Understanding and Engineering Thermostability in DNA Ligase from Thermococcus sp 1519

The physical chemical principles underlying,enzymatic thermostability are keys to understand the way evolution has shaped proteins,to adapt to a broad range of temperatures. Understanding the molecular determinants at the basis of protein thermostability is also important factor for engineering more thermoresistant enzymes to he used in the industrial-setting, such as, for instance, DNA, ligases, Which are important for DNA replication and repair and have been long used in molecular biology and biotechnology. Here, We fitst address the origin of thermostability in the thermophilic DNA ligase from archaeon Thermococcus sp. 1519 and identify thermosensitive regions using molecular modeling and simulations. In addition, we predict mutations that can enhance. thermostability of the enzyme through bioinformatics, analyses. We show that thermosensitive regions of this enzyme are stabilized at higher temperatures by optimization of charged groups on the surface, and we predict that thermostability can be further increased by further optimization of the network among these charged groups. Engineering this DNA ligase by introducing selected mutations (i.e., A287K, G304D, S364I, and A387K) eventually produced a significant and additive increase in the half-life of the enzyme when compared to that of the wild type.

Modeling and Engineering Proteins Thermostability

Hasan Pezeshgi Modarres

Enzymes have evolved during millions of years to become efficient catalysts for specific biochemical reactions within a specific range of working conditions in the cellular environment. The activity of an enzyme is directly related to its folded structure and even slight changes in its 3D conformation may cause irreversible, negative effects on its activity. The structure of enzymes are very sensitive to the environmental conditions and changes from their optimal conditions, like higher temperature, salt concentration, and pH, might result in denaturation and subsequently inactivation. In the past decades, natural enzymes extracted from different organisms have found a wide range of applications in the industrial and biotechnological setting. For the majority of the applications their activity at high temperatures is more favorable, however enzymes have evolved, in most of the cases, to optimally work in limited range of temperature of their native cellular environment. Therefore, enhancing enzyme thermostability will not only increase their application range, but could also shed light into new aspects of their evolution and chemical activity. The physical chemical principles underlying enzymatic thermostability are keys in fact to understand the way evolution has shaped proteins to adapt to a broad range of temperatures. Understanding the molecular determinants at the basis of protein thermostability, using both in silico methods and in vitro experiments, is also an important way for engineering more thermo-resistant enzymes to be used in the industrial setting, as for instance DNA ligases, which are important for DNA replication and repair and have been long used in molecular biology and biotechnology. In this thesis I used in silico techniques, like molecular modeling and simulation coupled with bioinformatics analyses, to assess existing methods and predict potential thermo-stabilizing mutations for target proteins. First, I studied a thermophilic protein and after exploring the origins of its thermostability I proposed mutations to further increase its thermostability. Then, I took advantage of what learned from this study to explore further thermostability engineering methods in order to develop faster, accurate, and easy-to-use methods that can be generally used for a broad array of proteins. 1. Understanding and engineering thermostability in the DNA ligase from Thermococcus sp. 1519 (LigTh1519). In this thesis, I first addressed the origin of thermostability in the thermophilic DNA ligase from archaeon Thermococcus sp. 1519, and identified thermo-sensitive regions using molecular modeling and simulations. In addition, I predicted mutations that can enhance thermostability of the enzyme through bioinformatics analyses. I showed that thermo-sensitive regions of this enzyme are stabilized at higher temperatures by optimization of charged groups on the surface, and predicted that thermostability can be further increased by further optimization of the network among these charged groups. Engineering this DNA ligase by introducing selected mutations (i.e., A287K, G304D, S364I and A387K) produced eventually a significant and additive increase in the half-life time of the enzyme when compared to the wild-type. Then, based on what I learned from thermostability analyses and improvement of LigTh1519, my aim was to design a general-purpose protein thermostability engineering protocol that can enable thermostability engineering [...]

