Force spectroscopy of barnase-barstar single molecule interaction

Results of the single molecule force spectroscopy study of specific interactions between ribonuclease barnase and its inhibitor barstar are presented. Experimental data obtained for the force loading rate ranging 2-70 nN/s are well approximated by a single straight line, from which the dissociation barrier of the width of 0.12 nm and height of 0.75-0.85X10(-19) J can be inferred. The measured value of specific interaction does not depend on the NaCl concentration. This apparently contradicts the well-known dependence of the binding energy of this pair on the salt concentration, but such a "contradiction" is explained by the insensitivity of the force spectroscopy data to the relatively long-range electrostatic interaction. The latter essentially contributes to the value of barnase-barstar binding energy revealed by biochemical measurements, and it is exactly this electrostatic interaction which is influenced by the salt concentration. Copyright (C) 2010 John Wiley & Sons, Ltd.

Cryo-AFM development for high resolution DNA imaging and spectroscopy

Susana del Carmen Tobenas Borron

The thesis presents the development of a custom made atomic force microscope (AFM) conceived to image biological molecules at low temperatures in ultrahigh vacuum conditions, and its application to DNA single molecule studies. Starting from a first prototype working in contact mode, our aim was to implement non-contact AFM in order to reduce possible sample damaging due to strong tip interactions. The first chapter of the thesis discusses the technical solutions developed to improve the previous system. A complete setup for in situ exchange of samples and tips was designed and implemented. This permits a continuous operation of the microscope under the experimental conditions of UHV. Thermal instabilities and temperature differences between the tip and the sample were solved with the design of a movable shield to avoid heating by thermal radiation. The introduction of a new high voltage signal generator with variable frequency and the development of a user-friendly LabVIEW interface to communicate with this device notably improved the piezomotors driving. These piezomotors perform the micro-positioning of all the microscope parts and are driven through this interface. Finally, the previous tip-holder design was modified to incorporate a piezo actuator behind the cantilever, which provides the necessary excitation for the implementation of dynamic AFM modes. The installation of a Phase Lock Loop (PLL) circuit to lock the cantilever frequency and the replacement of old feedback electronics for a digital SPM Controller, with advanced options of frequency-shift feedback, was another requirement for the correct implementation of non-contact AFM. The second chapter deals with DNA sample preparation for high resolution imaging by AFM. Based on our studies, the advantages and drawbacks of the most commonly used techniques for DNA adsorption are discussed in detail. Our measurements in contact mode demonstrate the convenience of using hydrophobic substrates for high resolution DNA imaging. At hydrophilic surfaces, like mica, the presence of a thin water layer could be responsible of strong adhesion forces between the cantilever and the sample. In order to overcome this limitation we tested HOPG as hydrophobic substrate. HOPG, chemically modified with amphiphilic compounds, allowed us to deposit DNA without molecular damage or stress. The HOPG surface treatment was significantly improved. New protocols were developed to control the formation of dodecylamine disordered monolayers or lamella structures on the substrate. The lamella formation induces a subsequent self assembly of DNA single molecules. In the last chapter we discuss our results on DNA imaging obtained with the different operation modes implemented in our microscope: contact mode, tapping mode and frequency modulation (non-contact) AFM. We conclude that the last one provides higher signal stability and the necessary sensitivity to observe DNA details at a single-molecule level. We also present a study of frequency shift versus distance dependence (force spectroscopy experiments) performed in DNA samples. We discuss for the first time a plausible explanation to the origin of the atomic force contrast between the substrate and the DNA molecules. Force spectroscopy measurements performed along DNA molecules reveal two distinguishable types of interaction. This result suggests that the chemical composition of the minor-major groove and/or of the sugar-phosphate backbone along the molecule can be detected, opening numerous possibilities for further studies and applications of this technique.

EPFL2008

Covalent attachment of proteins to calcite surface for the single molecule experiments

Two protocols of covalent attachment of proteins onto the calcite surface, viz. one using the metallochelat and second using the aminohexil, are elaborated. Single molecule force spectroscopy method has been used to test their efficiency and practical applicability. Experiments were performed measuring the specific interaction force between bovine serum albumin (BSA) fixed onto the freshly cleaved calcite single crystal surface (procedure under the study here) and its polyclonal antibody (Ab-BSA) immobilized onto an AFM tip using standard and well studied procedure. We found the conditions, when up to 3-3.5% of tip-sample approaches lead to the formation of a single specific bond. (C) 2007 Elsevier B.V. All rights reserved.

