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Google-Scholar Profile I am a biochemist by training with a mixed computational & experimental background. My early research focused on the biophysics of protein-protein interactions and structural dynamics using computational tools such as MD simulations, docking algorithms and lots of python programming. I later drifted back into the wet lab, helped developing standards and training programs for the emerging field of synthetic biology and started applying these principles to protein engineering. Ongoing collaborations with brilliant material scientists fuse our designer proteins with their designer materials and devices.

Bio(electronic) Sensors
in collaboration with Sahika Inal and her Organic Biolectronic Laboratory

What happens if you integrate a record-high-density layer of miniature antibodies (nanobodies) with a "wet transistor" technology (OECT) that amplifies electrical signals much better than any silicon device? You get bioelectronic sensors that can rapidly detect minute concentrations of target proteins in almost any kind of sample. Our latest "spyDirect" method self-assembles nanobodies onto gold electrodes to densities of close to 10 trillion (1013) molecules per cm2, all pointing "up" and ready to capture their target. We are constantly improving this technology and move it forward with partners in both clinic and industry.

*Guo K, *Wustoni S, *Koklu A, Diaz-Galicia E, Moser M, Hama A, Alqahtani AA, Ahmad AN, Alhamlan FS, Shuaib M, Pain A, McCulloch I, #Arold ST, #Grünberg R, #Inal S (2021) Rapid Single-Molecule Detection of COVID-19 and MERS Antigens via Nanobody-Functionalized Organic Electrochemical Transistors. Nature Biomedical Engineering

Guo K et al. #Arold ST, #Grünberg R, #Inal S (2023) SpyDirect enables autocatalytic and high-density nanobody functionalization for robust protein sensing. (submitted)

(* shared first co-authors; # shared corresponding)
additional papers in Advanced Materials and others, plus several patents
Therapeutic multi-enzyme Nanoreactors
in collaboration with Niveen Kashab and her Smart Hybrid Materials Lab as well as with Jasmmeen Merzaban and her team.

We are combining proteins with metal-organic frameworks (MOFs) to build multi-enzyme nanoreactors that can synthesize therapeutic molecules. Our nanoparticles operate in vitro but can also transplant a fully functional biosynthesis pathway into mammalian cells. The goal of this collaboration is to establish pathway MOFs as a platform for a novel class of intracellular "protein systems therapies".

*Sharip A, *Qutub SS, Baslyman W, Zhao L, #Khashab NM, #Arold ST, #Grünberg R (2023) Hierarchically engineered multi-enzyme nanoreactors for in vitro and intracellular drug biosynthesis. (submitted)
(* shared first co-authors; # shared corresponding)
Community
Synthetic Biology Open Language (SBOL)
Founding member, Editor (2015 - 2017)
iGEM
Team advisor to the Freiburg-Software team (2009)
and the Seattle robotics "team" (2009)
Chair of the iGEM software track (2014 - 2016)

EMBO Global Exchange "Introduction to Synthetic Biology" (Buenos Aires, 2012)
Co-organizer. The course and related activities helped kick-starting synbio in Latin America.
Lab automation

I have set up liquid handling systems in Montreal and, more recently, also at KAUST and train students and colleagues on their use. During the Covid pandemic, we implemented a custom RNA purification protocol for diagnostic RT-qPCR based on magnetic beads and reagents all produced in-house (by the lab of Mo Li).
Ongoing projects try to automate and scale up nanobody OECT measurements (with the Inal lab), automate the engineering of cell-free reactions (with the lab of Magnus Rueping) and implement a small-scale protein expression screen.

