Alchemical free energy
Energetics of ion conduction
through membrane channels
Free energy calculations
for protein design
Inhibition of peptide oligomer
formation with anle138b
Potassium channels:
mechanisms of ion conduction, selectivity and gating

Alchemical free energy calculations

In this project we are working on the method development and application of the approaches to calculate free energy differences by exploiting non-physical, alchemical, pathways across a thermodynamic cycle. In a number of applications we have been able to demonstrate a remarkable predictive accuracy of our methods in assessing changes in protein thermostability upon amino acid mutation. Further steps are made towards an accurate first principles based protein-protein and protein-peptide binding free energy estimation. Another branch of interest comprises protein-ligand interactions and automated alchemical calculation of the free energy differences for ligand modifications.


[1] Vytautas Gapsys, Servaas Michielssens, Daniel Seeliger, and Bert L. de Groot. pmx: Automated protein structure and topology generation for alchemical perturbations. J. Comput. Chem. 36:348-354 (2015)

[2] Vytautas Gapsys, Servaas Michielssens, Jan Henning Peters, Bert L. de Groot, Hadas Leonov. Calculation of Binding Free Energies. Molecular Modeling of proteins: 2nd edition. Book Series: Methods in Molecular Biology 1215: 173-209 (2015)

[3] Vytautas Gapsys, Daniel Seeliger, and Bert L. de Groot. New Soft-Core Potential Function for Molecular Dynamics Based Alchemical Free Energy Calculations. J. Chem. Theor. Comput. 8:2373-2382 (2012)

Vytautas Gapsys

Energetics of ion conduction through membrane channels

All animal cells are characterized by steep electrochemical gradients of Na+ and K+ ions across the plasma membrane. Membrane channels conduct selected ions downhill their gradients, and resulting ion flows underlie many important processes, such as cell volume regulation, hormone secretion and electrical impulse formation. Molecular simulations offer unique insights into the ion conduction process on the atomistic scale, e.g. using the computational electrophysiology approach developed earlier in our lab. However, the accuracy of such simulations is inherently limited by the quality of the underlying models, that are often not sufficient to realistically describe ion-protein interactions. Here, we aim to obtain a comprehensive picture of ion conduction by combing quantum mechanics (QM), quantum mechanic/molecular mechanics (QM/MM) and next generation force fields with existing computational methodologies.

Wojciech Kopec

Free energy calculations for protein design

Proteins are involved in all fundamental processes of living organisms, and have evolved into such precise and efficient machines that human-made ones can hardly compete. Thanks to their versatility, protein-based materials have the potential to tackle many challenges in biomedicine and nanotechnology, from efficient catalysis, to sensitive small molecule or light detection, to effective treatment of cancer, viral and bacterial infections. The use of computation for the design of synthetic proteins has been gaining momentum as a result of increasing availability of software and hardware resources, as well as a number of successes in different applications. In this project, we aim to assess the feasibility of using alchemical free energy calculations for the optimisation of proteins with specific target functions.

Matteo Aldeghi

Inhibition of peptide oligomer formation with anle138b

The di-phenyl-pyrazole compound anle138b is a known inhibitor of oligomeric aggregate formation in vitro and in vivo. Therefore, anle138b is considered a promising drug candidate for a number of neurodegenerative diseases in humans. The atomistic details of the aggregation inhibition mechanism, however, are to date unknown since the ensemble of small nonfibrillar aggregates is structurally heterogeneous and inaccessible to direct structural characterization. Here, we set out to elucidate anle138b's mode of action using all-atom molecular dynamics simulations on the multi-microsecond timescale.


[1] D. Matthes, V. Gaspys, C. Griesinger, B. L. de Groot. Resolving the atomistic modes of anle138b inhibitory action on peptide oligomer formation. ACS Chem. Neurosci. 2017.

