Biomedical subjects

Giovanni Zocchi

Publications and source records attributed to Giovanni Zocchi.

13 recordsLinked to original sources

PubMed2006

Guanylate kinase, induced fit, and the allosteric spring probe.

Since the introduction of the induced-fit theory by D. E. Koshland Jr., it has been established that conformational motion invariably accompanies the execution of protein function. The catalytic activity of kinases, specifically, is associated with large conformational changes ( approximately 1 nm amplitude). In the case of guanylate kinase, upon substrate binding, the LID and nucleotide-monophosphate-binding domains are brought together and toward the CORE with large concerted movements about the alpha3 (helix 3) axis. However, whether the change in conformation mostly affects the catalytic rate or mostly increases binding affinities for one or the other substrate is unclear. We investigate this question using a nanotechnology approach based on mechanical stress. Using an "allosteric spring probe", we bias conformational states in favor of the "open" (substrate-free) conformation of the enzyme; the result is that the binding constant for the substrate guanosine monophosphate (GMP) is reduced by up to a factor of 10, whereas the binding constant for adenosine triphosphate (ATP) and the catalytic rate are essentially unaffected. The results show that the GMP-induced conformational change, which promotes catalysis, does not promote ATP binding, consistent with previous mutagenesis studies. Furthermore, they show that this conformational change is of the induced-fit type with respect to GMP binding (but not ATP binding). We elaborate on this point by proposing a quantitative criterion for the classification of conformational changes with respect to the induced-fit theory. More generally, these results show that the allosteric spring probe can be used to affect enzymatic activity in a continuously controlled manner, and also to affect specific steps of the reaction mechanism while leaving others unaffected. It is presumed that this will enable informative comparisons with the results of future molecular dynamics or statistical mechanics computations.

Adenosine Triphosphate↗

PubMed2006

Mimicking cAMP-dependent allosteric control of protein kinase A through mechanical tension.

We report the activation of an enzyme complex by mechanical tension. Protein kinase A, a tetrameric enzyme that, in the cell, is allosterically controlled by cAMP, has been modified by the insertion of a "molecular spring" on the regulatory subunit. The spring is made of DNA, and its stiffness can be varied externally by hybridization to a complementary strand. This allows us to exert a controlled mechanical tension between the two points on the protein's surface where the spring is attached. We show that upon applying the tension, we can activate the enzyme with efficiency comparable to the activation by its natural regulatory molecule, cAMP.

Allosteric Regulation↗

PubMed2006

Mismatches and bubbles in DNA.

Single mismatches in the DNA double helix form nucleation sites for bubbles. Although the overall melting temperature of the duplex is affected to different degrees depending on the probe length, the statistical weights of the bubble states around the defect are always strongly affected. Here we show experimentally that a single mismatch has indeed a dramatic effect on the distribution of intermediate (bubble) states in the melting transition of DNA oligomers. For probe lengths in the range 20-40 bases, the mismatch transforms a transition with many intermediates into a nearly two-state transition. One surprising consequence is the existence of a regime where the sensitivity of a mismatch detection assay based on monitoring intermediate states would increase with probe length. Our results provide experimental constraints on how mismatches should be implemented in models of DNA melting, such as the widely used thermodynamic nearest neighbor model, to which we compare our data.

Base Pair Mismatch↗

PubMed2006

Analytical assays based on detecting conformational changes of single molecules.

One common strategy for the detection of biomolecules is labeling either the target itself or an antibody that binds to it. Herein, a different approach, based on detecting the conformational change of a probe molecule induced by binding of the target is discussed. That is, what is being detected is not the presence of the target or the probe, but the conformational change of the probe. Recently, a single-molecule sensor has been developed that exploits this mechanism to detect hybridization of a single DNA oligomer to a DNA probe, as well as specific binding of a single protein to a DNA probe. Biomolecular recognition often involves large conformational changes of the molecules involved, and therefore this strategy may be applicable to other assays.

DNA Probes↗

PubMed2005

Allosteric control through mechanical tension.

Conformational changes of proteins modulate their function. In allosteric control, the conformational change is induced by the binding of a signaling molecule. Here we insert a "molecular spring" on the enzyme guanylate kinase, to control the conformation of this protein. The stiffness of the spring can be varied externally, which allows one to exert a controlled mechanical tension between the two points on the protein's surface where the spring is attached. We show that by applying and releasing the tension we can reversibly turn the enzyme off and on.

Carrier Proteins↗

PubMed2005

Local cooperativity mechanism in the DNA melting transition.

We propose a statistical mechanics model for the melting transition of DNA. Base pairing and stacking are treated as separate degrees of freedom, and the interplay between pairing and stacking is described by a set of local rules which mimic the geometrical constraints in the real molecule. This microscopic mechanism intrinsically accounts for the cooperativity related to the free energy penalty of bubble nucleation. The model describes both the unpairing and unstacking parts of the spectroscopically determined experimental melting curves. Furthermore, the model explains the observed temperature dependence of the effective thermodynamic parameters used in models of the nearest neighbor type. We compute the partition function for the model through the transfer matrix formalism, which we also generalize to include nonlocal chain entropy terms.