2008

Statistical Study of the Unfolding of Multimodular Proteins and their Energy Landscape by Atomic Force Microscopy

Fabrizio Benedetti

The aim of the present thesis is to investigate several aspects of: the proteins mechanics, interprotein interactions and to study also new techniques, theoretical and technical, to obtain and analyze the force spectroscopy experiments. The first section is dedicated to the statistical properties of the unfolding forces in a chain of homomeric multimodular proteins. The basic idea of this kind of statistic is to divide the peaks observed in a force extension curve in separate groups and then analyze these groups considering their position in the force curves. In fact in a multimodular homomeric protein the unfolding force is related to the number of not yet unfolded modules (we call it "N"). Such effect yields to a linear dependence of the most probable unfolding force of a peak on ln(N). We demonstrate how such dependence can be used to extract the kinetic parameters and how, ignoring it, could lead to significant errors. Following this topic we continue with non kinetic methods that, using the resampling from the rupture forces of any peak, could reconstruct the rupture forces for all the other peaks in a chain. Then a discussion about the Monte Carlo simulation for protein pulling is present. In fact a theoretical framework for such methodology has to be introduced to understand the various simulations done. In this chapter we also introduce a methodology to study the ligand receptor interactions when we directly functionalize the AFM tip and the substrate. In fact, in many of our experiments, we see a "cloud of points" in the force vs loading rate graph. We have modeled a system composed by "N" parallel springs, and studying the distribution of forces obtained in the force vs loading rate graph we have establish a procedure to restore the kinetic parameters used. Such procedure has then been used to discuss real experiments similar to biotin-avidin interaction. In the following chapter we discuss a first order approximation of the Bell-Evans model where a more explicit form of the potential is considered. In particular the dependence of the curvature of the potential on the applied force at the minimum and at the metastable state is considered. In the well known Bell-Evans model the prefactors of the transition rate are fixed at any force, however this is not what happen in nature, where the prefactors (that are the second local derivative of the interacting energy with respect to the reaction coordinate in its minimum and maximum) depend on the force applied. The results obtained with the force spectroscopy of the Laminin-binding-protein are discussed, in particular this protein showed a phase transition when the pH was changed. The behavior of this protein changes, from a normal WLC behavior to a plateau behavior. The analysis of the force spectroscopy curves shows a distribution of length where the maximum of the first prominent peak correspond to the full length of the protein. However, length that could be associated with dimers and trymers are also present in this distribution. Later a new approach to study the lock and key mechanism, using "handles" with a specific force extension pattern, is introduced. In particular handles of (I27)3 and (I27–SNase)3 were biochemically attached to: strept-actin molecules, biotin molecules, RNase and Angiogenin. The main idea is to have a system composed by "handle-(molecule A)-(molecule B)-handle" where the handles are covalently attached to the respective molecules and the two molecules "A and B" are attached by secondary bonds. This approach allows a better recognition of the protein-protein interaction enabling us to filter out spurious events. Doing a statistic on the rupture forces and comparing this with the statistic of the detachments of the system of the bare handles, we are able to extract the information of the interaction between the molecule A and B. The two last chapters are of more preliminary character that the previous part of the thesis. A section is dedicated to the estimation of effective mass and viscous drag of the cantilevers studied by autocorrelation and noise power spectrum. Usually the noise power spectrum method is the most used, however the autocorrelation should give approximately the same information. The parameters obtained are important in high frequency modulation techniques. In fact, they are needed to interpret the results. The results of these two methods show a good agreement in the estimation of the mass and the viscous drag of the various cantilever used. Afterwards a chapter is dedicated to the discussion of the force spectroscopy experiments using a low frequency modulation of the cantilever base. Such experiments allow us to record the phase and the amplitude shift of the modulation signal used. Using the amplitude channel we managed to restore the static force signal with a lower level of noise. Moreover these signals give us direct information about the dynamic stiffness and the lose of energy in the system, information that, using the standard technique would be difficult (or even impossible) to obtain.