the Tans Biophysics LabAMOLF, Amsterdam

Key Publications

Small heat shock proteins sequester misfolding proteins in near-native conformation for cellular protection and efficient refolding
Sophia Ungelenk et al. | Nature Commun. 7, 13673: 1-8 (2016) | pdf & DOI: 10.1038/ncomms13673
Alternative modes of client binding enable functional plasticity of Hsp70
Alireza Mashaghi et al. | Nature 539, 448-451 (2016) | pdf & DOI: 10.1038/nature20137
Breaking evolutionary constraint with a tradeoff ratchet
Marjon G.J. de Vos et al. | PNAS 112, 14906-14911 (2015) | pdf & DOI: 10.1073/pnas.1510282112
High-throughput 3D tracking of bacteria on a standard phase contrast microscope
K.M. Taute et al. | Nature Commun. 6, 8776: 1-9 (2015) | pdf & DOI: 10.1038/ncomms9776
The Trigger Factor Chaperone Encapsulates and Stabilizes Partial Folds of Substrate Proteins
Kushagra Singhal et al. | PLoS Comput. Biol. 11, e1004444: 1-19 (2015) | pdf & DOI: 10.1371/journal.pcbi.1004444
Stochasticity of metabolism and growth at the single-cell level
Daniel J. Kiviet et al. | Nature 514, 376-379 (2014) | pdf & DOI: 10.1038/nature13582
Reshaping of the conformational search of a protein by the chaperone trigger factor
Alireza Mashaghi et al. | Nature 500, 98-101 (2013) | pdf & DOI: 10.1038/nature12293
Environmental dependence of genetic constraint
Marjon G. J. de Vos et al.| PLoS Genet. 9, e1003580 1-8 (2013) | pdf & DOI: 10.1371/journal.pgen.1003580
Tradeoffs and optimality in the evolution of gene regulation
Frank J. Poelwijk et al. | Cell 146, 462-470 (2011) | pdf & DOI:10.1016/j.cell.2011.06.035
Calcium modulates force sensing by the von Willebrand factor A2 domain
Arjen J. Jakobi et al. | Nat. Commun. 2, 385 1-9 (2011) | pdf & DOI:10.1038/ncomms1385
Direct observation of type 1 fimbrial switching
Aileen Adiciptaningrum et al. | EMBO Reports 10:527-532 (2009) | pdf & DOI:10.1038/embor.2009.25
Direct Observation of Chaperone-Induced Changes in a Protein Folding Pathway
Philipp Bechtluft1, Ruud van Leeuwen1 et al. | Science 318:1458-1461 (2007) | pdf & DOI:10.1126/science.1144972
Empirical fitness landscapes reveal accessible evolutionary paths
Frank Poelwijk1, Daan Kiviet1 et al. | Nature 445:383-386 (2007) | pdf & DOI:10.1038/nature05451
Evolutionary potential of a duplicated repressor-operator pair: simulating pathways using mutation data
Frank Poelwijk1, Daan Kiviet1, Sander J. Tans | PLoS Comput Biol. 2:e58 (2006) | pdf & DOI:10.1371/journal.pcbi.0020058
The bacteriophage straight phi29 portal motor can package DNA against a large internal force
Douglas E. Smith1, Sander J. Tans1 et al. | Nature. 413:748-52 (2001) | pdf & DOI:10.1038/35099581
Molecular transistors: Potential modulations along carbon nanotubes
Sander J. Tans, Cees Dekker. | Nature. 404:834-35 (2000) | pdf & DOI:10.1038/35009026
Imaging electron wave functions of quantized energy levels in carbon nanotubes
Liesbeth C. Venema et al. | Nature. 413:748-52 (1999) | pdf & DOI:10.1126/science.283.5398.52
Electron-electron correlations in carbon nanotubes
Sander J. Tans et al. | Nature. 394:761-64 (1998) | pdf & DOI:10.1038/29494
Room-temperature transistor based on a single carbon nanotube
Sander J. Tans, Alwin R. M. Verschueren & Cees Dekker | Nature. 393:49-52 (1998) | pdf & DOI:10.1038/29954
Individual single-wall carbon nanotubes as quantum wires
Sander J. Tans et al. | Nature. 386:474-77 (1997) | pdf & DOI:10.1038/386474a0
Fullerene 'crop circles'
Jie Liu et al. | Nature 385, 780-781 (1997) | pdf & DOI:10.1038/385780b0
1 joint first authors

All Publications

Small heat shock proteins sequester misfolding proteins in near-native conformation for cellular protection and efficient refolding
Sophia Ungelenk, Fatemeh Moayed, Chi-Ting Ho, Tomas Grousl, Annette Scharf, Alireza Mashaghi, Sander Tans, Matthias P. Mayer, Axel Mogk & Bernd Bukau
Nature Commun. 7, 13673: 1-8 (2016)
pdf & DOI: 10.1038/ncomms13673

Abstract

Small heat shock proteins (sHsp) constitute an evolutionary conserved yet diverse family of chaperones acting as first line of defence against proteotoxic stress. sHsps coaggregate with misfolded proteins but the molecular basis and functional implications of these interactions, as well as potential sHsp specific differences, are poorly explored. In a comparative analysis of the two yeast sHsps, Hsp26 and Hsp42, we show in vitro that model substrates retain near-native state and are kept physically separated when complexed with either sHsp, while being completely unfolded when aggregated without sHsps. Hsp42 acts as aggregase to promote protein aggregation and specifically ensures cellular fitness during heat stress. Hsp26 in contrast lacks aggregase function but is superior in facilitating Hsp70/Hsp100-dependent post-stress refolding. Our findings indicate the sHsps of a cell functionally diversify in stress defence, but share the working principle to promote sequestration of misfolding proteins for storage in native-like conformation.

Alternative modes of client binding enable functional plasticity of Hsp70
Alireza Mashaghi, Sergey Bezrukavnikov, David P. Minde, Anne S. Wentink, Roman Kityk, Beate Zachmann-Brand, Matthias P. Mayer, Günter Kramer, Bernd Bukau & Sander J. Tans
Nature 539, 448-451 (2016)
pdf & DOI: 10.1038/nature20137

Abstract

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation1–5, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins6-10. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid11–15. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70–substrate complexes raise the possibility of additional interaction mechanisms 15–18. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle19. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide- regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

Single-Cell Analysis of the Dps Response to Oxidative Stress
Michela de Martino, Dmitry Ershov, Peter J. van den Berg, Sander J. Tans & Anne S. Meyer
J. Bacteriol. 198, 11: 1662-1674 (2016)
pdf & DOI: 10.1128/JB.00239-16

Abstract

Microorganisms have developed an elaborate spectrum of mechanisms to respond and adapt to environmental stress conditions. Among these is the expression of dps, coding for the DNA-binding protein from starved cells. Dps becomes the dominant nucleoid-organizing protein in stationary-phase Escherichia coli cells and is required for robust survival under stress conditions, including carbon or nitrogen starvation, oxidative stress, metal exposure, and irradiation. To study the complex regulation of Dps in E. coli, we utilized time-lapse fluorescence microscopy imaging to examine the kinetics, input encoding, and variability of the Dps response in single cells. In the presence of an oxidative stressor, we observed a single pulse of activation of Dps production. Increased concentrations of H2O2 led to increased intensity and duration of the pulse. While lower concentrations of H2O2 robustly activated the Dps response with little effect on the growth rate, higher concentrations of H2O2 resulted in dramatically lower and highly varied growth rates. A comparison of cells exposed to the same concentration of H2O2 revealed that increased levels of Dps expression did not confer a growth advantage, indicating that recovery from stress may rely primarily upon variation in the amount of damage caused to individual cells.
IMPORTANCE: We show for the first time the response of the DNA-binding protein from starved cells ( Dps) to oxidative stress in single cells of E. coli. Through time-lapse fluorescence microscopy, a single pulse of Dps production is observed in cells exposed to H2O2, with a duration and intensity of induction proportional to the concentration of the applied stress. More intense Dps expression did not provide a growth benefit to the bacteria, suggesting that healing from oxidative stress may largely depend upon the amount of damage in each individual cell.