Dirk Matthes , Vytautas Gapsys

Potassium channels: mechanisms of ion conduction, selectivity and gating

All animal cells are characterized by steep electrochemical gradients of Na+ and K+ ions across the plasma membrane. Potassium channels facilitate the passage of K+ ions to near-diffusion limited rates, while reliably excluding smaller Na+ ions. A clear separation between K+ and Na+ currents is necessary to ensure sharp action potentials, facilitating the rapid propagation of electric signals in excitable cells such as neurons. In this project, we are aiming at elucidating the molecular mechanism of ion conduction of different ion through representative potassium channels (e.g. KcsA, MthK, Kv1.2) and their effect on spontaneous gating transitions. We are using the computational electrophysiology approach that enables us to directly observe hundreds of individual permeation events under voltage, closely mimicking electrophysiological recordings. A special attention is dedicated to force field calibration and validation.

Wojciech Kopec

~Previous projects~

Lipid-protein Interactions

In this project we focus on the effect of embedded peptides or proteins on the surrounding membrane and on the effect of the membrane on embedded peptides. Small peptides involve HIV-1 gp41 or gramicidin like channels. Membrane channel proteins involve VDAC (Voltage Dependent Anion Channel) [1] and Aquaporins [2] among others [3]. These systems are being studied in different types of model membranes (DMPC, DPPC, POPE, etc). This work is done with the close collaboration of NMR, EPR and electron crystallography groups. Tools for analyzing L-P complexes are also been developed [4].


[1] Saskia Villinger, Rodolfo Briones, Karin Giller, Ulrich Zachariae, Adam Lange, Bert L. de Groot, Christian Griesinger, Stefan Becker, Markus Zweckstetter. Functional dynamics in the voltage dependent anion channel. Proc. Nat. Acad. Sci. 107: 22546-22551 (2010).

[2] C. Aponte-Santamaria, R. Briones, A.Schenk, T. Walz, B. de Groot. Molecular driving forces defining lipid positions around aquaporin-0. PNAS 109: 44319-44325 (2012).

[3] Rasmus Linser, Nicola Salvi, Rodolfo Briones, Petra Rovo, Bert L. de Groot, Gerhard Wagner. The membrane anchor of the transcriptional activator SREBP is characterized by intrinsic conformational flexibility. Proc. Nat. Acad. Sci. 112: 12390-12395 (2015).

[4] Vytautas Gapsys, Bert L. de Groot, Rodolfo Briones. Computational analysis of local membrane properties. J. Comput. Aided Mol. Des. 27: 845-858 (2013).

[5] Rodolfo Briones, Camilo Aponte-Santamaria, Bert L de Groot. Localization and ordering of lipids around Aquaporin-0: Protein and lipid mobility effects Front. Physiol. 8: 124 (2017).

Rodolfo Briones

Insight into Ion Channel Gating by Computational Electrophysiology Simulations

Insight into the functional mechanisms of ion channels is of universal importance to enable drug design on the class of membrane proteins, and to enhance our understanding of some of the fundamental features of cells. We have recently developed a simulation protocol called Computational Electrophysiology (CompEL) [1], which facilitates the atomistic simulation of ion channels in action. The computational electrophysiology allows to simulate membrane embeded proteins in two compartments separated by lipid bilayers with a sustained ion flux. Thus far we have used CompEL to study several different membrane proteins ranging from highly ion-selective, narrow channels to wide diffusion pores [2-6].


[1] Carsten Kutzner, Helmut Grubmüller, Bert L. de Groot, Ulrich Zachariae. Computational Electrophysiology: The Molecular Dynamics of Ion Channel Permeation and Selectivity in Atomistic Detail. Biophys. J. 101: 809-817 (2011).

[2] Jan-Philipp Machtens, Rodolfo Briones, Claudia Alleva, Bert L. de Groot, Christoph Fahlke. Gating Charge Calculations by Computational Electrophysiology Simulations. Biophys. J. 112: 1396-1405 (2017).

[3] Carsten Kutzner, David A. Köpfer, Jan-Philipp Machtens, Bert L. de Groot, Chen Song, Ulrich Zachariae. Insights into the function of ion channels by computational electrophysiology simulations. BBA Biomembranes 1858:1741-1752 (2016).

[4] Jan-Philipp Machtens, Daniel Kortzak, Christine Lansche, Ariane Leinenweber, Petra Kilian, Birgit Begemann, Ulrich Zachariae, David Ewers, Bert L. de Groot, Rodolfo Briones and Christoph Fahlke. Mechanisms of Anion Conduction by Coupled Glutamate Transporters. Cell 160:542-553 (2015).

