Biomedical subjects

Marcelo Nöllmann

Publications and source records attributed to Marcelo Nöllmann.

10 recordsLinked to original sources

PubMed2006

Identification of the FtsK sequence-recognition domain.

FtsK is a prokaryotic multidomain DNA translocase that coordinates chromosome segregation and cell division. FtsK is membrane anchored at the division septum and, guided by highly skewed DNA sequences, translocates the chromosome to bring the terminus of replication to the septum. Here, we use in vitro single-molecule and ensemble methods to unveil a mechanism of action in which the translocation and sequence-recognition activities are performed by different domains in FtsK.

Base Sequence↗

PubMed2006

DNA overwinds when stretched.

DNA is often modelled as an isotropic rod, but its chiral structure suggests the possible importance of anisotropic mechanical properties, including coupling between twisting and stretching degrees of freedom. Simple physical intuition predicts that DNA should unwind under tension, as it is pulled towards a denatured structure. We used rotor bead tracking to directly measure twist-stretch coupling in single DNA molecules. Here we show that for small distortions, contrary to intuition, DNA overwinds under tension, reaching a maximum twist at a tension of approximately 30 pN. As tension is increased above this critical value, the DNA begins to unwind. The observed twist-stretch coupling predicts that DNA should also lengthen when overwound under constant tension, an effect that we quantitatively confirm. We present a simple model that explains these unusual mechanical properties, and also suggests a possible origin for the anomalously large torsional rigidity of DNA. Our results have implications for the action of DNA-binding proteins that must stretch and twist DNA to compensate for variability in the lengths of their binding sites. The requisite coupled DNA distortions are favoured by the intrinsic mechanical properties of the double helix reported here.

Biomechanical Phenomena↗

PubMed2006

Mechanochemical analysis of DNA gyrase using rotor bead tracking.

DNA gyrase is a molecular machine that uses the energy of ATP hydrolysis to introduce essential negative supercoils into DNA. The directionality of supercoiling is ensured by chiral wrapping of the DNA around a specialized domain of the enzyme before strand passage. Here we observe the activity of gyrase in real time by tracking the rotation of a submicrometre bead attached to the side of a stretched DNA molecule. In the presence of gyrase and ATP, we observe bursts of rotation corresponding to the processive, stepwise introduction of negative supercoils in strict multiples of two. Changes in DNA tension have no detectable effect on supercoiling velocity, but the enzyme becomes markedly less processive as tension is increased over a range of only a few tenths of piconewtons. This behaviour is quantitatively explained by a simple mechanochemical model in which processivity depends on a kinetic competition between dissociation and rapid, tension-sensitive DNA wrapping. In a high-resolution variant of our assay, we directly detect rotational pauses corresponding to two kinetic substeps: an ATP-independent step at the end of the reaction cycle, and an ATP-binding step in the middle of the cycle, subsequent to DNA wrapping.

Adenosine Triphosphate↗

PubMed2006

Giant proteins that move DNA: bullies of the genomic playground.

As genetic material DNA is wonderful, but as a macromolecule it is unruly, voluminous and fragile. Without the action of DNA replicases, topoisomerases, helicases, translocases and recombinases, the genome would collapse into a topologically entangled random coil that would be useless to the cell. We discuss the organization, movement and energetics of these proteins that are crucial to the preservation of a molecule that has such beautiful biological but challenging physical properties.

DNA↗

PubMed2005

Behavior of Tn3 resolvase in solution and its interaction with res.

The solution properties of Tn3 resolvase (Tn3R) were studied by sedimentation equilibrium, sedimentation velocity analytical ultracentrifugation, and small-angle neutron scattering. Tn3R was found to be in a monomer-dimer self-association equilibrium, with a dissociation constant of K(D)(1-2)=50 microM. Sedimentation velocity and small-angle neutron scattering data are consistent with a solution structure of dimeric Tn3R similar to that of gammadelta resolvase in a co-crystal structure, but with the DNA-binding domains in a more extended conformation. The solution conformations of sites I, II, and III were studied with small angle x-ray scattering and modeled using rigid-body and ab initio techniques. The structures of these sites do not show any distortion, at low resolution, from B-DNA. The equilibrium binding properties of Tn3R to the individual binding sites in res were investigated by employing fluorescence anisotropy measurements. It was found that site II and site III have the highest affinity for Tn3R, followed by site I. Finally, the affinity of Tn3R for nonspecific DNA was assayed by competition experiments.

Anisotropy↗

PubMed2005

SOMO (SOlution MOdeler) differences between X-Ray- and NMR-derived bead models suggest a role for side chain flexibility in protein hydrodynamics.