Generation and filtering of gene expression noise by the bacterial cell cycle
Noreen Walker, Philippe Nghe & Sander J. Tans
BMC Biol. 14, 11: 1-20 (2016)
pdf & DOI: 10.1186/s12915-016-0231-z

Abstract

BACKGROUND: Gene expression within cells is known to fluctuate stochastically in time. However, the origins of gene expression noise remain incompletely understood. The bacterial cell cycle has been suggested as one source, involving chromosome replication, exponential volume growth, and various other changes in cellular composition. Elucidating how these factors give rise to expression variations is important to models of cellular homeostasis, fidelity of signal transmission, and cell-fate decisions.
RESULTS: Using single-cell time-lapse microscopy, we measured cellular growth as well as fluctuations in the expression rate of a fluorescent protein and its concentration. We found that, within the population, the mean expression rate doubles throughout the cell cycle with a characteristic cell cycle phase dependent shape which is different for slow and fast growth rates. At low growth rate, we find the mean expression rate was initially flat, and then rose approximately linearly by a factor two until the end of the cell cycle. The mean concentration fluctuated at low amplitude with sinusoidal-like dependence on cell cycle phase. Traces of individual cells were consistent with a sudden two-fold increase in expression rate, together with other non-cell cycle noise. A model was used to relate the findings and to explain the cell cycle-induced variations for different chromosomal positions.
CONCLUSIONS: We found that the bacterial cell cycle contribution to expression noise consists of two parts: a deterministic oscillation in synchrony with the cell cycle and a stochastic component caused by variable timing of gene replication. Together, they cause half of the expression rate noise. Concentration fluctuations are partially suppressed by a noise cancelling mechanism that involves the exponential growth of cellular volume. A model explains how the functional form of the concentration oscillations depends on chromosome position.
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Individuality and universality in the growth-division laws of single E. coli cells
Andrew S. Kennard, Matteo Osella, Avelino Javer, Jacopo Grilli, Philippe Nghe, Sander J. Tans, Pietro Cicuta & Marco Cosentino Lagomarsino
Phys. Rev. E 19, 012408: 1-18 (2016)
pdf & DOI: 10.1103/PhysRevE.93.012408

Abstract

The mean size of exponentially dividing Escherichia coli cells in different nutrient conditions is known to depend on the mean growth rate only. However, the joint fluctuations relating cell size, doubling time, and individual growth rate are only starting to be characterized. Recent studies in bacteria reported a universal trend where the spread in both size and doubling times is a linear function of the population means of these variables. Here we combine experiments and theory and use scaling concepts to elucidate the constraints posed by the second observation on the division control mechanism and on the joint fluctuations of sizes and doubling times. We found that scaling relations based on the means collapse both size and doubling-time distributions across different conditions and explain how the shape of their joint fluctuations deviates from the means. Our data on these joint fluctuations highlight the importance of cell individuality: Single cells do not follow the dependence observed for the means between size and either growth rate or inverse doubling time. Our calculations show that these results emerge from a broad class of division control mechanisms requiring a certain scaling form of the “division hazard rate function,” which defines the probability rate of dividing as a function of measurable parameters. This “model free” approach gives a rationale for the universal body-size distributions observed in microbial ecosystems across many microbial species, presumably dividing with multiple mechanisms. Additionally, our experiments show a crossover between fast and slow growth in the relation between individual-cell growth rate and division time, which can be understood in terms of different regimes of genome replication control.

Stochasticity and homeostasis in the E. coli replication and division cycle
Aileen M. Adiciptaningrum, Matteo Osella, M.Charl Moolman, Marco Cosentino Lagomarsino & S.J. Tans
Sci. Rep. 5, 18261: 1-8 (2015)
pdf & DOI: 10.1038/srep18261

Abstract

How cells correct for stochasticity to coordinate the chromosome replication and cellular division cycle is poorly understood. We used time-lapse microscopy and fluorescently labelled SeqA to determine the timing of birth, initiation, termination, and division, as well as cell size throughout the cell cycle. We found that the time between birth and initiation (B-period) compensates for stochastic variability in birth size and growth rate. The time between termination and division (D-period) also compensates for size and growth variability, invalidating the notion that replication initiation is the principal trigger for cell division. In contrast, the time between initiation and termination (C-period) did not display such compensations. Interestingly, the C-period did show small but systematic decreases for cells that spontaneously grew faster, which suggests a coupling between metabolic fluctuations and replication. An auto-regressive theoretical framework was employed to compare different possible models of sub-period control.

Breaking evolutionary constraint with a tradeoff ratchet
Marjon G.J. de Vos, Alexandre Dawid, Vanda Sunderlikova & Sander J. Tans
PNAS 112, 14906-14911 (2015)
pdf & DOI: 10.1073/pnas.1510282112

Abstract

Epistatic interactions can frustrate and shape evolutionary change. Indeed, phenotypes may fail to evolve when essential mutations are only accessible through positive selection if they are fixed simultaneously. How environmental variability affects such constraints is poorly understood. Here, we studied genetic constraints in fixed and fluctuating environments using the Escherichia coli lac operon as a model system for genotype-environment interactions. We found that, in different fixed environments, all trajectories that were reconstructed by applying point mutations within the transcription factor-operator interface became trapped at suboptima, where no additional improvements were possible. Paradoxically, repeated switching between these same environments allows unconstrained adaptation by continuous improvements. This evolutionary mode is explained by pervasive cross-environmental tradeoffs that reposition the peaks in such a way that trapped genotypes can repeatedly climb ascending slopes and hence, escape adaptive stasis. Using a Markov approach, we developed a mathematical framework to quantify the landscape-crossing rates and show that this ratchet-like adaptive mechanism is robust in a wide spectrum of fluctuating environments. Overall, this study shows that genetic constraints can be overcome by environmental change and that cross-environmental tradeoffs do not necessarily impede but also, can facilitate adaptive evolution. Because tradeoffs and environmental variability are ubiquitous in nature, we speculate this evolutionary mode to be of general relevance.