[5] David A. Köpfer, Chen Song, Tim Gruene, George M. Sheldrick, Ulrich Zachariae and Bert L. de Groot. Ion permeation in K+ channels occurs by direct Coulomb knock-on. Science 346: 352-355 (2014).

[6] Chen Song, Conrad Weichbrodt, Evgeniy S. Salnikov, Marek Dynowski, Björn O. Forsberg, Burkhard Bechinger, Claudia Steinem, Bert L. de Groot, Ulrich Zachariae, and Kornelius Zeth. Crystal structure and functional mechanism of a human antimicrobial membrane channel Proc. Nat. Acad. Sci. 110: 4586-4591 (2013)

David A. Köpfer, Chen Song, Ulrich Zachariae, Rodolfo Briones

Steric zipper peptide aggregation

We employ molecular dynamics simulations in an explicit solvent environment to study the spontaneous aggregation of steric zipper peptides at atomistic detail. These short model peptides were recently shown to yield detailed structural insights into aggregates, known as amyloid fibrils, which are related to life threatening conditions in vivo (e.g. Alzheimers disease). Our aim is to unveil the energetic and structural determinants that drive the formation of amyloidogenic peptide assemblies, and also stabilize the formed aggregates.


[1] Dirk Matthes, Vytautas Gapsys, Julian Tim Brennecke and Bert L. de Groot. An Atomistic View of Amyloidogenic Self-assembly: Structure and Dynamics of Heterogeneous Conformational States in the Pre-nucleation Phase. Sci. Rep. 6: 33156 (2016).

[2] D. Matthes, V. Daebel, K. Meyenberg, D. Riedel, G. Heim, U. Diederichsen, A. Lange, B. L. de Groot. Spontaneous Aggregation of the Insulin-Derived Steric Zipper Peptide VEALYL Results in Different Aggregation Forms with Common Features. J. Mol. Biol., 426: 362-376 (2014).

[3] D. Matthes, V. Gapsys, B. L. de Groot, Driving forces and structural determinants of steric zipper peptide oligomer formation elucidated by atomistic simulations, J. Mol. Bio., 421: 390-416 (2012).

[4] D. Matthes, V. Gapsys, V. Daebel, B. L. de Groot, Mapping the conformational dynamics and pathways of spontaneous steric zipper peptide oligomerization, PLoS ONE, 6(5): e19129 (2011).

Dirk Matthes , Vytautas Gapsys

Thermodynamic Integration for Enhanced Protein Crystallization

In this project aim to rigorously calculate the changes in free energy and entropy that result from protein mutations by using alchemical free energy simulations. This method allows us to enhance protein crystallization in one of two ways. First, we can selectively stabilize a specific protein conformation through mutation. Having one dominant protein structure encourages the formation of regular crystals. Second, we can mutate high-entropy surface residues to lower entropy residues. This reduces the loss in entropy during protein crystallization, thus making the process more thermodynamically favorable.

Vance Jaeger

Regulation of Permeation in Aquaporins

Aquaporins and related aquaglyceroporins are versatile water channels that serve diverse purposes including the permeation of other solutes like glycerol and ammonia. The aim of this project is to investigate the mechanisms by which aquaporins regulate the permeation of water and alternate substrates. In this project we use diverse techniques such as umbrella sampling, Principle Component Analysis (PCA), Functional Mode Analysis (FMA) and Essential Dynamics to understnad how to relate the dynamics of the aquaporins structure to their function.


[1] Shreyas Kaptan, Mette Assentoft, Hans Peter Schneider, Robert A. Fenton, Joachim W. Deitmer, Nanna MacAulay, and Bert L. de Groot. H95 is a pH-dependent gate in aquaporin 4. Structure. 23:2309-2318 (2015).

[2] Mette Assentoft, Shreyas Kaptan, Robert A. Fenton, Susan Z. Hua, Bert L. Groot, Nanna MacAulay. Phosphorylation of rat aquaporin-4 at Ser111 is not required for channel gating Glia 61: 1101-1112 (2013).

