Universal two-step crystallization of DNA-functionalized nanoparticles

We examine the crystallization dynamics of nanoparticles reversibly tethered by DNA hybridization. We show that the crystallization happens readily only in a narrow temperature "slot," and always proceeds via a two-step process, mediated by a highly-connected amorphous intermediate. For lower temperature quenches, the dynamics of unzipping strands in the amorphous state is sufficiently slow that crystallization is kinetically hindered. This accounts for the well-documented difficulty of forming crystals in these systems. The strong parallel to the crystallization behavior of proteins and colloids suggests that these disparate systems crystallize in an apparently universal manner.Comment: Accepted for publication in Soft Matte

Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches

Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (KD). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions.

Crystallization and preliminary crystallographic analysis of the DNA gyrase B protein from B-stearothermophilus

DNA gyrase B (GyrB) from B. stearothermophilus has been crystallized in the presence of the non-hydrolyzable ATP analogue, 5'-adenylpl-beta-gamma-imidodiphosphate (ADPNP), by the dialysis method. A complete native data set to 3.7 Angstrom has been collected from crystals which belonged to the cubic space group I23 with unit-cell dimension a = 250.6 Angstrom. Self-rotation function analysis indicates the position of a molecular twofold axis. Low-resolution data sets of a thimerosal and a selenomethionine derivative have also been analysed. The heavy-atom positions are consistent with one dimer in the asymmetric unit

Crystallization of a Mos1 transposase-inverted-repeat DNA complex: biochemical and preliminary crystallographic analyses

A complex formed between Mos1 transposase and its inverted-repeat DNA has been crystallized. The crystals diffract to 3.25 Å resolution and exhibit monoclinic (P2(1)) symmetry, with unit-cell parameters a = 120.8, b = 85.1, c = 131.6 Å, β = 99.3°. The X-ray diffraction data display noncrystallographic twofold symmetry and characteristic dsDNA diffraction at ∼3.3 Å. Biochemical analyses confirmed the presence of DNA and full-length protein in the crystals. The relationship between the axis of noncrystallographic symmetry, the unit-cell axes and the DNA diffraction pattern are discussed. The data are consistent with the previously proposed model of the paired-ends complex containing a dimer of the transposase

Crystallization of the regulatory and effector domains of the key sporulation response regulator Spo0A

The key response-regulator gene of sporulation, spo0A, has been cloned from Bacillus stearothermophilus and the encoded protein purified. The DNA-binding and phospho-acceptor domains of Spo0A have been prepared by tryptic digestion of the intact protein and subsequently crystallized in forms suitable for X-ray crystallographic studies. The DNA-binding domain has been crystallized in two forms, one of which diffracts X-rays to beyond 2.5 Angstrom spacing. The crystals of the phospho-acceptor domain diffract X-rays beyond 2.0 Angstrom spacing using synchrotron radiation

The conserved XPF:ERCC1-like Zip2:Spo16 complex controls meiotic crossover formation through structure-specific DNA binding.

In eukaryotic meiosis, generation of haploid gametes depends on the formation of inter-homolog crossovers, which enable the pairing, physical linkage, and eventual segregation of homologs in the meiosis I division. A class of conserved meiosis-specific proteins, collectively termed ZMMs, are required for formation and spatial control of crossovers throughout eukaryotes. Here, we show that three Saccharomyces cerevisiae ZMM proteins-Zip2, Zip4 and Spo16-interact with one another and form a DNA-binding complex critical for crossover formation and control. We determined the crystal structure of a Zip2:Spo16 subcomplex, revealing a heterodimer structurally related to the XPF:ERCC1 endonuclease complex. Zip2:Spo16 lacks an endonuclease active site, but binds specific DNA structures found in early meiotic recombination intermediates. Mutations in multiple DNA-binding surfaces on Zip2:Spo16 severely compromise DNA binding, supporting a model in which the complex's central and HhH domains cooperate to bind DNA. Overall, our data support a model in which the Zip2:Zip4:Spo16 complex binds and stabilizes early meiotic recombination intermediates, then coordinates additional factors to promote crossover formation and license downstream events including synaptonemal complex assembly