EPFL2015

Effects of base-pair sequence, nicks and gaps on DNA minicircle shapes

Arnaud Amzallag

DNA is a long polymer with the form of a double helix of about two nanometers diameter (2.10-9 meters). It is composed of nucleotides whose sequence carries the information of heredity. The four possible nucleotides have slightly different geometries, and their sequence along the DNA molecule are thought to influence the shape and the stiffness of the double helix on a scale of few tens to a few hundred base pairs, and thereby its biological activity. DNA minicircles (or miniplasmids) are closed loops with lengths of the order of a few tens or hundreds of base pairs in which the double helix bends around to close on its own tail with some number of twists. Its minimal energy shape and its energy of formation depend on the sequence of base pairs, and can be efficiently computed by polymer and rod models, if shape and stiffness parameters of DNA are provided as inputs. This structure is therefore an interesting experimental motif to test sequence-dependent mechanical properties of the DNA molecule. The purpose of this thesis is to explore the experimental methods to determine the shape and free energy of formation of DNA minicircles. The shapes have been be determined from cryo-electron micrographs. Efficiency of formation has been investigated by a novel method called annealing-cyclization. Cryo-electron microscopy allows observation of very small molecules (here 17 or 11 nm diameter) in vitrified water, in order to keep the 3D shape of the molecules as close as possible to the shape they had in solution. In Chapter 1, I used stereo cryo-electron micrographs to determine and compare the three-dimensional shape of 95 individual DNA minicircles of 158 base pairs, which were identical in sequence except within a 18 bp block which contained either a TATA box sequence or a CAP site. I defined the notion of shape-distance which I used to estimate the error of reconstruction, and I detected clusters of shapes using an appropriate sorting algorithm. However the cluster did not seem to be associated with the variable sequence (TATA or CAP). I then analyzed (in Chapter 2) two-dimensional shapes of shorter DNA minicircles (94 bp) determined by negative staining electron microscopy, designed with either two nicks (breaks in one of the strands) or two gaps (missing nucleotides) at diametrically opposite sites of the minicircles. I observed that the gapped minicircles have an elongated shape with respect to the nicked minicircles, and I used this result together with the results of atomic level molecular dynamics simulations to conclude that the gap flexibility, perhaps together with base unpairing at the gap site, is responsible for this elongated minicircle shape. Finally, I proposed a ligase-free assay to measure the minicircle formation efficiency, which can give a high yield of nicked minicircles. The method avoids the use of ligase and the associated concerns about the effect of ligase concentration on the measurements. I determined an equation from a chemical model of the reaction that fits the experimental data, and I defined the Ja factor which gives a measure of the cyclization yield independent of DNA initial concentration. This method seems to confirm that minicircles with two gaps cyclize more efficiently that minicircles with two nicks, probably because of the gap flexibility. As a perspective, the conclusions of this thesis could be used for the design of minicircle constructs whose shape would be sensitive enough to sequence mutation in order to be detected by electron microscopy. Such shapes could be then compared, thanks to the shape-distance tool defined herein, to shapes computed with known or putative DNA models.

EPFL2008

The conformational flexibility of the carboxy terminal residues 105-114 is a key modulator of the catalytic activity and stability of macrophage migration inhibitory factor

Michele Cascella, Farah El Turk, Hilal Lashuel, Hajer Ouertatani-Sakouhi, Ursula Röthlisberger, Magdalena Zweckstetter

Macrophage migration inhibitory factor (MIF) is a multifunctional protein and a major mediator of innate immunity. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo and the factors governing its oligomerization and stability remain poorly understood. The C-terminal region of MIF is highly conserved and participates in several intramolecular interactions that suggest a role in modulating the stability and biochemical activity of MIF. To determine the importance of these interactions, point mutations (A48P, L46A), insertions (P107) at the monomer-monomer interfaces, and C-terminal deletion (Delta 110-114NSTFA and Delta 105-114NVGWNNSTFA) variants were designed and their structural properties, thermodynamic stability, oligomerization state, catalytic activity and receptor binding were characterized using a battery of biophysical methods. The C-terminal deletion mutants DeltaC5 huMIF 1-109 and DeltaC10 huMIF 1-104 were enzymatically inactive and thermodynamically less stable than wild type MIF. Analytical ultracentrifugation studies demonstrate that both C-terminal mutants sediment as trimers and exhibit similar binding to CD74 as the wild type protein. Disrupting the conformation of the C-terminal region 105-114 and increasing its conformational flexibility through the insertion of a proline residue at position 107 was sufficient to reproduce the structural, biochemical and thermodynamic properties of the deletion mutants. P107 MIF forms an enzymatically inactive trimer and exhibits reduced thermodynamic stability relative to the wild type protein. To provide a rationale for the changes induced by these mutations at the molecular level, we also performed molecular dynamics simulations on these mutants in comparison to the wild type MIF. Together, our studies demonstrate that intersubunit interactions involving the C-terminal region 105-114, including a salt-bridge interaction between Arg73 of one monomer and the carboxy terminus of a neighboring monomer, play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties. Our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity.

American Chemical Society2008