High-throughput 3D tracking of bacteria on a standard phase contrast microscope
K.M. Taute, S. Gude, S.J. Tans & T.S. Shimizu
Nature Commun. 6, 8776: 1-9 (2015)
pdf & DOI: 10.1038/ncomms9776

Abstract

Bacteria employ diverse motility patterns in traversing complex three-dimensional (3D) natural habitats. 2D microscopy misses crucial features of 3D behaviour, but the applicability of existing 3D tracking techniques is constrained by their performance or ease of use. Here we present a simple, broadly applicable, high-throughput 3D bacterial tracking method for use in standard phase contrast microscopy. Bacteria are localized at micron-scale resolution over a range of 350x300x200 μm by maximizing image cross-correlations between their observed diffraction patterns and a reference library. We demonstrate the applicability of our technique to a range of bacterial species and exploit its high throughput to expose hidden contributions of bacterial individuality to population-level variability in motile behaviour. The simplicity of this powerful new tool for bacterial motility research renders 3D tracking accessible to a wider community and paves the way for investigations of bacterial motility in complex 3D environments.

The Trigger Factor Chaperone Encapsulates and Stabilizes Partial Folds of Substrate Proteins
Kushagra Singhal, Jocelyne Vreede, Alireza Mashaghi, Sander J. Tans & Peter G. Bolhuis
PLoS Comput. Biol. 11, e1004444: 1-19 (2015)
pdf & DOI: 10.1371/journal.pcbi.1004444

Abstract

How chaperones interact with protein chains to assist in their folding is a central open question in biology. Obtaining atomistic insight is challenging in particular, given the transient nature of the chaperone-substrate complexes and the large system sizes. Recent single-molecule experiments have shown that the chaperone Trigger Factor (TF) not only binds unfolded protein chains, but can also guide protein chains to their native state by interacting with partially folded structures. Here, we used all-atom MD simulations to provide atomistic insights into how Trigger Factor achieves this chaperone function. Our results indicate a crucial role for the tips of the finger-like appendages of TF in the early interactions with both unfolded chains and partially folded structures. Unfolded chains are kinetically trapped when bound to TF, which suppresses the formation of transient, non-native end-to-end contacts. Mechanical flexibility allows TF to hold partially folded structures with two tips (in a pinching configuration), and to stabilize them by wrapping around its appendages. This encapsulation mechanism is distinct from that of chaperones such as GroEL, and allows folded structures of diverse size and composition to be protected from aggregation and misfolding interactions. The results suggest that an ATP cycle is not required to enable both encapsulation and liberation.

Non-monotonic dynamics and crosstalk in signaling pathways and their implications for pharmacology
Roeland J. van Wijk, Sander Tans, Pieter Rein ten Wolde & Alireza Mashaghi
Sci. Rep. 5, 11376 1-13 (2015)
pdf & DOI: 10.1038/srep11376

Abstract

Currently, drug discovery approaches commonly assume a monotonic dose-response relationship. However, the assumption of monotonicity is increasingly being challenged. Here we show that for two simple interacting linear signaling pathways that carry two different signals with different physiological responses, a non-monotonic input-output relation can arise with simple network topologies including coherent and incoherent feed-forward loops. We show that non-monotonicity of the response functions has severe implications for pharmacological treatment. Fundamental constraints are imposed on the effectiveness and toxicity of any drug independent of its chemical nature and selectivity due to the specific network structure.

Random fluctuations, metabolism and growth at the single-cell level
Philippe Nghe, Sarah Boulineau & Sander J. Tans
Med. Sci. 31, 233-235 (2015)
pdf & DOI: 10.1051/medsci/20153103002

Circuit topology of self-interacting chains: implications for folding and unfolding dynamics
Andrew Mugler, Sander J. Tans & Alireza Mashaghi
Phys. Chem. Chem. Phys. 16, 22537-22544 (2014)
pdf & DOI: 10.1039/c4cp03402c

Abstract

Understanding the relationship between molecular structure and folding is a central problem in disciplines ranging from biology to polymer physics and DNA origami. Topology can be a powerful tool to address this question. For a folded linear chain, the arrangement of intra-chain contacts is a topological property because rearranging the contacts requires discontinuous deformations. Conversely, the topology is preserved when continuously stretching the chain while maintaining the contact arrangement. Here we investigate how the folding and unfolding of linear chains with binary contacts is guided by the topology of contact arrangements. We formalize the topology by describing the relations between any two contacts in the structure, which for a linear chain can either be in parallel, in series, or crossing each other. We show that even when other determinants of folding rate such as contact order and size are kept constant, this 'circuit' topology determines folding kinetics. In particular, we find that the folding rate increases with the fractions of parallel and crossed relations. Moreover, we show how circuit topology constrains the conformational phase space explored during folding and unfolding: the number of forbidden unfolding transitions is found to increase with the fraction of parallel relations and to decrease with the fraction of series relations. Finally, we find that circuit topology influences whether distinct intermediate states are present, with crossed contacts being the key factor. The approach presented here can be more generally applied to questions on molecular dynamics, evolutionary biology, molecular engineering, and single-molecule biophysics.

Stochasticity of metabolism and growth at the single-cell level
Daniel J. Kiviet, Philippe Nghe, Noreen Walker, Sarah Boulineau, Vanda Sunderlikova & Sander J. Tans
Nature 514, 376-379 (2014)
pdf & DOI: 10.1038/nature13582

Abstract

Elucidating the role of molecular stochasticity in cellular growth is central to understanding phenotypic heterogeneity and the stability of cellular proliferation. The inherent stochasticity of metabolic reaction events should have negligible effect, because of averaging over the many reaction events contributing to growth. Indeed, metabolism and growth are often considered to be constant for fixed conditions. Stochastic fluctuations in the expression level of metabolic enzymes could produce variations in the reactions they catalyse. However,whether suchmolecular fluctuations can affect growth is unclear, given the various stabilizing regulatory mechanisms, the slow adjustment of key cellular components such as ribosomes, and the secretion and buffering of excess metabolites. Here we use time-lapse microscopy to measure fluctuations in the instantaneous growth rate of single cells of Escherichia coli, and quantify time-resolved cross-correlations with the expression oflac genes and enzymes in central metabolism. We show that expression fluctuations of catabolically active enzymes can propagate and cause growth fluctuations, with transmission depending on the limitation of the enzyme to growth. Conversely, growth fluctuations propagate back to perturb expression. Accordingly, enzymes were found to transmit noise to other unrelated genes via growth.Homeostasis is promoted by a noise-cancelling mechanism that exploits fluctuations in the dilution of proteins by cell-volume expansion. The results indicate thatmolecular noise is propagatednot only by regulatory proteins but also by metabolic reactions. They also suggest that cellular metabolism is inherently stochastic, and a generic source of phenotypic heterogeneity.

Misfolding of Luciferase at the Single-Molecule Level
Alireza Mashaghi, Samaneh Mashaghi and Sander J. Tans
Ang. Chem., Int. Ed. 25, 10390-10393 (2014)
pdf & DOI: 10.1002/anie.201405566

Abstract

The folding of complex proteins can be dramatically affected by misfolding transitions. Directly observing misfolding and distinguishing it from aggregation is challenging. Experiments with optical tweezers revealed transitions between the folded states of a single protein in the absence of mechanical tension. Nonfolded chains of the multidomain protein luciferase folded within seconds to different partially folded states, one of which was stable over several minutes and was more resistant to forced unfolding than other partially folded states. Luciferase monomers can thus adopt a stable misfolded state and can do so without interacting with aggregation partners. This result supports the notion that luciferase misfolding is the cause of the low refolding yields and aggregation observed with this protein. This approach could be used to study misfolding transitions in other large proteins, as well as the factors that affect misfolding.

