Projects

The labeling of the projects (Forschungsmodul, BSc and MSc) are suggestive. If a project interest you for a Forschungsmodul but has a MSc thesis classification, you are still strongly encouraged to contact the relevant supervisor to discuss the project.



The Evolutionary Origins of Novel Enzyme Functions

The projects in this particular section are a mix of theory and experiment. The balance between the two is flexible depending on preference and experience.

Main research topics:

  • What makes an enzyme evolvable?
  • How do the presence and levels of secondary activities depend on selection pressure for the primary function?
  • How do new functions evolve?

Approaches:

  • Characterization of ancestral proteins, which are the origin of many ‘successful’ existing enzymes and should therefore be more evolvable.
  • Test the evolvability of existing enzymes.
BSc or MSc thesis

I. Are there Limitations to the Degree of Functional Specialization?

Catalytic promiscuity, the ability for one enzyme to catalyze more than one reaction, is believed to play an important role in enzyme evolution and is increasingly shown to be widespread in nature, in contrast to the previously held paradigm of ‘one enzyme = one activity’. However for an enzyme to become extremely proficient at their primary function, it is believed that they have to be extremely specific. Recent data for sulfatases in the alkaline phosphatase (AP) superfamily of enzymes undermine this statement and suggest that there are limits to how specific an enzyme can be between two related reactions. The goal of this project is to pin down the underlying cause of this phenomenon using artificial laboratory evolution. The main goal of this project would be to test if the aforementioned limit to specificity is also present in a sulfatase from the metallo-beta-lactamase (MBL) superfamily in order to investigate the role of a particular protein fold on the limits of enzyme evolution.

Techniques used:

  • Standard molecular cloning techniques
  • Mutant library construction and screening
  • Protein expression, purification and characterization.

References:

  • Baier & Tokuriki (2014) J. Mol. Biol. 426, 2442-56
  • Kintses et al (2012) Chem. Biol. 19, 1001-9
  • van Loo et al (2010) Proc. Natl. Acad. Sci. U. S. A. 107, 2740-45
  • van Loo et al (2017), manuscript(s) submitted/in preparation (available on request)
  • Zinchenko et al (2014) Anal. Chem. 86, 2526-33

contact: Bert van Loo

Forschungsmodul

II. Ancestral Sequence Reconstruction: The origin of TEM-1 beta-lactamases

Ancestral sequence reconstruction can provide insight into the origin of novel functions. For this purpose a phylogeny has to be constructed that allows the inference of an ancestral node. The minimum tree required for this should consist of at least three taxa (or in practice three distinct clades), with a minimum of two different primary functions. In the ideal case the phylogenetic distances between these taxa and the common ancestor is as small as possible. In practice if more than 2 functionally distinct taxa are known, the phenotypic distances are usually long. The main goal of this project is to define and execute the optimal strategy to obtain a tree with the minimal phenotypic distance from the last common ancestor of two functionally distinct enzymes. We have several candidate enzyme pairs available for these type of studies, or a protein pair can be selected based on preference.Ancestral sequence reconstruction can provide insight into the origin of diverging biological functions, i.e. two related proteins with different functions evolved out of a common ancestor. In order to reliably reconstruct this common ancestor, a densely populated phylogenetic tree representing the relevant functional divergence has to be created. Often two known related proteins described in the literature are not nescecarily the closest relatives that are functionally different within the protein superfamily that they belong to. Populating the sequence space between these two members is therefore key to identifying the 'minimum' distance in sequence space associated with the functional divergence of interest. The goal of this project is to identify the best possiblke phylogenetic relationship for reconstructing the common ancestor of the pencillin binding domain proteins (PBDs) found in several cyanobacteria and the class A TEM-1 beta-lactamases.

References

  • Urbach et al. (2008) J. Biol. Chem. 83, 32516-26.
  • Urbach et al. (2009) J. Mol. Biol. 386, 109-120.
  • Harms & Thornton (2010) Curr. Opin. Struct. Biol. 20, 360-6.

Contact: Bert van Loo

BSc or MSc thesis

III. The Origins of Dehalogenating Enzymes

There is a wide variety of enzymes that can catalyze the breaking of carbon-halogen bonds. At the time of the major boom of their discovery in the late 1980’s and first half of the 1990’s their ubiquity was largely explained by the assumption that naturally occurring halogenated compounds were the actual substrates of these enzymes prior to the mass-introduction of halogenated pollutants in the environment. In recent years catalytic promiscuity has been suggested as the main reason for the natural resilience towards xenobiotic compounds, which in principle indicates that the absence of selective pressure does not exclude the possibility for an enzyme to be able to catalyze reactions. Based on these recent assumptions it would be interesting to resurrect ancestral enzymes that eventually evolved into dehalogenases.
The fact that there are a variety of ‘catalytic solutions’ for biological dehalogenation, i.e. dehalogenases occur in a diverse set of structurally and mechanistically unrelated enzyme families, suggest that the potential for current enzymes to convert halogenated compounds, albeit at low rates, should be high. The existing knowledge on current dehalogenases can be used to identify likely progenitors, which should be tested for their ability to catalyze carbon-halogen bond breaking.