Colloids with key-lock interactions: non-exponential relaxation, aging and anomalous diffusion

The dynamics of particles interacting by key-lock binding of attached biomolecules are studied theoretically. Experimental realizations of such systems include colloids grafted with complementary single-stranded DNA (ssDNA), and particles grafted with antibodies to cell-membrane proteins. Depending on the coverage of the functional groups, we predict two distinct regimes. In the low coverage localized regime, there is an exponential distribution of departure times. As the coverage is increased the system enters a diffusive regime resulting from the interplay of particle desorption and diffusion. This interplay leads to much longer bound state lifetimes, a phenomenon qualitatively similar to aging in glassy systems. The diffusion behavior is analogous to dispersive transport in disordered semiconductors: depending on the interaction parameters it may range from a finite renormalization of the diffusion coefficient to anomalous, subdiffusive behavior. We make connections to recent experiments and discuss the implications for future studies.Comment: v2: substantially revised version, new treatment of localized regime, 19 pages, 10 figure

Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants

The crystallization of three catalytically inactive mutants of penicillin Vacylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide threedimensional structures of precursor PVA forms, plus open a route to the study of enzyme-substrate complexes for this industrially important enzyme

Cloning and expression of the Propionibacterium shermanii methylmalonyl-CoA epimerase gene in Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Genomic DNA was isolated from Propionibacterium shermanii (52W). A 454 bp DNA fragment coding for the methylmalonyl-CoA epimerase (EC 5.1.99.1, subsequently referred to as epimerase) was amplified from genomic DNA by the polymerase chain reaction using primers designed from the known DNA sequence of the gene. The P. shermanii epimerase gene was ligated into the 2.47 kbp expression vector pT7-7. The ligation reaction mixture was transformed into electroporation competent E.coli XL1-Blue cells. Plasmid DNA prepared from several transformants was analysed, by agarose gel electrophoresis of restriction enzyme digestions, and transformed into E.coli SRP84/pGP1-2 cells to identify potential epimerase expression constructs (pTEEX) by heat shock induction. The insert DNA of one of the putative pTEEX epimerase constructs was fully sequenced and shown to be identical to the known DNA sequence of the epimerase gene described by Davis (1987). Using the sequenced expression construct pTEEX, recombinant epimerase was expressed to 20-35% of the total cell protein in the protease deficient E.coli strain SRP84 using the dual plasmid expression system of Tabor and Richardson (1985). The recombinant epimerase was ~95-100% soluble in E.coli. The recombinant epimerase and the 'wild-type' epimerase produced by P. shermanii were purified using the procedures developed for the 'wild-type' epimerase. The addition of a heat-treatment step (70°C for 15 min) early in the purification of the recombinant enzyme successfully exploited the unusually high thermostability of the epimerase protein. The epimerase protein was found to have an anomalously low electrophoretic mobility in a modified Laemmli discontinuous Tris-glycine alkaline buffer system for SDS-PAGE gels compared to the Weber and Osborn continuous phosphate buffer system. Using the latter system, a subunit molecular weight of 16.6 kDa was obtained. This is consistent with the molecular weight of 16.72 kDa (methionine on) calculated from the inferred amino acid sequence. The N-terminal sequence of the purified 'wild-type' and recombinant epimerases were identical although only half of N-terminal methionine residues were removed from the recombinant protein. The subunit molecular weight, specific activity, activation by divalent metal ions and behaviour in crystallization trials of the 'wild-type' and recombinant epimerases were very similar. Recombinant epimerase crystals were grown in a buffer containing 0.2 M ammonium acetate and 0.1 M citrate, pH 5.6, containing 30% PEG 4000 as precipitant. These crystals were relatively poorly ordered and diffracted to only 4.5 Ǻ resolution, but crystals of the recombinant epimerase that diffract to 2.6Ǻ can be grown under appropriate conditions