[3] Mette Assentoft, Shreyas Kaptan, Hans Peter Schneider, Joachim W. Deitmer, Bert L. de Groot, and Nanna MacAulay. Aquaporin 4 as a NH3 Channel. J. Biol. Chem. 291: 19184-19195 (2016).

[4] Andreas Kirscht, Shreyas S. Kaptan, Gerd Patrick Bienert, Francois Chaumont, Poul Nissen, Bert L. de Groot, Per Kjellbom, Pontus Gourdon and Urban Johanson. Crystal Structure of an Ammonia Permeable Aquaporin. PLOS Biol. 14:e1002411 (2016).

Shreyas Kaptan

Ubiquitin Dynamics, Multispecificity, and Allostery

Ubiquitin is involved in many cellular processes, most notably proteasome-mediated proteolysis. To facilitate all of these functions, it needs to interact with hundreds of other proteins. In order to bind these many different molecular partners, ubiquitin must adopt different conformations. We have previously shown that ubiquitin takes these conformations not only when in contact with its binding partners, but also when free in solution (Lange et al., Science, 2008), suggesting a conformational selection binding mechanism. This conformational equilibrium can be rationally shifted to selectively destabilize binding to particular partners (Michielssens et al., Angew. Chem. Int. Ed., 2014). While most of the structural rearrangements can happen in solution prior to binding, some parts of the structure, particularly side chains, only change upon binding (Peters and de Groot, PLOS Comput. Biol., 2012), a process we refer to as “residual induced fit”. Importantly, ubiquitin side chain motions are not independent of the backbone, but are kinetically interrelated and form a hierarchy of timescales called “population shuffling” [1]. Through a detailed investigation into these kinetics, we have uncovered an allosteric network that enables multispecific binding through a conformational switch in a region of the protein that does not directly interact with the binding partners. We are continuing our investigations into the conformational dynamics and kinetics of ubiquitin [2]. In particular we are interested in how subdividing structural ensembles into a hierarchy of timescales can improve agreement with experimental data and give a more accurate representation of its structural properties.


[1] Colin A. Smith, David Ban, Supriya Pratihar, Karin Giller, Claudia Schwiegk, Bert L. de Groot, Stefan Becker, Christian Griesinger, Donghan Lee Population Shuffling of Protein Conformations Angew. Chem. Int. Ed. 54:207-210 (2015).

[2] Colin A. Smith, David Ban, Supriya Pratihar, Karin Giller, Maria Paulat, Stefan Becker, Christian Griesinger, Donghan Lee and Bert L. de Groot. Allosteric switch regulates protein-protein binding through collective motion. Proc. Nat. Acad. Sci. 113:3269-3274 (2016).

Colin Smith

Understanding the Molecular Machinery of Aquaporins through Molecular Dynamics Simulations

Aquaporins are protein channels responsible for the permeation of water and other solutes through biological membranes in response to osmotic pressure. The main goal is to expand our understanding on the molecular machinery of aquaporins by employing molecular dynamics simulations and related computational techniques. We provide a mechanism for the permeation of solutes through the Plasmodium falciparum aquaglyceroporin, a promising antimalarial drug target. In this mechanism, hydrophobic regions in the middle of the channel are the main water rate limiting barriers. Furthermore, the replacement of water-arginine interactions and solute-matching at the narrowest region of the channel are the main determinants underlying selectivity for the permeation of solutes like glycerol and urea (1). We also investigate the molecular determinants governing aquaporin gating, which has emerged as an efficient regulatory mechanism for organisms to quickly counteract sudden osmotic shocks. Our simulations, together with structural and functional studies, suggest that the yeast aquaporin-1 may be gated by both serine phosphorylation or mechanosensing (2). Furthermore, we observed voltage regulation of the single-channel water permeability of human AQP1 and AQP4 in silico, attributed to gating transitions of the arginine residue at the aromatic/arginine region. Our results suggests that voltage sensitivity may be a general feature of aquaporins, a hypothesis to be tested experimentally (3).


[1] Camilo Aponte-Santamaria, Jochen S. Hub and Bert L. de Groot. Dynamics and energetics of solute permeation through the Plasmodium falciparum aquaglyceroporin. PCCP. 12:10246-10254 (2010).

