in solution that may explain the ability of this short peptide to mimic the structure of the protein surface, the peptide has been studied by NMR in aqueous solution as well as under denaturing conditions. The peptide is shown to be a random coil in solution. However, the sequence forming a 3(10)helix in the complex is biased into a helical conformation according to NOE intensity data as well as from urea and pH titrations. This sequence adopts the same conformation in an unrelated protein. NOE data suggest that a beta-turn found in the complex may be sampled in solution. Also, Glu4, interacting with Arg6 in the crystal, has a reduced pKa value in solution. It is concluded that the local structure present in the random coil state of VP2(156-170) contains enough information to direct the production of antibodies that bind to and neutralize HRV2.

Mucins in airway secretions from healthy and chronic bronchitic subjects

Little is known about whether the properties of respiratory mucins are altered as a result of airway irritation, but histochemical studies of respiratory tract secretory cells show a more 'acidic' staining pattern after exposure to tobacco smoke. Furthermore it has been suggested that proteoglycans are the major glycoconjugates in 'normal' respiratory secretions, whereas mucins predominate in sputum. To investigate these observations further, mucins from secretions collected from the tracheal surface of healthy non-smoking 'normal' subjects and sputum from patients with chronic bronchitis were compared. All samples contained one major mucin population after density-gradient centrifugation, and a small amount of 'denser' mucin was present in some chronic bronchitic and one of the 'normal' samples. Proteoglycans were not a major component of 'normal' secretions. The major mucin population from chronic bronchitic samples had molecular masses between 10 and 30 MDa and behaved as random coils in solution. Whole mucins from 'normal' individuals and chronic bronchitic patients were excluded from Sepharose CL-2B, whereas reduced subunits were included. Proteolysis of subunits yielded two populations of high-molecular-mass glycopeptides differing in size, suggesting the presence of two different tandem repeat regions in the mucins. Finally, mucins from patients with chronic bronchitis are less, rather than more, acidic than those from 'normal' individuals. Mucins from bronchitic sputum and 'normal' secretions are thus similar in their macromolecular properties, but differ slightly in charge density.

Iodine-induced uncoiling of polyisoprene random coils in solution

Recently, we demonstrated that conjugated sequences of unsaturated double bonds can be introduced into 1,4-polyisoprene backbone through conjugation reactions induced by iodine [1]. Here, we report a structural investigation on the conjugated polyisoprene chains in solution by means of small angle X-ray scattering (SAXS). The SAXS results indicate a conformational transition from a random coil, characteristic of the pristine polyisoprene chains in solution [2], to a rod-like structure by uncoiling of the macromolecular coils due to a remarkable increase in chain rigidity associated with the formation of rigid conjugated sequences via the I 2-induced conjugation reaction.

Secondary structure of proteins associated in thin films

The solid state secondary structure of myoglobin, RNase A, concanavalin A (Con A), poly(L-lysine), and two linear heterooligomeric peptides were examined by both far-uv CD spectroscopy1 and by ir spectroscopy. The proteins associated from water solution on glass and mica surfaces into noncrystalline, amorphous films, as judged by transmission electron microscopy of carbon-platinum replicas of surface and cross-fractured layer. The association into the solid state induced insignificant changes in the amide CD spectra of all alpha-helical myoglobin, decreased the molar ellipticity of the alpha/beta RNase A, and increased the molar ellipticity of all-beta Con A with no change in the positions of the bands' maxima. High-temperature exposure of the films induced permanent changes in the conformation of all proteins, resulting in less alpha-helix and more beta-sheet structure. The results suggest that the protein alpha-helices are less stable in films and that the secondary structure may rearrange into beta-sheets at high temperature. Two heterooligomeric peptides and poly(L-lysine), all in solution at neutral pH with "random coil" conformation, formed films with variable degrees of their secondary structure in beta-sheets or beta-turns. The result corresponded to the protein-derived Chou-Fasman amino acid propensities, and depended on both temperature and solvent used. The ir and CD spectra correlations of the peptides in the solid state indicate that the CD spectrum of a "random" structure in films differs from random coil in solution. Formic acid treatment transformed the secondary structure of the protein and peptide films into a stable alpha-helix or beta-sheet conformations. The results indicate that the proteins aggregate into a noncrystalline, glass-like state with preserved secondary structure. The solid state secondary structure may undergo further irreversible transformations induced by heat or solvent.