Reduced numbers of frictional/scattering centers are essential for tractable hydrodynamic and small-angle scattering data modeling. We present a method for generating medium-resolution models from the atomic coordinates of proteins, basically by using two nonoverlapping spheres of differing radii per residue. The computed rigid-body hydrodynamic parameters of BPTI, RNase A, and lysozyme models were compared with a large database of critically assessed experimental values. Overall, very good results were obtained, but significant discrepancies between X-ray- and NMR-derived models were found. Interestingly, they could be accounted for by properly considering the extent to which highly mobile surface side chains differently affect translational/rotational properties. Models of larger structures, such as fibrinogen fragment D and citrate synthase, also produced consistent results. Foremost among this method's potential applications is the overall conformation and dynamics of modular/multidomain proteins and of supramolecular complexes. The possibility of merging data from high- and low-resolution structures greatly expands its scope.

Algorithms↗

PubMed2004

Solution structure of the Tn3 resolvase-crossover site synaptic complex.

Tn3 resolvase is a site-specific DNA recombinase, which catalyzes strand exchange in a synaptic complex containing twelve resolvase subunits and two res sites. Hyperactive mutants of resolvase can form a simpler complex (X synapse) containing a resolvase tetramer and two shorter DNA segments at which strand exchange takes place (site I). We have solved the low-resolution solution structure of the purified, catalytically competent X synapse from small-angle neutron and X-ray scattering data, using methods in which the data are fitted with models constructed by rigid body transformations of a published crystallographic structure of a resolvase dimer bound to site I. Our analysis reveals that the two site I fragments are on the outside of a resolvase tetramer core and provides some information on the quaternary structure of the tetramer. We discuss implications of our structure for the architecture of the natural synaptic complex and the mechanism of strand exchange.

Bacterial Proteins↗

PubMed2004

Low-resolution reconstruction of a synthetic DNA holliday junction.

We have studied the low-resolution solution conformation of a Holliday (or four-way) DNA junction by using small-angle x-ray scattering, sedimentation velocity, and computational modeling techniques. The scattering data were analyzed in two independent ways: firstly, by rigid-body modeling of the scattering data using previously suggested models for the Holliday junction (HJ), and secondly, by ab initio reconstruction methods. The models found by both methods agree with experimentally determined sedimentation coefficients and are compatible with the results of previous studies using different techniques, but provide a more direct and accurate determination of the solution conformation of the HJ. Our results show that addition of Mg(2+) alters the conformation of the HJ from an extended to a stacked arrangement.

Algorithms↗

PubMed2004

The role of cholesterol in the activity of pneumolysin, a bacterial protein toxin.

The mechanism via which pneumolysin (PLY), a toxin and major virulence factor of the bacterium Streptococcus pneumoniae, binds to its putative receptor, cholesterol, is still poorly understood. We present results from a series of biophysical studies that shed light on the interaction of PLY with cholesterol in solution and in lipid bilayers. PLY lyses cells whose walls contain cholesterol. Using standard hemolytic assays we have demonstrated that the hemolytic activity of PLY is inhibited by cholesterol, partially by ergosterol but not by lanosterol and that the functional stoichiometry of the cholesterol-PLY complex is 1:1. Tryptophan (Trp) fluorescence data recorded during PLY-cholesterol titration studies confirm this ratio, reveal a significant blue shift in the Trp fluorescence peak with increasing cholesterol concentrations indicative of increasing nonpolarity in the Trp environment, consistent with cholesterol binding by the tryptophans, and provide a measure of the affinity of cholesterol binding: K(d) = 400 +/- 100 nM. Finally, we have performed specular neutron reflectivity studies to observe the effect of PLY upon lipid bilayer structure.

Amino Acid Motifs↗

PubMed2004

The solution structure and oligomerization behavior of two bacterial toxins: pneumolysin and perfringolysin O.

Pneumolysin (PLY), an important protein virulence factor of the human bacterial pathogen Streptococcus pneumoniae, could be a candidate for inclusion in a new anti-streptococcal vaccine. PLY solution species from monomer via multimeric intermediates to ring-shaped oligomers were studied with time-dependent sedimentation velocity in the analytical ultracentrifuge (AUC). Hydrodynamic bead modeling was used to interpret the data obtained. PLY remained mostly monomeric in solution; intermediate PLY multimers were detected in small quantities. Current understanding of PLY molecular mechanism is guided by a model built on the basis of its homology with perfringolysin O (PFO) for which there is an atomic structure. PFO, a virulence factor of the organism Clostridium perfringens, has almost the same molecular mass as PLY and shares 48% sequence identity and 60% sequence similarity with PLY. We report a comparative low-resolution structural study of PLY and PFO using AUC and small-angle x-ray scattering (SAXS). AUC data demonstrate that both proteins in solution are mostly monodisperse but PLY is a monomer whereas PFO is mostly dimeric. Ab initio dummy atom and dummy residue models for PFO and PLY were restored from the distance distribution function derived from experimental small-angle x-ray scattering curves. In solution, PLY is elongated, consistent with the shape predicted by its high-resolution homology model. The PFO dimer is also an elongated particle whose shape and volume are consistent with a staggered antiparallel dimer.

Bacterial Proteins↗