Biological molecules under non-natural conditions

March 10, 2014 to March 12, 2014
Location : University of Stuttgart, Germany


  • Maria Fyta (Institute for Computational Physics, University of Stuttgart, Germany)
  • Jens Smiatek (Institute for Computational Physics, University of Stuttgart, Germany)
  • Johannes Kästner (Institute of Theoretical Chemistry, University of Stuttgart, Germany)
  • Jürgen Pleiss (Institute for Technical Biochemistry, University of Stuttgart, Germany)






Biomacromolecules such as DNA, RNA, and proteins play an extremely important role in biological and technical processes. These processes can either sustain life or are linked to novel biotechnological applications. At the cellular level, biomacromolecules like proteins and DNA are flexible and can be manipulated to function variably. In this respect, the conditions in which these biomacromolecules 'live' are essential for expressing the biomacromolecule's functions either in a living cell or in a functional biomaterial. Until recently, it was believed that the conditions in which the biomacromolecules reside should be the so called 'natural conditions'. However, over the last decades, the interest in the properties of biological molecules under non-natural conditions has grown enormously. Extremophiles, for example, are organisms that exist in high temperatures, high acidity, extreme cold, extreme pressure, or extreme salt concentrations. Guided by technological developments which allow the researchers to gain novel biological insights, it has been recognized that natural materials show a rich diversity of functionality and behavior under varying conditions.

Typical examples for non-natural conditions are high salinity, low or high temperature, high pressure and varying solvent qualities. The famous DNA B- to A-form transition is a prominent example of the latter. The transition is essential for biological function, as shown by the existence of the A-form in many protein–DNA complexes. The resulting DNA form can also be affected by applying external forces which stretch DNA. In this way B-DNA assumes an S-DNA conformation [1,2]. Stretching forces are in this respect also considered as non-natural conditions. It is, thus, important to understand the mechanics of conformational changes which take us into the rarely studied area far from the canonical forms of the double helix. This double helix has also the potential to be combined with inorganic materials or self-assemble in order to give rise to unique bionanotechnological applications.

The non-natural conditions also considerably affect another type of widely studied and used biomacromolecules, the enzymes. Enzymes are versatile catalysts with amazing rate accelerations of up to 1019 for chemical reactions [3], high regio-, chemo-, and stereoselectivity, and are straightforward to be produced as recombinant protein by fermentation of a host organism such as bacteria or fungi. Most importantly, molecular biology techniques, especially gene synthesis, allow to modify natural enzymes easily, and even produce synthetic genes de novo [4]. In most cases, the major challenge for developing optimized biocatalysts for desired reactions is our lack of understanding of the relationship between sequence and biochemical properties rather than enzyme production. All these are affected by the environment surrounding the enzymes, non-natural conditions included. A thorough interdisciplinary investigation of all these factors affecting biomolecules by joining together theorists and experimentalists defines the novelty of the proposed workshop.

Biomacromolecules have been intensively studied. Many of the investigations involve the behavior, structure and function, of these biomacromolecules under normal physiological conditions. On the other hand, the specific investigation of macromolecular conformations under non-natural conditions and the evolutionary strategies to prevent denaturation of proteins, as an example is a growing research topic. It has been recognized that often the presence of co-solutes like TMAO, proline, or ectoine is an important factor to preserve the cell metabolism [5]. Recent studies have illustrated, that specific osmolytes like TMAO are able to protect the proteins from denaturation even under high pressure [6]. A point which is of particular importance for deep sea organisms. It has been assumed that environmental circumstances have also lead to the presence of anti-freeze proteins. Detailed computer simulations have shown that these proteins avoid the formation of ice phases [7].

Non-natural conditions involve also high temperature, which is found to highly affect the structural conformation of biomacromolecules. DNA can unzip and wrap at a sufficiently high temperature, a structural transition which leads to a separation of double stranded-DNA into two single strands and is known as DNA melting or DNA denaturation [8]. The melting stability has been shown to depend on the DNA sequence [9,10]. In proteins a high temperature can lead to destruction of the amino acid packing and a loss of the secondary and tertiary structure [11].