Circuit topology of proteins and nucleic acids
Alireza Mashaghi, Roeland J. van Wijk and Sander J. Tans
Structure 22, 1227-1237 (2014)
pdf & DOI: 10.1016/j.str.2014.06.015

Abstract

Folded biomolecules display a bewildering structural complexity and diversity. They have therefore been analyzed in terms of generic topological features. For instance, folded proteins may be knotted, have beta-strands arranged into a Greek-key motif, or display high contact order. In this perspective, we present a method to formally describe the topology of all folded linear chains and hence provide a general classification and analysis framework for a range of biomolecules. Moreover, by identifying the fundamental rules that intrachain contacts must obey, the method establishes the topological constraints of folded linear chains. We also briefly illustrate how this circuit topology notion can be applied to study the equivalence of folded chains, the engineering of artificial RNA structures and DNA origami, the topological structure of genomes, and the role of topology in protein folding.

Trehalose facilitates DNA melting : a single-molecule optical tweezers study
Sergey Bezrukavnikov, Alireza Mashaghi, Roeland J. van Wijk, Chan Gu, Li Jian Yang, Yi Quin Gao and Sander J. Tans
Soft Matter 10, 7269–7277 (2014)
pdf & DOI: 10.1039/c4sm01532k

Abstract

Using optical tweezers, here we show that the overstretching transition force of double-stranded DNA (dsDNA) is lowered significantly by the addition of the disaccharide trehalose as well as certain polyol osmolytes. This effect is found to depend linearly on the logarithm of the trehalose concentration. We propose an entropic driving mechanism for the experimentally observed destabilization of dsDNA that is rooted in the higher affinity of the DNA bases for trehalose than for water, which promotes base exposure and DNA melting. Molecular dynamics simulation reveals the direct interaction of trehalose with nucleobases. Experiments with other osmolytes confirm that the extent of dsDNA destabilization is governed by the ratio between polar and apolar fractions of an osmolyte.

Evolutionary constraints in variable environments, from proteins to networks
Katja Taute, Sebastian Gude, Philippe Nghe and Sander J. Tans
Trends Genet. 30, 192-198 (2014)
pdf & DOI: 10.1016/j.tig.2014.04.003

Abstract

Environmental changes can not only trigger a regulatory response, but also impose evolutionary pressures that can modify the underlying regulatory network. Here, we review recent approaches that are beginning to disentangle this complex interplay between regulatory and evolutionary responses. Systematic genetic reconstructions have shown how evolutionary constraints arise from epistatic interactions between mutations in fixed environments. This approach is now being extended to more complex environments and systems. The first results suggest that epistasis is affected dramatically by environmental changes and, hence, can profoundly affect the course of evolution. Thus, external environments not only define the selection of favored phenotypes, but also affect the internal constraints that can limit the evolution of these phenotypes. These findings also raise new questions relating to the conditions for evolutionary transitions and the evolutionary potential of regulatory networks.

An improved Escherichia coli strain to host gene regulatory networks involving both the AraC and Lacl inducible transcription factors
Manjunatha Kogenaru and Sander J. Tans
J. Biol. Eng. 8, 2 1-5 (2014)
pdf & DOI: 10.1186/1754-1611-8-2

Abstract

Many of the gene regulatory networks used within the field of synthetic biology have extensively employed the AraC and LacI inducible transcription factors. However, there is no Escherichia coli strain that provides a proper background to use both transcription factors simultaneously. We have engineered an improved E. coli strain by knocking out the endogenous lacI from a strain optimal for AraC containing networks, and thoroughly characterized the strain both at molecular and functional levels. We further show that it enables the gradual and independent induction of both AraC and LacI in a simultaneous manner. This construct will be of direct use for various synthetic biology applications.

Chaperone Action at the Single-Molecule Level
Alireza Mashaghi, Günter Kramer, Don C. Lamb, Matthias P. Mayer and Sander J. Tans
Chem. Rev. 114, 660-676 (2014)
pdf & DOI: 10.1021/cr400326k

Abstract

Considerable knowledge has been amassed over the past decades on the molecular mechanisms of chaperones, yet many fundamental questions remain unanswered. Specifically, we know little about how chaperones influence the conformation of client proteins during folding, which is considered to be central to chaperone function. Chaperones are thought to act both as suppressors of aggregation and as folding catalysts, but distinguishing both roles remains a challenge. The physical principles that chaperones exploit are also often unclear. Chaperones may affect the entropy of unfolded chains, lower reaction barriers by stabilizing intermediates, or guide the conformation of the protein chain (e.g., by preventing off-pathway intermediates). Questions also remain on the precise role of energy input, the dynamics of chaperone conformational changes, its relation to protein client dynamics, and how chaperones can act in a generic manner on many different proteins. These questions are difficult to address with bulk techniques because the underlying molecular mechanisms involve interactions that are often transient and heterogeneous. Single-molecule approaches have allowed the study of isolated proteins in real time, thereby revealing the rich conformational dynamics of proteins during the folding process. This capability makes single-molecule methods ideally suited to address many of the open questions with respect to chaperone function. In recent years, a start has been made to study chaperone-assisted protein folding using single-molecule techniques. Many excellent reviews on unassisted protein folding and on bulk studies of chaperones have been published. However, the emerging single-molecule studies of chaperones and assisted protein folding have so far received little attention. Here, we review a number of studies that address the folding of proteins aided by chaperones at the single molecule level. This will be preceded by an overview of the main chaperone systems and a discussion of the nature of single molecule assays as compared to bulk measurement techniques. We highlight how single-molecule methods can be used to answer the key questions related to assisted-protein folding reactions.

Microfabricated Polyacrylamide Devices for the Controlled Culture of Growing Cells and Developing Organisms
Philippe Nghe, Sarah Boulineau, Sebastian Gude, Pierre Recouvreux, Jeroen S. van Zon, Sander J. Tans
PLoS One 8, e75537 1-11 (2013)
pdf & DOI: 10.1371/journal.pone.0075537

Abstract

The ability to spatially confine living cells or small organisms while dynamically controlling their aqueous environment is important for a host of microscopy applications. Here, we show how polyacrylamide layers can be patterned to construct simple microfluidic devices for this purpose. We find that polyacrylamide gels can be molded like PDMS into micron-scale structures that can enclose organisms, while being permeable to liquids, and transparent to allow for microscopic observation. We present a range of chemostat-like devices to observe bacterial and yeast growth, and C. elegans nematode development. The devices can integrate PDMS layers and allow for temporal control of nutrient conditions and the presence of drugs on a minute timescale. We show how spatial confinement of motile C. elegans enables for time-lapse microscopy in a parallel fashion.