*Ramos-Mandujano G, *Grünberg R et al., Li M (2023) An Open-Source, Automated, and Cost-Effective Platform for COVID-19 Diagnosis and Rapid Portable Genomic Surveillance Using Nanopore Sequencing. (submitted) [GitHub]
(* shared first co-authors)
Software Development

Completed Projects

KAUST (2018-2020)
Cell-free multi-protein droplet microreactors
King Abdullah University of Science & Technology (KAUST)
Abstract We set up a multi-protein expression platform that combined microfluidic droplet generation, cell-free system, magnetic beads and rapid on-bead ligation or recombineering of multi-gene DNA. Cell-free droplet microreactors had been previously heralded as as a promising platform for protein engineering. In fact, standard microfluidics systems can quite easily generate millions of droplets which could, in theory, become the vessel for massively parallel in vitro evolution experiments. From a synthetic biology perspective, the co-expression of several proteins in a single droplet would then allow for the engineering of "protein systems".
In practice however, cell-free expression requires high DNA concentrations. It was not clear how one could reach the necessary quantity of DNA while at the same providing different DNA templates to different droplets. Our solution was to couple single- or multi-gene DNA fragments to micro-beads, which we then co-encapsulated with a commercial cell-free system. We demonstrate that up to three different proteins can be co-expressed to high concentrations in a single droplet but that we can also create "genetic diversity" between droplets.
Paper Restrepo Sierra, A.M., Arold, S.A., Grünberg, R. (2022) Efficient multi-gene expression in cell-free droplet microreactors. PLoS ONE
 

research associate (2013-2015)
Automated DNA Assembly
IRIC, University of Montreal
Talk A Field Guide to Automated Cloning
(COMBINE, October 2015, Salt Lake City, Utah) [Slides]
Poster Cellular and Molecular Biotechnology, 12/2015, Paris [view]
Abstract The assembly of larger DNA constructs, often from a mix of gene synthesized and in-house fragments, is the starting point for most synthetic biology projects. Despite major technical advances, DNA assembly remains a bottleneck in many laboratories.
We have implemented a robotic synthetic biology workstation that automates the complete multi-fragment DNA assembly work flow. This includes fragment PCR setup, cleanup, Gibson assembly, transformation, spreading on standard microplates, robust colony picking, colony PCR, DNA miniprep and auxiliary steps. Usability and user-adoption is facilitated by a strictly modular design as well as by convenient configuration through MS-Excel tables.
Paper Tecan Application Note (not peer reviewed): Grünberg R, van der Sloot A., Xu X., Tyers M. (2015) Fully integrated plating and colony picking for synthetic biology workflows. [PDF] [PDF via Tecan]
(We are working on a proper paper. The Tecan folks also show-cased our platform in their Tecan Journal)
 

postdoc / research associate (2010-2013)
hiFRET -- the Power of Weak Interactions
Center for Genomic Regulation (CRG), Barcelona
IRIC, University of Montreal
Poster
Abstract FRET (Fluorescent Resonance Energy Transfer) is currently the only practical method to localize and image protein interactions in live cells. But FRET sensor design is complicated by the need to bring the two fluorescent proteins (donor and acceptor) very close together. To overcome this limitation, we engineered very weak affinities between donor and acceptor probes. Not unlike the magnetic cover of a tablet computer, once donor and acceptor are brought together by the interaction of interest, our helper affinity is pulling them into a better orientation for strong FRET.
We realized these helper interactions in two different ways. First, we created an electrostatic "encounter complex" between FRET probes using computational design. In a second approach we instead attached specific but very weak "protein interaction modules" from natural signalling proteins. This second variant can be immediately applied to any of a wide variety of modern fluorescent proteins.
We tested hiFRET probes for the detection of different protein interactions in vitro and in live cells. Thanks to hiFRET, we were the first to image the interaction between BRaf and CRaf proteins in response to a new anti-cancer drug. This interaction offers tumors a way to escape the cancer treatment and is therefore also of medical interest.
Paper Grünberg R, Burnier JV, Ferrar T, Beltran-Sastre V, Stricher F, van der Sloot A, Garcia-Olivas R, Mallabiabarrena A, Sanjuan X, Zimmermann T, Serrano L (2013): Engineering of weak helper interactions for high-efficiency FRET probes. Nature Methods.