Main objectives:

  • Identification and resurrection of ancestral ‘dehalogenases’
  • Identification of promiscuous dehalogenating activity in existing enzymes.

References

  • Copley (2009) Nat. Chem. Biol. 5, 559-66.
  • De Jong & Dijkstra (2003) Curr. Opin. Struct. Biol. 13, 722-30.
  • Poelarends et al. (2008) Cell. Mol. Life Sci. 65, 3606-18.

contact: Bert van Loo

BSc or MSc thesis

IV. Can neutral be beneficial? Unraveling the importance of single mutations and epistasis in evolution of new enzymatic functions.

Not all the mutations appearing at a genomic level result in significant and measurable functional changes. Many of them are deleterious or permissive – they might become beneficial only upon occurrence of further mutations. This phenomenon, called the epistatic ratchet, plays an important role in the evolution of new functions.

The alkaline phosphatase superfamily, comprising a large group of promiscuous (multifunctional) enzymes profiles has been subjected to phylogenetic study in which the ancestral enzymes were resurrected. These studies, aimed at understanding the origin and evolution of enzyme specificity, allowed the identification of the protein intermediates leading from the last common ancestor of two distinct protein subfamilies – arylsulfatases and phosphonate monoester hydrolases - to the extant enzymes that share a common fold and a high level of sequence conservation but exhibit different activity profiles. The resurrected intermediates reflect step-by-step evolution toward the extant proteins and show relatively little difference at the sequence level (usually several amino-acids).

The main goal of this project is to identify the role of epistatic mutations in enzyme evolution by examining a set of intermediate ancient proteins. This will be done by characterizing the effect of mutations (separately and in combination) that occur between two ancestral states (i.e. ancestral enzymes), on enzyme activity (and specificity).

Approaches:

  • Characterization of ancestral proteins, which are the origin of many ‘successful’ existing enzymes and should therefore be more evolvable.
  • Testing the effect of single/combined mutations that occur between two known active enzymes (e.g. between ancestor and current enzyme), on the enzymatic function, identification of permissive mutations and alternative but convergent paths

References:

  • Bridgham et al (2006) Science, 312, 97-101.
  • Harms & Thornton (2010) Curr. Opin. Struct. Biol. 20, 360-6
  • Kaltenbach et al (2015) eLife. 06492
  • van Loo et al (2017), manuscript(s) submitted/in preparation (available on request)

Contact: Bert van Loo



Domain projects

Bsc/Msc projects

Background

One of the research areas in our group is the analysis of proteomes, pathways etc. based on domain content. For this purpose we have and still are developing algorithms and programs. We implemented a set of basic functions and classes for the domain analysis in a central C++ library that can than be used for the development of the different programs to perform domain analyses. We currently plan to implement a range of programs/algorithms which would be good opportunity for biologist with a strong interest in programming or a computer scientist with some interest in biology to work on some of our programs. The available projects are listed below. Please contact Carsten Kemena [c.kemena[guesswhat]uni-muenster.de] (tel: 8321086) for more information.

Domain arrangement clustering

Bsc

Domain arrangement clustering is an important step in many analysis. Therefore in our group a program called porthoDom has been developed. This algorithm should now be extended and reimplemented using the library described above.

References

  • Bitard-Feildel et al., BMC Bioinformatics 2015

Domain arrangement expression change

Bsc

Domains, as for example defined in the Pfam database, are building blocks that are reused in different proteins to produce proteins with different functions. Paralogous sequences can have the same or similar domain arrangements. Often paralogs have different expression profiles due to adaption to new functions. This project has the goal to analyse the differential expression of paralogous sequences with special focus on paralos that suffered domain loss. The results can be compared to the expression profiles from orthologous sequences in closely related species. Further analyses might be the inclusion of the gene age as an important factor in the changes in differential expression.

Domain arrangement clustering is an important step in many analysis. Therefore in our group a program called porthoDom has been developed. This algorithm should now be extended and reimplemented using the library described above.

Domain-Exon overlap

Bsc

Domains are building blocks of proteins that have a specific structure or function. Due to the combination of different domains, proteins can obtain different functions. Due to alternative splicing (creating different proteins from the same gene e.g. by alternative exon inclusion) the domain order of the "original" gene can be changed. The objective of this project is to analyse the influence of alternative splicing on domain arrangements. For this purpose the different events (e.g. exon removal → domain loss) should be identified and quantified.

Domain Database

FM

The student has a look at the domain database overlap in uniprot, the basis of the new RADS database. The idea is to see which domain database covers how much of the database, what the increase of coverage is for each single database and which databases contributes the most. One should check the GO term distribution over the different databases as well.