Optimality in evolution: new insights from synthetic biology
Marjon G. J. de Vos, Frank J. Poelwijk and Sander J. Tans
Curr. Opin. Biotechnol. 24, 797-802 (2013)
pdf & DOI: 10.1016/j.copbio.2013.04.008

Abstract

Whether organisms evolve to perform tasks optimally has intrigued biologists since Lamarck and Darwin. Optimality models have been used to study diverse properties such as shape, locomotion, and behavior. However, without access to the genetic underpinnings or the ability to manipulate biological functions, it has been difficult to understand an organism's intrinsic potential and limitations. Now, novel experiments are overcoming these technical obstacles and have begun to test optimality in more quantitative terms. With the use of simple model systems, genetic engineering, and mathematical modeling, one can independently quantify the prevailing selective pressures and optimal phenotypes. These studies have given an exciting view into the evolutionary potential and constraints of biological systems, and hold the promise to further test the limits of predicting future evolutionary change.

Reshaping of the conformational search of a protein by the chaperone trigger factor
Alireza Mashaghi, Günter Kramer, Philipp Bechtluft, Beate Zachmann-Brand, Arnold J. M. Driessen, Bernd Bukau and Sander J. Tans
Nature 500, 98-101 (2013)
pdf & DOI: 10.1038/nature12293

Abstract

Protein folding is often described as a search process, in which polypeptides explore different conformations to find their native structure. Molecular chaperones are known to improve folding yields by suppressing aggregation between polypeptides before this conformational search starts, as well as by rescuing misfolds after it ends. Although chaperones have long been speculated to also affect the conformational search itself - by reshaping the underlying folding landscape along the folding trajectory - direct experimental evidence has been scarce so far. In Escherichia coli, the general chaperone trigger factor (TF) could play such a role. TF has been shown to interact with nascent chains at the ribosome, with polypeptides released from the ribosome into the cytosol, and with fully folded proteins before their assembly into larger complexes. To investigate the effect of TF from E. coli on the conformational search of polypeptides to their native state, we investigated individual maltose binding protein (MBP) molecules using optical tweezers. Here we show that TF binds folded structures smaller than one domain, which are then stable for seconds and ultimately convert to the native state. Moreover, TF stimulates native folding in constructs of repeated MBP domains. The results indicate that TF promotes correct folding by protecting partially folded states from distant interactions that produce stable misfolded states. As TF interacts with most newly synthesized proteins in E. coli, we expect these findings to be of general importance in understanding protein folding pathways.

Environmental dependence of genetic constraint
Marjon G. J. de Vos, Frank J. Poelwijk, Nico Battich, Joseph D. T. Ndika and Sander J. Tans
PLoS Genet. 9, e1003580 1-8 (2013)
pdf & DOI: 10.1371/journal.pgen.1003580

Abstract

The epistatic interactions that underlie evolutionary constraint have mainly been studied for constant external conditions. However, environmental changes may modulate epistasis and hence affect genetic constraints. Here we investigate genetic constraints in the adaptive evolution of a novel regulatory function in variable environments, using the lac repressor, LacI, as a model system. We have systematically reconstructed mutational trajectories from wild type LacI to three different variants that each exhibit an inverse response to the inducing ligand IPTG, and analyzed the higher-order interactions between genetic and environmental changes. We find epistasis to depend strongly on the environment. As a result, mutational steps essential to inversion but inaccessible by positive selection in one environment, become accessible in another. We present a graphical method to analyze the observed complex higher-order interactions between multiple mutations and environmental change, and show how the interactions can be explained by a combination of mutational effects on allostery and thermodynamic stability. This dependency of genetic constraint on the environment should fundamentally affect evolutionary dynamics and affects the interpretation of phylogenetic data.

Single-cell dynamics reveals sustained growth during diauxic shifts
Sarah Boulineau, Filipe Tostevin, Daniel J. Kiviet, Pieter Rein ten Wolde, Philippe Nghe and Sander J. Tans
PLoS One 8, e61686 1-9 (2013)
pdf & DOI:10.1371/journal.pone.0061686

Abstract

Stochasticity in gene regulation has been characterized extensively, but how it affects cellular growth and fitness is less clear. We study the growth of E. coli cells as they shift from glucose to lactose metabolism, which is characterized by an obligatory growth arrest in bulk experiments that is termed the lag phase. Here, we follow the growth dynamics of individual cells at minute-resolution using a single-cell assay in a microfluidic device during this shift, while also monitoring lac expression. Mirroring the bulk results, the majority of cells displays a growth arrest upon glucose exhaustion, and resume when triggered by stochastic lac expression events. However, a significant fraction of cells maintains a high rate of elongation and displays no detectable growth lag during the shift. This ability to suppress the growth lag should provide important selective advantages when nutrients are scarce. Trajectories of individual cells display a highly non-linear relation between lac expression and growth, with only a fraction of fully induced levels being sufficient for achieving near maximal growth. A stochastic molecular model together with measured dependencies between nutrient concentration, lac expression level, and growth accurately reproduces the observed switching distributions. The results show that a growth arrest is not obligatory in the classic diauxic shift, and underscore that regulatory stochasticity ought to be considered in terms of its impact on growth and survival.

Hydrophobic collapse of trigger factor monomer in solution
Kushagra Singhal, Jocelyne Vreede, Alireza Mashaghi, Sander J. Tans and Peter G. Bolhuis
PLoS One 8, e59683 1-11 (2013)
pdf & DOI:10.1371/journal.pone.0059683

Abstract

Trigger factor (TF) is a chaperone, found in bacterial cells and chloroplasts, that interacts with nascent polypeptide chains to suppress aggregation. While its crystal structure has been resolved, the solution structure and dynamics are largely unknown. We performed multiple molecular dynamics simulations on Trigger factor in solution, and show that its tertiary domains display collective motions hinged about inter-domain linkers with minimal or no loss in secondary structure. Moreover, we find that isolated TF typically adopts a collapsed state, with the formation of domain pairs. This collapse of TF in solution is induced by hydrophobic interactions and stabilised by hydrophilic contacts. To determine the nature of the domain interactions, we analysed the hydrophobicity of the domain surfaces by using the hydrophobic probe method of Acharya et al., as the standard hydrophobicity scales predictions are limited due to the complex environment. We find that the formation of domain pairs changes the hydrophobic map of TF, making the N-terminal and arm2 domain pair more hydrophilic and the head and arm1 domain pair more hydrophobic. These insights into the dynamics and interactions of the TF domains are important to eventually understand chaperone-substrate interactions and chaperone function.

A polypeptide-DNA hybrid with selective linking capability applied to single molecule nano-mechanical measurements using optical tweezers
Fatemeh Moayed, Alireza Mashaghi and Sander J. Tans
PLoS One 8, e54440 1-7 (2013)
pdf & DOI:10.1371/journal.pone.0054440

Abstract

Many applications in biosensing, biomaterial engineering and single molecule biophysics require multiple non-covalent linkages between DNA, protein molecules, and surfaces that are specific yet strong. Here, we present a novel method to join proteins and dsDNA molecule at their ends, in an efficient, rapid and specific manner, based on the recently developed linkage between the protein StrepTactin (STN) and the peptide StrepTag II (ST). We introduce a two-step approach, in which we first construct a hybrid between DNA and a tandem of two STs peptides (tST). In a second step, this hybrid is linked to polystyrene bead surfaces and Maltose Binding Protein (MBP) using STN. Furthermore, we show the STN-tST linkage is more stable against forces applied by optical tweezers than the commonly used biotin-Streptavidin (STV) linkage. It can be used in conjunction with Neutravidin (NTV)-biotin linkages to form DNA tethers that can sustain applied forces above 65 pN for tens of minutes in a quarter of the cases. The method is general and can be applied to construct other surface-DNA and protein-DNA hybrids. The reversibility, high mechanical stability and specificity provided by this linking procedure make it highly suitable for single molecule mechanical studies, as well as biosensing and lab on chip applications.