News and Views by Kees Jalink. (requires subsription)
 

postdoc (2006-2010)
Protein Synthetic Biology
Center for Genomic Regulation (CRG), Barcelona
Poster HFSP Fellows meeting, Tokyo, June 2009
Abstract Can synthetic proteins and sophisticated protein circuits be built from standard parts? Can we decompose dynamic protein networks into re-usable devices? What are the best strategies and methods for protein systems engineering? These are some of the questions that I have been working on since switching back to the wet lab bench. Despite all their complexity (see my previous projects below), I think that proteins are, in fact, a very good material for systems engineering. A focus on modular protein-protein interactions may be our best starting point for the manipulation of natural and the design of synthetic protein networks. In a proof-of-concept study, we have constructed 25 synthetic two-domain proteins from BioBrick-formatted standardized "parts", purified many of them and characterized different combinations of interaction input and output "devices" biophysically (using FRET and surface plasmon resonance). Our parts are available for free re-use from the Registry of Standard Biological Parts and a list of protocols is maintained on OpenWetware.
Paper Grünberg R, Ferrar T, van der Sloot A, Constante M, Serrano L (2010) Building blocks for protein interaction devices. Nucleic Acids Research doi: 10.1093/nar/gkq152. [Full text]

Grünberg R, Serrano L (2010): Strategies for Protein Synthetic Biology. (review) Nucleic Acids Research.
 

postdoc (2007-2009)
(Voronoi-) Shelling of Protein Interfaces
Collaboration with Frederic Cazals (INRIA Sophia-Antipolis), Benjamin Bouvier (CNRS Lyon) and Michael Nilges (Institut Pasteur)
Abstract Protein-protein interfaces are, traditionally, defined by different "ad-hoc" criteria such as atom contacts or changes in surface areas. However, these descriptions are rather arbitrary and complicate the systematic comparison of protein interface architectures. We here extend a fast, parameter-free and purely geometric definition of protein interfaces and introduce the shelling order of Voronoi facets (VSO) as a novel measure for an atom's depth inside the interface. VSO can predict the solvent exchange in different interface regions, that is, the occurence of "dry" or "wet" residues. In fact, the seemingly complex water dynamics at protein interfaces appears largely controlled by geometry. Also the sequence conservation and residue composition of interfaces is often "radially" structured. Voronoi Shelling Order thus reveals clear geometric patterns in protein interface composition, function and dynamics and facilitates the comparative analysis of protein-protein interactions.
Paper Bouvier B*, Grünberg R*, Nilges M, Cazals F (2009): Shelling the Voronoi interface of protein-protein complexes reveals patterns of residue conservation, dynamics, and composition. Proteins 15;76(3):677-92. [Abstract]
(* shared first co-authors)
 

PhD project (2001 - 2005)
The dynamics of protein-protein binding
Institut Pasteur, Paris
Talk Protein flexibility and entropy on the edge of binding
(International Biophysics Congress, August 2005, Montpellier)
Poster Conference on "Modeling of Protein Interactions in Genomes", June 2003, Stony Brook, New York; June 2005, Kansas
Abstract The interplay between protein-protein binding and structural dynamics is poorly understood. We selected a set of 17 protein-protein complexes for which the three-dimensional structures of both free components and the complex are available. Based on the analysis of molecular dynamics simulations in explicit water for each of these 51 systems, we find: (1) most uncomplexed binding sites are more flexible than the remaining surface; (2) as expected, they loose conformational freedom upon complex formation; (3) however, in the majority of cases, binding does not restrict the overall motion of proteins. We calculated the change in conformational entropy from longer simulations on 7 complexes (21 systems) using a new method based on quasiharmonic analysis. Two small complexes and an antibody-antigen system exhibited a significant loss whereas three larger complexes showed increased or unchanged conformational entropy.
Molecular fluctuations excert important influence both on the speed of recognition and the stability of protein complexes. Structure dynamics may be the missing link in our understanding of this fundamental process. This is the largest study so far on the influence of complex formation on protein flexibility and conformational entropy.
Paper Grünberg, R., M. Nilges, J. Leckner (2006): Flexibility and entropy in protein-protein binding. Structure 14(4), 683-93 [PDF]
 