The solvent or the salt concentration is also known to influence in various ways the structures of biomacromolecules. Proteins are more soluble in dilute salt solutions than in pure water. At a high enough concentration, the proteins become sufficiently dehydrated and loose solubility, a process known as salting-out [12,13]. A high enough salt concentration can result in a salt-induced helicity reversal, forming left-handed RNA double helices [14]. In DNA, high salt concentration can induce DNA double-strand breaks [15]. A high salt concentration is technologically also an efficient method to extract high quality genomic DNA from different organisms [16].

Conformational changes of macromolecules under non-natural conditions, for example by the variation of the pH value, are also technologically used. Prominent examples are specific DNA molecules that work as nanomachines [17] or as pH sensors [18]. Although a lot of experimental work has been spent to investigate non-natural conditions and their influence on the conformational behavior in more detail, the theoretical understanding of the underlying processes is rather sparse. This can be related to the fact that the exchange of knowledge between the two communities is often delayed.

The application of external forces which are able to stretch biomacromolecules is often essential in understanding the structural details of these molecules. Pulling proteins by means of two cantilevers leads to an understanding of the forces needed and the shapes that a protein undertakes when it tries to unfold [19,20,21]. Proteins have also been stretched by atomic force microscopy [22] or flow [23,24]. In real life, proteins, either in recognizing the double helix or as part of their functional role, may induce important local distortions and multiple protein binding. This was seen in the case of RecA [25,26], which can extend such deformations over long DNA lengths. Single DNA molecules have been successfully visualized and manipulated by means of various methods. The electrophoresis of DNA in gels [27] or microlithographic arrays [28] has been studied, as well as the stretching of DNA with a receding meniscus [29]. Fluorescent DNA molecules grafted to beads can be manipulated by optical tweezers [30,31], force measuring laser tweezers [32,33] or more recently, by electromagnetic torque tweezers [34]. One of the most promising relevant applications is DNA translocation through nanopores, in which DNA is being stretched as it passes through nanopores allowing for a read-out of its sequence [35,36,37,38,39,40].

While most natural enzymes operate at ambient conditions (temperature between 20 and 40°C, pressure of 1 bar, water near to pH=7 as solvent), they are generally not compatible with conditions of technical processes such as organic solvents, high temperature, or high pressure. As a consequence, enzymatic steps cannot be integrated into reaction cascades including non-enzymatic catalysts. However, there are also enzymes from extremophilic organisms that are active at temperatures beyond 100°C, high pressure, high salt concentrations, extreme pH, or in organic solvents [41]. Protein engineering and de novo protein design are promising strategies to develop robust [42], active, and selective biocatalysts [43]. Thus, harsh "non-natural" conditions are not per se incompatible with biocatalysis, and it is mainly our lack of insight into the effect of the environment to stability, activity, specificity, and selectivity of enzymes that still restricts the use of enzymes in a broad range of synthetic applications.
To bridge the gap between the need of enzymes with desired properties under non-natural conditions and the straightforward production or biocatalysts from a gene sequence, atomistic simulations of the biochemical properties of enzymes in organic solvents [44,45], elevated temperature [46], and high pressure [47,48] have been successfully applied to understand experimentally observed properties and to design improved biocatalysts.
Finally, tuning the conditions in which biomacromolecules live in can result in novel biomolecular structures prone to novel biotechnological applications. DNA for example, can self-assemble or can be folded to create nanoscale shapes and patterns, in what is known as DNA origami [49]. DNA origami can be used as a delivery vehicle for anti-cancer drugs [50], to assemble gold nanoparticles [51] or can be self-organized on lithographically patterned surfaces [52], to be used as a scaffold for electronic components [53]. DNA self-assembled nanogrids can be used as scaffolds on which periodic arrays of proteins were templated, and the resulted nanoribbon can act as a scaffold for highly conductive silver nanowires [54]. In the end, DNA might be a biological molecule, but can be further exploited for material purposes as it can direct the assembly of highly structured materials with specific nanoscale features [55].


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