Noise reduction by signal combination in Fourier space applied to drift correction in optical tweezers
Alireza Mashaghi, Peter J. Vach and Sander J. Tans
Rev. Sci. Instr. 82, 115103 (2011)
pdf & DOI:10.1063/1.3658825

Abstract

A general method is proposed to reduce noise by combining signals. Different measurements of the same physical quantity often exhibit different noise levels in different frequency ranges. Hence, a single high-fidelity signal can be constructed by combining the low-noise parts of the signals in Fourier space. We demonstrate this method by reducing noise in the measured bead-to-bead distance in an optical tweezers setup.

Optimality and evolution of transcriptionally regulated gene expression
Frank J. Poelwijk, Philip D. Heyning, Marjon G.J. de Vos, Daniel J. Kiviet and Sander J. Tans
BMC Syst. Biol. 5, 128 1-34 (2011)
pdf & DOI:10.1186/1752-0509-5-128

Abstract

How transcriptionally regulated gene expression evolves under natural selection is an open question. The cost and benefit of gene expression are the driving factors. While the former can be determined by gratuitous induction, the latter is difficult to measure directly.

Tradeoffs and optimality in the evolution of gene regulation
Frank J. Poelwijk, Marjon G.J. de Vos and Sander J. Tans
Cell 146, 462-470 (2011)
pdf & DOI:10.1016/j.cell.2011.06.035

Abstract

Cellular regulation is believed to evolve in response to environmental variability. However, this has been difficult to test directly. Here, we show that a gene regulation system evolves to the optimal regulatory response when challenged with variable environments. We engineered a genetic module subject to regulation by the lac repressor (LacI) in E. coli, whose expression is beneficial in one environmental condition and detrimental in another. Measured tradeoffs in fitness between environments predict the competition between regulatory phenotypes. We show that regulatory evolution in adverse environments is delayed at specific boundaries in the phenotype space of the regulatory LacI protein. Once this constraint is relieved by mutation, adaptation proceeds toward the optimum, yielding LacI with an altered allosteric mechanism that enables an opposite response to its regulatory ligand IPTG. Our results indicate that regulatory evolution can be understood in terms of tradeoff optimization theory.

Calcium modulates force sensing by the von Willebrand factor A2 domain
Arjen J. Jakobi, Alireza Mashaghi, Sander J. Tans and Eric G. Huizinga
Nat. Commun. 2, 385 1-9 (2011)
pdf & DOI:10.1038/ncomms1385

Abstract

von Willebrand factor (VWF) multimers mediate primary adhesion and aggregation of platelets. VWF potency critically depends on multimer size, which is regulated by a feedback mechanism involving shear-induced unfolding of the VWF-A2 domain and cleavage by the metalloprotease ADAMTS-13. Here we report crystallographic and single-molecule optical tweezers data on VWF-A2 providing mechanistic insight into calcium-mediated stabilization of the native conformation that protects A2 from cleavage by ADAMTS-13. Unfolding of A2 requires higher forces when calcium is present and primarily proceeds through a mechanically stable intermediate with non-native calcium coordination. Calcium further accelerates refolding markedly, in particular, under applied load. We propose that calcium improves force sensing by allowing reversible force switching under physiologically relevant hydrodynamic conditions. Our data show for the first time the relevance of metal coordination for mechanical properties of a protein involved in mechanosensing.

Taming membranes: functional immobilization of biological membranes in hydrogels
Ilja Kusters, Nobina Mukherjee, Menno R. de Jong, Sander J. Tans, Armağan Koçer and Arnold J.M. Driessen
PLoS One 6, e20435 1-8 (2011)
pdf & DOI:10.1371/journal.pone.0020435

Abstract

Insight into the ruggedness of adaptive landscapes is central to understanding the mechanisms and constraints that shape the course of evolution. While empirical data on adaptive landscapes remain scarce, a handful of recent investigations have revealed genotype-phenotype and genotype-fitness landscapes that appeared smooth and single peaked. Here, we used existing in vivo measurements on lac repressor and operator mutants in Escherichia coli to reconstruct the genotype-phenotype map that details the repression value of this regulatory system as a function of two key repressor residues and four key operator base pairs. We found that this landscape is multipeaked, harboring in total 19 distinct optima. Analysis showed that all direct evolutionary pathways between peaks involve significant dips in the repression value. Consistent with earlier predictions, we found reciprocal sign epistatic interactions at the repression minimum of the most favorable paths between two peaks. These results suggest that the occurrence of multiple peaks and reciprocal epistatic interactions may be a general feature in coevolving systems like the repressor-operator pair studied here.

Reciprocal sign epistasis is a necessary condition for multi-peaked fitness landscapes
Frank J. Poelwijk, Sorin Tanase-Nicola, Daniel J. Kiviet and Sander J. Tans
J. Theor. Biol. 272, 141-144 (2011)
pdf & DOI:10.1016/j.jtbi.2010.12.015

Abstract

Having multiple peaks within fitness landscapes critically affects the course of evolution, but whether their presence imposes specific requirements at the level of genetic interactions remains unestablished. Here we show that to exhibit multiple fitness peaks, a biological system must contain reciprocal sign epistatic interactions, which are defined as genetic changes that are separately unfavorable but jointly advantageous. Using Morse theory, we argue that it is impossible to formulate a sufficient condition for multiple peaks in terms of local genetic interactions. These findings indicate that systems incapable of reciprocal sign epistasis will always possess a single fitness peak. However, reciprocal sign epistasis should be pervasive in nature as it is a logical consequence of specificity in molecular interactions. The results thus predict that specific molecular interactions may yield multiple fitness peaks, which can be tested experimentally.

Multiple peaks and reciprocal sign epistatis in an empirically determinded genotype-phenotype landscape
Alexandre Dawid, Daniel J. Kiviet, Manjunatha Kogenaru, Marjon G.J. de Vos and Sander J. Tans
Chaos 20, 026105 1-7 (2010)
request reprint here & DOI:10.1063/1.3453602

Abstract

Insight into the ruggedness of adaptive landscapes is central to understanding the mechanisms and constraints that shape the course of evolution. While empirical data on adaptive landscapes remain scarce, a handful of recent investigations have revealed genotype-phenotype and genotype-fitness landscapes that appeared smooth and single peaked. Here, we used existing in vivo measurements on lac repressor and operator mutants in Escherichia coli to reconstruct the genotype-phenotype map that details the repression value of this regulatory system as a function of two key repressor residues and four key operator base pairs. We found that this landscape is multipeaked, harboring in total 19 distinct optima. Analysis showed that all direct evolutionary pathways between peaks involve significant dips in the repression value. Consistent with earlier predictions, we found reciprocal sign epistatic interactions at the repression minimum of the most favorable paths between two peaks. These results suggest that the occurrence of multiple peaks and reciprocal epistatic interactions may be a general feature in coevolving systems like the repressor-operator pair studied here.