[2] Gerhard Fischer, Urszula Kosinska-Eriksson, Camilo Aponte-Santamaria, Madelene Palmgren, Cecilia Geijer, Kristina Hedfalk, Stefan Hohmann, Bert L. de Groot, Richard Neutze, Karin Lindkvist-Petersson. Crystal Structure of a Yeast Aquaporin at 1.15 Angstrom Reveals a Novel Gating Mechanism. PLoS Biology. 7: e1000130 (2009).

[3] Jochen S. Hub, Camilo Aponte-Santamaria, Helmut Grubmüller and Bert L. de Groot. Voltage-regulated water flux through aquaporin channels in silico. Biophys. J. 99:L97-L99 (2010)

[4] Camilo Aponte-Santamaria, Gerhard Fischer, Petra Bath, Richard Neutze, Bert L. Groot. Temperature dependence of protein-water interactions in a gated yeast aquaporin. Sci. Rep. 7: 4016 (2017).

Camilo Aponte-Santamaria

Ubiquitin Dynamics in Complexes

Protein-protein interactions play an important role in all metabolic processes. However, the principles underlying these interactions are only beginning to be understood. Ubiquitin is a small signalling protein that is covalently attached to proteins to mark them for degradation, regulate transport or induce other functions. As such, it interacts with and is recognized by a multitude of binding partners.

We use molecular dynamics simulations to investigate the effect of binding on ubiquitin by comparing simulation ensembles of ubiquitin bound to different binding partners with ensembles of unbound ubiquitin. Both collective structural behaviour and local conformational differences are being considered to identify the principles of ubiquitin binding and determine the influence of complex formation on the dynamic properties of this protein. Particularly the question of induced fit versus conformational selection scenarios both on a global and local level is investigated.


[1] Servaas Michielssens, Jan Henning Peters, David Ban, Supriya Pratihar, Daniel Seeliger, Monika Sharma, Karin Giller, Thomas Michael Sabo, Stefan Becker, Donghan Lee, Christian Griesinger and Bert L. de Groot. A Designed Conformational Shift To Control Protein Binding Specificity Angew. Chem. Int. Ed. 53: 10367-10371 (2014).

[2] Jan-Henning Peters and Bert L. de Groot. Ubiquitin dynamics in complexes reveal molecular recognition mechanisms beyond induced fit and conformational selection. PLOS Comp. Biol. 8: e1002704 (2012).

Jan Henning Peters, Servaas Michielssens


Allostery is essential for regulation in many biological systems. In allosteric systems the binding affinity of one binding site depends on the binding in a distant binding site. The information flow between these sites is assumed to be communicated through a conformational change of the system. It is still a challenging question to find this collective motion on an atomistic level. We currently focus on Hemoglobin, ABCE1 and GroEL/ES extracting collective motions from Molecular Dynamics simulations using Principal Component Analysis and related methods.


[1] Martin Vesper and Bert L. de Groot. Collective Dynamics Underlying Allosteric Transitions in Hemoglobin. PLoS Comp. Biol. 9: e1003232 (2013).

Martin Vesper

Inhibition of Membrane Channel Proteins

Membrane channel proteins, like ion and water channels, facilitate the permeation of water and solutes (e.g. ions) across cell membranes. They are important for many physiological processes as for example nerve conduction or the osmotic balance of cells. Dysfunction of these channels can cause severe diseases as Multiple sclerosis or Diabetes. Therefore, these channels are potential targets for future drugs. We investigate molecular determinants of channel blockage of druglike molecules using state-of-the-art computer simulations.


[1] Sören J. Wacker, Camilo Aponte-Santamaria, Per Kjellbom, Soren Nielsen, Bert L. de Groot, Michael Rützler. The identification of novel, high affinity AQP9 inhibitors in an intracellular binding site. Molec. Membrane Biol. 30:246-260 (2013).

[1] Sören J. Wacker, Wiktor Jurkowski, Katie J. Simmons, Colin W. G. Fishwick, Peter Johnson, David Madge, Erik Lindahl, Jean-Francois Rolland, Bert L. de Groot. Identification of Selective Inhibitors of the Potassium Channel Kv1.1-1.2(3) by High-Throughput Virtual Screening and Automated Patch Clamp. ChemMedChem 7:1775-1783 (2012)

Sören Wacker