Contact: Carsten Kemena [c.kemena[guesswhat]uni-muenster.de] (tel: 8321633)



Previous Projects

These projects are already been set to someone but if you are interested in one of them you are strongly encourage to contact the supervisor of the project.

Analysis of the blood clotting cascade

Forschungsmodul

Introduction

The blood clotting cascade of vertebrates consists of several multi-domain proteins that interact with each other. Due to its important role, the pathway exists in all vertebrates.

Project

The project proposes to explore the evolutionary changes taking place in the evolution of the blood clotting cascade, with a focus on the domain changes. The student will have to analyse the appearing domain arrangements and analyse the appearance of the different domains.

contact: Carsten Kemena



Resurrection of Ancestral Sulfatases/Phosphonate Monoester Hydrolases

One of the main questions in evolution is how new enzymatic functions emerge. The widely accepted model is that one gene can evolve in to many by duplication and subsequent specialization for the new functions, however very little is known about what makes one enzyme more evolvable than another. Today’s enzymes have evolved from a common ancestor. Although the sequence of this common ancestor is difficult to pin down, many of the intermediate ancestors that have developed over the years can sometimes be determined computationally by inference from the sequences of present enzymes. The ancestral sequences can be synthesized and their related gene products can than be expressed and characterized. Ancestors from which two enzyme classes have evolved that differ in primary function are of course particularly interesting. We have defined such a functional split-point in the phylogenetic relationship between sulfatases and phosphonate monoester hydrolases in the AP-superfamily. The aim of this project is to determine the most likely sequence of the common ancestor computationally, have the gene synthesized and characterize the resulting enzyme in the laboratory. The latter may give insight into the properties of more evolvable enzymes.

Techniques used:

Multiple sequence alignments, construction of phylogenetic trees, standard molecular cloning techniques, protein purification and characterization.

References:

  • Bridgham et al (2006) Science, 312, 97-101.
  • Jonas et al (2008) J. Mol. Biol. 384, 120-36.
  • van Loo et al (2010) Proc. Natl. Acad. Sci. U. S. A. 107, 2740-45..
  • van Loo et al (2016), manuscript(s) in preparation (available on request)

contact: Bert van Loo

BSc or MSc thesis

Are the Nature and Level of Secondary Protein Activities Predictable Based on the Nature of Their Primary Function?

Enzymes have long been thought of as both extremely proficient and specific. In recent years an increasing number of enzymes are reported to have secondary or promiscuous activities besides their main one. These promiscuous activities are believed to play an important role in the evolution of new enzyme functions. Promiscuous activities are usually thought of as either accidental or as relics of the evolutionary history of the enzyme in which they occur. The latter could explain why many promiscuous functions are the primary functions of related enzymes and vice versa, i.e. the common ancestor of the related enzymes had both activities and the current enzymes are specialized toward either one of the activities, with a low level of the other activity remaining. However, certain so-called crosswise activity pairs, e.g. sulfatase and phosphoesterase activity, are observed in two or more unrelated enzyme families. This could suggest that the nature of the promiscuous activities observed in an enzyme are the result of the selective pressure it is under, rather than its evolutionary history. This phenomenon is a case of exaptation, since the appearance of a new trait, i.e. the promiscuous activity, is the result of selection pressure of another trait, i.e. the primary activity. Promiscuous sulfatase activity has thus far only been observed in phosphoesterases that are related to enzymes that have sulfate hydrolysis as their primary function. Phosphoesterase activity has likewise been observed as a promiscuous activity in many sulfatases. However there have been several reports on promiscuous phosphoesterase activity in other hydrolases (e.g. peptidases, beta-lactamases). This raises two important questions:

  1. Are enzymes that show phosphoesterase activity, either as a primary or promiscuous function, intrinsically able to catalyze sulfate ester hydrolysis?
  2. Are the nature and the levels of promiscuous activity observed in enzymes linked to the nature of the selective pressure they are under?

Approach

  • Identification of as many enzymes as possible from as many different enzyme families as possible that catalyze either phosphoester or sulfoester hydrolysis, either as primary or promiscuous activity, and characterize their substrate specificity.
  • Study the effect of artificial selection pressure for increased phosphodiesterase activity, e.g. in a directed evolution experiment, on the presence and levels of sulfatase activity.

References

  • Baier & Tokuriki (2014) J. Mol. Biol. 426, 2442-56
  • Ercan et al (2006) Biochemistry 45, 13779-93
  • van Loo et al (2010) Proc. Natl. Acad. Sci. U. S. A. 107, 2740-45
  • van Loo & Hollfelder (2010) pages 524-538 in Baltz et al. (ed.) Manual of Industrial Microbiology and Biotechnology, 3rd edition, (pdf available on request)

contact: Bert van Loo