SecB a chaperone dedicated to protein translocation
Philipp Bechtluft, Nico Nouwen, Sander J. Tans and Arnold J.M. Driessen
Mol. BioSyst. 6, 620-627 (2010)
pdf & DOI:10.1039/b915435c

Abstract

SecB is a molecular chaperone in Gram-negative bacteria dedicated to the post-translational translocation of proteins across the cytoplasmic membrane. The entire surface of this chaperone is used for both of its native functions in protein targeting and unfolding. Single molecule studies revealed how SecB affects the folding pathway of proteins and how it prevents the tertiary structure formation and aggregation to support protein translocation.

Tight hydrophobic contacts with the SecB chaperone prevent folding of substrate proteins
Philipp Bechtluft, Alexej Kedrov, Dirk-Jan Slotboom, Nico Nouwen, Sander J. Tans and Arnold J.M. Driessen
Biochemistry 49: 2380-2388 (2010)
pdf & DOI:10.1021/bi902051e

Abstract

The molecular chaperone SecB binds to hydrophobic sections of unfolded secretory proteins and thereby prevents their premature folding prior to secretion by the translocase of Escherichia coli. Here, we have investigated the effect of the single-residue mutation of leucine 42 to arginine (L42R) centrally positioned in the polypeptide binding pocket of SecB on its chaperonin function. The mutant retains its tetrameric structure and SecA targeting function but is defective in its holdase activity. Isothermal titration calorimetry and single-molecule optical tweezer studies suggest that the SecB(L42R) mutant exhibits a reduced polypeptide binding affinity allowing for partial folding of the bound polypeptide chain rendering it translocation-incompetent.

Revealing evolutionary pathways by fitness landscape reconstruction
Manjunatha Kogenaru, Marjon G.J. de Vos, and Sander J. Tans
Crit Rev Biochem Mol Biol. 44:169-174 (2009)
pdf & DOI:10.1080/10409230903039658

Abstract

The concept of epistasis has since long been used to denote non-additive fitness effects of genetic changes and has played a central role in understanding the evolution of biological systems. Owing to an array of novel experimental methodologies, it has become possible to experimentally determine epistatic interactions as well as more elaborate genotype-fitness maps. These data have opened up the investigation of a host of long-standing questions in evolutionary biology, such as the ruggedness of fitness landscapes and the accessibility of mutational trajectories, the evolution of sex, and the origin of robustness and modularity. Here we review this recent and timely marriage between systems biology and evolutionary biology, which holds the promise to understand evolutionary dynamics in a more mechanistic and predictive manner.

Internal Dynamics of Supercoiled DNA Molecules
Thomas Kalkbrenner, Axel Arnold, Sander J. Tans.
Biophysical Journal 96:4951-4955 (2009)
pdf & DOI:10.1016/j.bpj.2009.03.056

Abstract

The intramolecular diffusive motion within supercoiled DNA molecules is of central importance for a wide array of gene regulation processes. It has recently been shown, using fluorescence correlation spectroscopy, that plasmid DNA exhibits unexpected acceleration of its internal diffusive motion upon supercoiling to intermediate density. Here, we present an independent study that shows a similar acceleration for fully supercoiled plasmid DNA. We have developed a method that allows fluorescent labeling of a 200-bp region, as well as efficient supercoiling by Escherichia coli gyrase. Compared to plain circular or linear DNA, the submicrosecond motion within the supercoiled molecules appears faster by up to an order of magnitude. The mean-square displacement as a function of time reveals an additional intermediate regime with a lowered scaling exponent compared to that of circular DNA. Although this unexpected behavior is not fully understood, it could be explained by conformational constraints of the DNA strand within the supercoiled topology in combination with an increased apparent persistence length.

Direct observation of type 1 fimbrial switching
Aileen Adiciptaningrum, Ian Blomfield, Sander J. Tans.
EMBO Reports 10:527-532 (2009)
pdf & DOI:10.1038/embor.2009.25

Abstract

The defining feature of bacterial phase variation is a stochastic 'all-or-nothing' switching in gene expression. However, direct observations of these rare switching events have so far been lacking, obscuring possible correlations between switching events themselves, and between switching and other cellular events, such as division and DNA replication. We monitored the phase variation of type 1 fimbriae in individual Escherichia coli in real time and simultaneously tracked the chromosome replication process. We observed distinctive patterns of fim (fimbriae) expression in multiple genealogically related lineages. These patterns could be explained by a model that combines a single switching event with chromosomal fim replication, as well as the epigenetic inheritance of expressed fim protein and RNA, and their dilution by growth. Analysis of the moment of switching at sub-cell-cycle resolution revealed a correlation between fim switching and cell age, which challenges the traditional idea of phase variation as a random Poissonian phenomenon.

Direct Observation of Chaperone-Induced Changes in a Protein Folding Pathway
Philipp Bechtluft1, Ruud van Leeuwen1, Matthew Tyreman1, Danuta Tomkiewicz, Nico Nouwen, Harald Tepper, Arnold Driessen, Sander J. Tans.
1 joint first authors
Science 318:1458-1461 (2007)
pdf & DOI:10.1126/science.1144972

Abstract

How chaperone interactions affect protein folding pathways is a central problem in biology. With the use of optical tweezers and all-atom molecular dynamics simulations, we studied the effect of chaperone SecB on the folding and unfolding pathways of maltose binding protein (MBP) at the single-molecule level. In the absence of SecB, we find that the MBP polypeptide first collapses into a molten globulelike compacted state and then folds into a stable core structure onto which several α helices are finally wrapped. Interactions with SecB completely prevent stable tertiary contacts in the core structure but have no detectable effect on the folding of the external α helices. It appears that SecB only binds to the extended or molten globulelike structure and retains MBP in this latter state. Thus during MBP translocation, no energy is required to disrupt stable tertiary interactions.

Empirical fitness landscapes reveal accessible evolutionary paths
Frank Poelwijk1, Daan Kiviet1, Daniel Weinreich, Sander J. Tans.
1 joint first authors
Nature 445:383-386 (2007)
pdf & DOI:10.1038/nature05451

Abstract

When attempting to understand evolution, we traditionally rely on analysing evolutionary outcomes, despite the fact that unseen intermediates determine its course. A handful of recent studies has begun to explore these intermediate evolutionary forms, which can be reconstructed in the laboratory. With this first view on empirical evolutionary landscapes, we can now finally start asking why particular evolutionary paths are taken.

Evolutionary potential of a duplicated repressor-operator pair: simulating pathways using mutation data
Frank Poelwijk1, Daan Kiviet1, Sander J. Tans.
1 joint first authors
PLoS Comput Biol. 2:e58 (2006)
pdf & DOI:10.1371/journal.pcbi.0020058

Abstract

Ample evidence has accumulated for the evolutionary importance of duplication events. However, little is known about the ensuing step-by-step divergence process and the selective conditions that allow it to progress. Here we present a computational study on the divergence of two repressors after duplication. A central feature of our approach is that intermediate phenotypes can be quantified through the use of in vivo measured repression strengths of Escherichia coli lac mutants. Evolutionary pathways are constructed by multiple rounds of single base pair substitutions and selection for tight and independent binding. Our analysis indicates that when a duplicated repressor co-diverges together with its binding site, the fitness landscape allows funneling to a new regulatory interaction with early increases in fitness. We find that neutral mutations do not play an essential role, which is important for substantial divergence probabilities. By varying the selective pressure we can pinpoint the necessary ingredients for the observed divergence. Our findings underscore the importance of coevolutionary mechanisms in regulatory networks, and should be relevant for the evolution of protein-DNA as well as protein-protein interactions.