The phase behaviour and gelation of a rod-like polymer in solution and implications for microcellular foam morphology

The phase separation and gelation of the rod-like macromolecule, poly(γ-benzyl- l-glutamate), were studied in an effort to understand the mechanism by which microcellular materials are made via thermally induced phase separation processes. Previous workers have studied similarly prepared materials from molecules which exist as random coils in solution. The microcellular materials were formed by lowering the solution temperature until phase separation and solvent freezing occurred. The solvent was removed by vacuum sublimation. An emphasis was placed on dilute isotropic solutions (<5 wt%) which yield low-density materials or ‘foams’. Both one- (e.g. benzene) and two-component (e.g. dioxane/water) solvent systems were employed. The morphology produced by liquid-liquid phase separation and gelation was an open-celled, fibrous structure, which resembles a three-dimensional lattice. The cell diameters were in the range of 1–10 μm and the fibres which comprise the struts were 0.2 to 2.0 μm thick. Various experimental observations are discussed in terms of the theories and proposed mechanisms of phase separation in solutions of rod-like macromolecules. Calculations of the spinodal for the Flory theory of rod-like particles were also made to assess the possibility of placing the isotropic solutions into an unstable region.

A repeated decapeptide motif in the C-terminal domain of the ribosomal RNA methyltransferase from the erythromycin producer Saccharopolyspora erythraea

Re-analysis of the primary structure of the ribosomal RNA N-methyltransferase that confers self-resistance on the erythromycin-producing bacterium Saccharopolyspora erythraea has confirmed the presence of a C-terminal domain containing extensive repeat sequences. Nine tandem repeats can be discerned, with a decapeptide consensus sequence GGRx(H/R)GDRRT, although no single residue is wholly invariant. This highly polar, potentially flexible domain, which is predicted to adopt either a random coil or a structure with β turns, has a counterpart in the erythromycin methyltransferase of an erythromycin-producing species of Arthrobacter. It also significantly resembles a portion of the C-tenninal region of the eukaryotic protein nucleolin, which is unusually rich in dimethylarginine and glycine, and which is also predicted to behave as a random coil in solution. This resemblance, despite the very different roles of these proteins in ribosome biogenesis, strengthens the idea that in both rRNA methyltransferases and nucleolin these C-tenninal sequences might contribute to rRNA binding.

Crystallization and Preliminary X-ray Diffraction Data of an Anti-angiotensin II Fab and of the Peptide-Fab Complex

mAb-131 is a monoclonal antibody that binds with high affinity (K alpha = 7.4 x 10(9) M-1) to the 8-residue peptide hormone angiotensin II, the major effector of the renin/angiotensin system. mAb-131 is a member of a well characterized idiotypic antibody network since it was raised as an anti-anti-idiotype of an antibody raised against angiotensin II. mAb-131 Fabs prepared with papain contain four major charge isoforms that can be separated by pH gradient elution from an anion-exchange column. Diffraction quality isomorphous crystals of two of the isoforms and of the Fab.peptide complexes have been grown. The crystals diffract to 3.5 A resolution, are tetragonal, space group P4(1) (or P4(3] with cell dimensions a = b = 78.6 A, c = 125.2 A, and have two Fab molecules per asymmetric unit. By using a different buffer, a second crystal form has been grown which diffracts to 3.3 A. It also belongs to space group P4(1) (or P4(3] but has cell dimensions of a = b = 109.6 A and c = 125.2 A. Knowledge of the three-dimensional structure of this Fab and of the peptide.Fab complex will give insight into two problems: 1) the recognition of small peptide hormones (which exist as random coils in solution) with high affinity by proteins, and 2) the nature of conservation of antibody combining sites in idiotypic networks.