PhD project (2001 - 2004)
A model of flexible protein-protein recognition
Institut Pasteur, Paris
Talk Complementarity of structure ensembles in protein-protein binding
(CECAM workshop on "Flexible Macromolecular Docking", April 2004, Lyon)
Abstract Protein-protein association is often accompanied by changes in receptor and ligand structure. This interplay between protein flexibility and protein-protein recognition is currently the largest obstacle to our understanding and also to the reliable prediction of protein complexes. We performed two sets of molecular dynamics simulations for the unbound receptor and ligand structures of 17 protein complexes and applied shape-driven rigid body docking to all combinations of representative snapshots. The cross docking of structure ensembles increased the chances to find near native solutions. The free ensembles appeared to contain multiple complementary conformations. These were in general not related to the bound structure. We suggest that protein-protein binding follows a three-step mechanism of diffusion, free conformer selection and folding. This model combines previously conflicting ideas and is in better agreement with the current data on interaction forces, time scales, and kinetics.
More Details...
Paper *Grünberg, R., *J. Leckner, M. Nilges (2004): Complementarity of structure ensembles in protein-protein binding. Structure 12, 2125-2136. [PubMed] [pre-print] (with minor differences) (* shared first co-authors)

Comment by M. Eisenstein in the same issue: Introducing a 4th dimension to protein-protein docking. [PDF]

Quote of our model in a commentary by Boehr and Wright in Science: How do proteins interact?

 

PhD project (2002 - 2004)
CROW - A data integration platform for the semantic web
Institut Pasteur, Paris
Talk Crowsing the semantic web
(Journee bioinformatique, February 2004, Institut Pasteur)
Poster Pacific Symposium on Biocomputing, January 2004, Hawaii
Abstract Crow (Computational Representation Of Whatever) is a (alpha-stage) Java library for an anarchist approach to data integration (i.e. link whatever you want whenever you want to whereever you want). The library is tailored (but not restricted) to the work with semantic web documents. It allows the mining and manipulation of distributed, heterogeneous, chaotically cross-linked data. The design is multi-threaded, event-based, and supports plugins and third-party data types. It containes a simple Python (Jython) interface for interactive work. The library is freely available under the GPL. We currently use it to select targets for protein-protein docking from sets of predicted protein interactions and various other information.
More details...
SourceForge project...
Paper I somehow never got to publish this...but the code is on sourceforge and well documented. If anybody wants to join me converting this into a Python library... send me an e-mail ;-)
 

PhD project (warm up) (06/2000 - 12/2001)
The dynamic topology of spectrin repeats
EMBL, Heidelberg and Institut Pasteur, Paris
Talk Stretching a molecular shock absorber
(Congress des jeunes chercheurs, 18/06/2003, Institut Pasteur)
Poster Gordon conference on "Protein folding dynamics", January 2002, Ventura, U.S.
Abstract Spectrin repeats are triple-helical domains found in many proteins that are regularly subjected to mechanical stress. My collaborators studied the behavior of a wild-type spectrin repeat and two mutants with atomic force microscopy. I interpreted the results of these single molecule experiments with steered molecular dynamics simulations. The experiments indicated that spectrin repeats can form stable unfolding intermediates when subjected to external forces. In the simulations the unfolding proceeded via a variety of pathways. Stable intermediates were associated to kinking of the central helix close to a proline residue. A mutant stabilizing the central helix showed no intermediates in experiments, in agreement with the simulations. Simulations and experiments hence suggest a "hyphenation point" within the domain and hint at the existence of force-resistant intermediates with non-native topology. The simulations also explained the broad variance of experimental unfolding lenghts. The combination of both effects would allow for a smooth and elastic response to external forces over a wide range of extensions. Spectrin molecules seem to be optimized for elasticity on all scales of their molecular architecture.
More Details...
Watch it unfolding...Movie :)
Paper *Altmann S.M., R. *Grünberg, P.F. Lenne, J. Ylänne, A. Raae, K. Herbert, M. Saraste, M. Nilges, J.K. Hörber (2002): Pathways and intermediates in forced unfolding of spectrin repeats. Structure 10, 1085-1096. [PubMed] (* shared first co-authors)
 