The bacteriophage straight phi29 portal motor can package DNA against a large internal force
Douglas E. Smith1, Sander J. Tans1, Steven B. Smith, Shelley Grimes, Dwight L. Anderson, Carlos Bustamante
1 joint first authors
Nature. 413:748-52 (2001)
pdf & DOI:10.1038/35099581

Abstract

As part of the viral infection cycle, viruses must package their newly replicated genomes for delivery to other host cells. Bacteriophage Φ29 packages its 6.6-microm long, double-stranded DNA into a 42 x 54 nm capsid1 by means of a portal complex that hydrolyses ATP2. This process is remarkable because entropic, electrostatic and bending energies of the DNA must be overcome to package the DNA to near-crystalline density. Here we use optical tweezers to pull on single DNA molecules as they are packaged, thus demonstrating that the portal complex is a force-generating motor. This motor can work against loads of up to 57 pN on average, making it one of the strongest molecular motors reported to date. Movements of over 5 μm are observed, indicating high processivity. Pauses and slips also occur, particularly at higher forces. We establish the force-velocity relationship of the motor and find that the rate-limiting step of the motor's cycle is force dependent even at low loads. Notably, the packaging rate decreases as the prohead is filled, indicating that an internal force builds up to ≈50 pN owing to DNA confinement. Our data suggest that this force may be available for initiating the ejection of the DNA from the capsid during infection.

Molecular transistors: Potential modulations along carbon nanotubes
Sander J. Tans, Cees Dekker.
Nature. 404:834-35 (2000)
pdf & DOI:10.1038/35009026

Abstract

True molecular-scale transistors have been realized using semiconducting carbon nanotubes, but no direct measurements of the underlying electronic structure of these have been made. Here we use a new scanning-probe technique to investigate the potential profile of these devices. Surprisingly, we find that the potential does not vary in a smooth, monotonic way, but instead shows marked modulations with a typical period of about 40 nm. Our results have direct relevance for modelling this promising class of molecular devices.

Imaging electron wave functions of quantized energy levels in carbon nanotubes
Liesbeth C. Venema, Jeroen W. G. Wildoer, Jorg W. Janssen, Sander J. Tans, Hinne L. J. Temminck Tuinstra, Leo P. Kouwenhoven, Cees Dekker
Nature. 413:748-52 (1999)
pdf & DOI:10.1126/science.283.5398.52

Abstract

Carbon nanotubes provide a unique system for studying one-dimensional quantization phenomena. Scanning tunneling microscopy was used to observe the electronic wave functions that correspond to quantized energy levels in short metallic carbon nanotubes. Discrete electron waves were apparent from periodic oscillations in the differential conductance as a function of the position along the tube axis, with a period that differed from that of the atomic lattice. Wave functions could be observed for several electron states at adjacent discrete energies. The measured wavelengths are in good agreement with the calculated Fermi wavelength for armchair nanotubes.

Electron-electron correlations in carbon nanotubes
Sander J. Tans, Michel H. Devoret, Remco J. A. Groeneveld & Cees Dekker
Nature. 394:761-64 (1998)
pdf & DOI:10.1038/29494

Abstract

Single-wall carbon nanotubes1,2 are ideally suited for electron-transport experiments on single molecules because they have a very robust atomic and electronic structure and are sufficiently long to allow electrical connections to lithographically defined metallic electrodes. The electrical transport properties of single nanotubes3 and bundles of nanotubes4 have so far been interpreted by assuming that individual electrons within the nanotube do not interact, an approximation that is often well justified for artificial mesoscopic devices such as semiconductor quantum dots5. Here we present transport spectroscopy data on an individual carbon nanotube that cannot be explained by using independent-particle models and simple shell-filling schemes. For example, electrons entering the nanotube in a low magnetic field are observed to all have the same spin direction, indicating spin polarization of the nanotube. Furthermore, even when the number of electrons on the nanotube is fixed, we find that variation of an applied gate voltage can significantly change the electronic spectrum of the nanotube and can induce spin flips. The experimental observations point to significant electron–electron correlations. We explain our results phenomenologically using a model that assumes that the capacitance of the nanotube depends on its many-body quantum state.

Room-temperature transistor based on a single carbon nanotube
Sander J. Tans, Alwin R. M. Verschueren & Cees Dekker
Nature. 393:49-52 (1998)
pdf & DOI:10.1038/29954

Abstract

The use of individual molecules as functional electronic devices was first proposed in the 1970s (ref. 1). Since then, molecular electronics has attracted much interest, particularly because it could lead to conceptually new miniaturization strategies in the electronics and computer industry. The realization of single-molecule devices has remained challenging, largely owing to difficulties in achieving electrical contact to individual molecules. Recent advances in nanotechnology, however, have resulted in electrical measurements on single molecules. Here we report the fabrication of a field-effect transistor—a three-terminal switching device—that consists of one semiconducting single-wall carbon nanotube connected to two metal electrodes. By applying a voltage to a gate electrode, the nanotube can be switched from a conducting to an insulating state. We have previously reported5 similar behaviour for a metallic single-wall carbon nanotube operated at extremely low temperatures. The present device, in contrast, operates at room temperature, thereby meeting an important requirement for potential practical applications. Electrical measurements on the nanotube transistor indicate that its operation characteristics can be qualitatively described by the semiclassical band-bending models currently used for traditional semiconductor devices. The fabrication of the three-terminal switching device at the level of a single molecule represents an important step towards molecular electronics.

Individual single-wall carbon nanotubes as quantum wires
Sander J. Tans, Michel H. Devoret, Hongjie Dai, Andreas Thess, Richard E. Smalley, L. J. Geerligs & Cees Dekker
Nature. 386:474-77 (1997)
pdf & DOI:10.1038/386474a0

Abstract

Carbon nanotubes have been regarded since their discovery1 as potential molecular quantum wires. In the case of multi-wall nanotubes, where many tubes are arranged in a coaxial fashion, the electrical properties of individual tubes have been shown to vary strongly from tube to tube2,3, and to be characterized by disorder and localization4. Single-wall nanotubes5,6 (SWNTs) have recently been obtained with high yields and structural uniformity7. Particular varieties of these highly symmetric structures have been predicted to be metallic, with electrical conduction occurring through only two electronic modes8–10. Because of the structural symmetry and stiffness of SWNTs, their molecular wavefunctions may extend over the entire tube. Here we report electrical transport measurements on individual single-wall nanotubes that confirm these theoretical predictions. We find that SWNTs indeed act as genuine quantum wires. Electrical conduction seems to occur through well separated, discrete electron states that are quantum-mechanically coherent over long distance, that is at least from contact to contact (140nm). Data in a magnetic field indicate shifting of these states due to the Zeeman effect.