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I am using a combination of structural (e.g. X-ray crystallography), biophysical (e.g. isothermal titration calorimetry), computational (e.g. molecular dynamics simulations and computational docking) and evolution-based (e.g. consensus design & ancestral sequence reconstruction) approaches to understand and engineer a range of molecular interactions. This page provides an overview of some of my current research interests and related publications.

I am working under the amazing guidance of Professor Colin Jackson at the Australian National University, and have the pleasure of working with some amazing collaborators.  

Lab Bench, RSC
 

Protein Evolution


Similar to the process of evolution at the level of organisms, proteins gain new functions and characteristics through gene-duplication events, the accumulation of mutations (genetic diversification) and natural selection. Understanding the fundamental mechanisms through which proteins gain new functions can provide insight into the factors that drive functional diversification of protein families, but also can guide protein engineering efforts by highlighting the factors that drive or hinder the emergence and improvement of new functions and/or properties.

The evolution of an enzyme from a non-catalytic binding protein
I have had the pleasure of working on a project studying the natural evolution of an enzyme, cyclohexadienyl dehydratase, from an ancestral protein that was non-catalytic. Earlier evolutionary biochemistry studies had highlighted the transition of one enzyme to another enzymatic activity using directed evolution and ancestral reconstruction approaches, but no one had really studied the mechanisms underlying the emergence of new enzyme activity in proteins folds that are non-catalytic.

Lab Bench, RSC
 

In a study led by Ben Clifton, we showed that the enzyme cyclohexadienyl dehydratase evolved from an ancestral solute-binding protein, and that the new enzyme activity emerged and improved through distinct steps including (i) the introduction of key catalytic residues into the binding site of the ancestral protein, (ii) the reshaping of the active site cavity and development of hydrogen bonding networks around the core of the protein, and (iii) the accumulation of remote mutations that likely altered the conformational sampling of the enzyme (favouring the more closed, catalytically-relevant state).

  • Clifton BE, Kaczmarski JA, Carr PD, Gerth ML, Tokuriki N, Jackson CJ. 2018. Evolution of cyclohexadienyl dehydratase from an ancestral solute-binding protein. Nat Chem Biol 14:542–547.
  • In a follow up study, we collaborated with Gottfried Otting (ANU) and Daniella Goldfarb (Weizman Institute of Science, Isreal), to explore how the sampling of open and closed states changed during the evolution from a non-catlytic binding protein to an enzyme. By comparing enzyme kinetic data with molecular dynamics simulations and double electron-electron resonance spectroscopy measurements (on tagged variants of the proteins), we showed that: (i) the ancestral solute-binding protein sampled both open and closed states, typical of proteins with the periplasmic binding protein fold; (ii) the intermediate ancestors sampled mainly wide open states which were likely unproductive to catalysis and (iii) the modern enzyme sampled more compact states, which likely contributed to the observed increase in catalytic efficiency.

  • Kaczmarski JA, Mahawaththa MC, Feintuch A, Clifton BE, Adams LA, Goldfarb D, Otting G, Jackson CJ. 2020. Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme. Nature Communications 11:5945
  • Our on-going work on this project is exploring, in more detail, the role that oligomerisation has had during the evolution of this enzyme (the ancestors are monomers, while the modern day enzyme is a trimer).

     

    Protein Dynamics


    My life changed the moment that I started seeing proteins as dynamic species, rather than the static blobs that were portrayed in my high-school text books. Recognising the important role that dynamics has in determining the properties of proteins (including thermostability, activity, interactions with other proteins, involvement in signalling pathways etc) is vital when studying and engineering proteins - and makes studying these molecules even more fascinating.

    As described above, our work on cyclohyexadienyl dehydratase showed how the sampling of different conformational states has been important during the evolution of a new enzyme activity. Many of the other projects that I have been involved in have had a particular focus on studying how properties of a protein are influenced by the way a protein moves.

  • Clifton BE, Kaczmarski JA, Carr PD, Gerth ML, Tokuriki N, Jackson CJ. 2018. Evolution of cyclohexadienyl dehydratase from an ancestral solute-binding protein. Nat Chem Biol 14:542–547.
  • Kaczmarski JA, Mahawaththa MC, Feintuch A, Clifton BE, Adams LA, Goldfarb D, Otting G, Jackson CJ. 2020. Altered conformational sampling along an evolutionary trajectory changes the catalytic activity of an enzyme. Nature Communications 11:5945
  • In my first publication, I worked under the supervision of Ben Corry (ANU) to study the dynamics of the fenestrations of voltage-gated sodium channels. These fenestrations (channels that lead from the membrane to the central cavity of the protein) are important for allowing the passage of small molecule drugs that are used to block sodium channels. Our molecular dynamics simulation study highlighted the dynamic nature of these fenestrations, and highlighted key residues that lined the tunnels which likely control access to the central cavity of the protein. Interestingly, we saw that lipids from the membrane often protruded into these fenestrations.

  • Kaczmarski JA, Corry B. 2014. Investigating the size and dynamics of voltage-gated sodium channel fenestrations: A molecular dynamics study. Channels 8:264–277.

  • Structures and dynamics of non-ribosomal peptide synthesis proteins


  • Izoré, T, Candace H, Kaczmarski JA, Gavriilidou A, Chow KH, Steer DL, Goode RJA, Schittenhelm RB, Tailhades J, Tosin M, Challis GL, Krenske EH, Ziemert N, Jackson CJ & Cryle MJ. 2021. Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity.Nat Commun 12: 2511.
  • Greule A, Izoré T, Iftime D, Tailhades J, Schoppet M, Zhao Y, Peschke M, Ahmed I, Kulik A, Adamek M, Goode RJA, Schittenhelm RB, Kaczmarski JA, Jackson CJ, Ziemert N, Krenske EH, De Voss JJ, Stegmann E, Cryle MJ. 2019. Kistamicin biosynthesis reveals the biosynthetic requirements for production of highly crosslinked glycopeptide antibiotics. Nat Commun 10:2613.
  • Izoré T, Tailhades J, Hansen MH, Kaczmarski JA, Jackson CJ, Cryle MJ. 2019. Drosophila melanogaster nonribosomal peptide synthetase Ebony encodes an atypical condensation domain. Proc Natl Acad Sci USA 116:2913–2918.

  • Structural Immunology


  • Fisher CR, Sutton HJ, Kaczmarski JA, McNamara HA, Clifton B, Mitchell J, Cai Y, Dups JN, D’Arcy NJ, Singh M, Chuah A, Peat TS, Jackson CJ, Cockburn IA. 2017. T-dependent B cell responses to Plasmodium induce antibodies that form a high-avidity multivalent complex with the circumsporozoite protein. PLoS Pathog 13:1–23.
  • Chatterjee D, Lewis FJ, Sutton HJ, Kaczmarski JA, Gao X, Cai Y, McNamara HA, Jackson CJ, Cockburn IA. 2021. Avid binding by B cells to the Plasmodium circumsporozoite protein repeat suppresses responses to protective subdominant epitopes. Cell Reports 35(2):108996
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    Protein Engineering


    Protein-based Sensor Design
    Cyanobacterial carbon-concentrating mechanisms