Computer Simulation↗

PubMed2005

Mechanics of binding of a single integration-host-factor protein to DNA.

We report on a single-molecule experiment where we directly observe local bending of a 76 base pair DNA oligomer caused by specific binding of a single integration-host-factor (IHF) protein. The conformational change of the DNA is detected by optically monitoring the displacement of a micron size bead tethered to a surface by the DNA. Since in the bound state the DNA loops around the IHF, a mechanical tension on the DNA tends to eject the protein. We measure how the rate for the protein to fall off the DNA depends on the mechanical tension in the DNA, gaining insight into the energy landscape for this molecular bond. Our method further demonstrates a new paradigm of molecular detection, where ligand binding is detected through the conformational change induced in the probe molecule. Here this allows the detection of single, unlabeled proteins.

Chemical Phenomena↗

PubMed2005

Artificial allosteric control of maltose binding protein.

We demonstrate the allosteric control of a protein based on mechanical tension. When substrate binding is accompanied by a significant change of conformation of the protein, a mechanical tension favoring one or the other conformation will alter the binding affinity for the substrate. We have constructed a chimera where the two lobes of the maltose-binding protein are covalently coupled to the ends of a DNA oligomer. The mechanical tension on the protein is controlled externally by exploiting the difference in stiffness between single stranded and double stranded DNA. We report that the binding affinity of the protein for its substrates is significantly altered by the tension.

Binding Sites↗

PubMed2004

Statistical mechanics of base stacking and pairing in DNA melting.

We propose a statistical mechanics model for DNA melting in which base stacking and pairing are explicitly introduced as distinct degrees of freedom. Unlike previous approaches, this model describes thermal denaturation of DNA secondary structure in the whole experimentally accessible temperature range. Base pairing is described through a zipper model, base stacking through an Ising model. We present experimental data on the unstacking transition, obtained exploiting the observation that at moderately low pH this transition is moved down to experimentally accessible temperatures. These measurements confirm that the Ising model approach is indeed a good description of base stacking. On the other hand, comparison with the experiments points to the limitations of the simple zipper model description of base pairing.

Base Pairing↗

PubMed2004

Bubble nucleation and cooperativity in DNA melting.

Bubbles in DNA are related to fundamental processes such as duplication and transcription. Using a new ensemble technique to trap intermediate states, we present direct measurements of the average length of the denaturation bubble and the statistical weights of the bubble states in the temperature-driven melting of DNA oligomers. For a bubble flanked by double-stranded regions, we find a nucleation size of approximately 20 bases, and a broad distribution of bubble sizes. However, for bubbles opening at the ends of the molecule there is no nucleation threshold. The measured statistical weights of different conformations agree with the predictions of the thermodynamic models in the case of unzipping from the ends; however, internal bubble states are not completely described by the models. The measurements further show that, due to end effects, the melting transition becomes a two-state process only in the limit of a molecule length L approximately 1 bp.

Chemical Phenomena↗

PubMed2003

Length and statistical weight of bubbles in DNA melting.

We present measurements of the length of the bubble versus temperature in the melting transition of DNA oligomers. The sequences are clamped at the ends of GC pairs (strong binding), and have AT rich (weaker binding) middle regions of variable length B relative to the molecule size. We use a quenching technique to trap intermediate states. We find that the average relative bubble size grows from zero for increasing temperature, but reaches a plateau at a length of order B, producing a discontinuous transition with finite bubble size. The statistical weight of the bubble states decreases as B is reduced, the transition becoming more cooperative.

Base Sequence↗

PubMed2003

Single-molecule detection of DNA hybridization.

We demonstrate the detection of nanometer-scale conformational changes of single DNA oligomers through a micromechanical technique. The quantity monitored is the displacement of a micrometer-size bead tethered to a surface by the probe molecule undergoing the conformational change. This technique allows probing of conformational changes within distances beyond the range of fluorescence resonance energy transfer. We apply the method to detect single hybridization events of label-free target oligomers. Hybridization of the target is detected through the conformational change of the probe.

Biophysical Phenomena↗

PubMed2002

Plastic deformation of protein monolayers.

Globular proteins are peculiar solids that display both local stability of their conformation and the ability to undergo large cooperative changes of shape (conformational changes). If one forces a large deformation of the molecule, such that the structure is necessarily changed, it is not obvious whether the deformed globule can still remain a solid or whether it will melt. Is it possible to plastically deform a protein? Here we investigate this question with a micro-mechanical experiment on a small region (approximately 10 molecules) of a protein monolayer adsorbed on a rigid surface. For the two proteins studied, albumin and myoglobin, we observed that the molecules can be substantially deformed (approximately 1-2 nm deformation) by comparatively small stresses applied for sufficiently long times. The deformation is irreversible, and the protein remains in the solid state (i.e., displays a nonzero shear modulus). The dynamics of the deformation is approximately logarithmic in time, similar to creep in solids. These results show that globular proteins adsorbed on a surface can be plastically deformed.

Animals↗