Diploma Project (03/99 - 10/99)
Rational Design of Trypsin Variants for Peptide Synthesis
University of Leipzig, under the guidance of Dr. Frank Bordusa
Talk Hacking Trypsin (given on June 25st 1999 in Leipzig)
Abstract Based on their catalytic mechanism it is possible to employ serine proteases like trypsin for enzymatic peptide bond formation. Especially, substrate mimetics - a new class of artificial substrates, allow for efficient ligations of amino acid derivatives and peptides, as Bordusa et al. demonstrated in recent years. However, if product or starting material contain specific cleavage sites recognised by the protease, this ligation is counteracted by the (natural) proteolytic reaction.
A promising approach to overcome this problem is the direct manipulation of the enzyme's natural specifity. I prepared and isolated two variants of rat-trypsin with altered S1-binding pocket (D189A and D189A/S190A). The proteins were examined by HPLC-based acyl-transfer experiments revealing promising catalytic properties as well as an impaired specifity towards natural trypsin and chymotrypsin substrates.
Parallely, I employed the program AutoDock to simulate the binding of several substrates to the altered enzymes in silico. With these docking studies I could elucidate, in part unexpected, experimental results on a structural basis.
More Details...
Paper Grünberg, R., I. Domgall, R. Günther, H.-J. Hofmann, and F. Bordusa (2000): Peptide Bond Formation Mediated by Substrate Mimetics: Structure-guided Optimisation of Trypsin for Synthesis. Eur J Biochem 267(24), 7024-30.
 

undergrad project (02/98 - 06/98)
Analysing the adc promoter in Clostridium beijerinckii
with the gusA reporter gene
University of Wales, Aberystwyth, under the guidance of Dr. Adriana Ravagnani and Prof. Michael Young
Talk A reporter gene for Chlostridium (given in April 1998 in Aberystwyth)
Abstract I introduced ß-D-glucuronidase (gusA) of Escherichia coli as reporter gene in Clostridium beijerinckii, so far lacking a versatile reporter system, by constructing the promoter probe vector pRGus. This vector was employed to evaluate the significance of three putative "OA box" sequence motifs within the promoter of the acetoacetate decarboxylase gene (adc) in vivo. I fused the adc promoter, as well as a variant with destroyed OA boxes, to the gusA gene in pRGus and introduced them into C. beijerinckii by electroporation.
Preliminary tests revealed a high ß-D-glucuronidase (GUS) activity in transformants containing the wild type adc promoter compared with no detectable GUS activity in case of the construct with destroyed OA boxes. Since OA boxes are recognition sequences for the Spo0A transcription factor, these findings indicate that the expression of the adc gene is directly regulated by Spo0A.
Acetoacetate decarboxylase is involved in the metabolic conversion of starches (and various other substrates) into commercially significant solvents like acetone and butanol. The gusA reporter gene will facilitate further investigation into the trancsiptional regulation of this pathway.
Paper Ravagnani, A., K. C. B. Jennert, E. Steiner, R. Grünberg, J. R. Jefferies, S. R. Wilkinson, D. I. Young, E. C. Tidswell, D. P. Brown, P. Youngman, J. G. Morris & M. Young. (2000): Spo0A directly controls the switch from acid to solvent production in solvent-forming clostridia. Molecular Microbiology 37(5), 1172-85.