Biological superstructures: the materials of biology

Nuclear Instruments and Methods in Physics Research B 97 (1995) 295-302

Beem Interactions with Materials & Atoms

EISEVIER

Biological superstructures:

the materials of biology

M.H.J. Koch * European Molecular Biology Laboratory, Hamburg Outstation, EMBL c/o

DESy Notkestrasse 85, D-22603 Hamburg, Germany.

Abstract A few examples of the contribution of X-ray scattering with synchrotron radiation to the study of biological materials are given to illustrate some specific characteristics of these materials. It is shown that at the scale of sizes of biological systems the materials and the devices are inseparable. The photocycle of bacteriorhodopsin illustrates the separation between (fast) reactions and (slow) dissipative steps that must necessarily exist in a molecular machine. It is also shown that the potential for nonlinear behaviour built into oligomeric proteins can lead to phenomena like switching or temporal and spatial oscillations. The latter may provide a mechanism for macroscopic morphogenesis.

1. Introduction During the past decades biochemistry and materials science have both made spectacular progress, albeit largely in “splendid” isolation. The gap between the two is best illustrated by the fact that the index of a recent, very influential, biochemistry textbook does not contain any entry to such concepts as epitaxy, mineralization, interfaces, autocatalysis or Gouy-Chapman layer. Before we embark on an attempt to find some connections between materials science and biology, let me make clear that I am not going to talk about biomaterials (i.e. synthetic materials used for biomedical applications). These are economically very important with an estimated annual sale of $24 billion in the USA alone, excluding pharmaceuticals [l]. As it is, fashionable to relate one’s research to some profitable branch of economy I shall, however, mention that we have some projects involving the characterization of potential nanoparticle drug carriers for which synchrotron radiation (SR) X-ray scattering is an indispensable tool [2,3]. I shall thus rather discuss some characteristics of the materials of biology (i.e. the materials found in biological systems). Obviously, this can only be superficial and several topics like gels, lipids and protein structures at atomic resolution are completely left out. From the point of view of materials science the most interesting work in the latter field concerns not so much the analysis of structures as the ab initio design of synthetic proteins [4] or the production of catalytic antibodies, whereby the reagent produces its own catalyst [5]. I shall also not consider motor systems,

* Corresponding author. Tel. +49-40-89902113, fax +49-4089902149. 0168-583X/95/$09.50

0 1995 Elsevier Science

SSDI 0168-583X(94)00360-2

of which muscle provides the best example. Its study motivated the pioneering work using X-ray SR [6] and it is still an area of active research at several SR sources (for a review see Ref. [7]). One of the main connections between materials science and biology is the field of molecular recognition and biomineralization. This involves the controlled nucleation and crystallization at interfaces between a macromolecular substrate and another (inorganic) phase, usually a solution. This subject has been recently reviewed [8,9]. The control of crystal texture is one of the ways of nature to adapt the structure of its materials to specific requirements and this has been investigated using SR [lo]. The usefulness of such observations comes from the fact that biomimetic processing allows, for example, to produce thin ceramic films on other materials at temperatures below 100°C [ll]. Along those lines one should also mention studies of the properties and pathologies of collagen (for a review, see Ref. [12]). Polymer scientists are trying hard to mimic the hierarchical structure of collagen [13] which easily adapts to various mechanical (e.g. tendon, teeth, bones, cartilage) or optical (e.g. transparency in cornea and opacity of the sclera) requirements. Such observation have inspired the fabrication of synthetic inorganic/organic composites [14] and the use of oriented thin films of polymers as substrates for oriented growth of materials [15]. We shall come back later to the fact that in nature the crystal morphologies depend on the kinetics of the processes leading to their formation [16]. The view that biological systems have sophisticated control mechanisms is well established. It is not so easy, however, to recognize how the basic functions of abstract control systems are implemented (e.g. rectification, amplification, capacity, resistance and interconnection) because at the scale of sizes of biological objects the materials and

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the devices become inseparable. I shall try to illustrate this, by considering some aspects of the visual system. This will be followed by a brief introduction to some nonlinear properties of protein assemblies.

2. Materials and devices in vision The optical components of the eye of higher mammals are the cornea, the lens, the vitreous and the retina. The cornea consists essentially of collagen fibrils with a uniform diameter (20 nm) packed with short range order in a ground substance with centre to centre distances around 100 nm [17]. The transmission spectrum of the cornea which has a sharp cutoff around 350 nm [18] suggests that the cornea acts as a long pass interference filter that protects the lens and the photopigments from damage by the UV. The sclera, where there is a nonuniform distribution of diameters of the collagen fibrils, is opaque and completes the camera obscura of the eye. The cornea which has a fixed curvature for every type of eye is responsible for a considerable part of the focusing but the fine focusing is done by the lens. The latter has one of the highest concentrations of proteins, the crystallins, in the body (300 mg/ml), packed with short range order so that no light is scattered as established using SR [19]. This packing gives rise to a characteristic maximum in the X-ray scattering pattern of lenses as illustrated in Fig. 1. As a consequence of aging, or diseases such as diabetes, the arrangement of the crystallins is disturbed, light scatter-

Fig. 1. A: Scattering age (from [201X

ing occurs and above the age of 55 or so things start looking more yellowish and then progressively loose their colour, especially for those suffering from cataract. A combined UV fluorescence and SR X-ray scattering study of lenses as a function of age [20] suggests that the alteration of the packing of crystallins is due to the reaction of sugars with the proteins resulting in cross-linking. This reaction, the Maillard reaction, which causes browning, is also a well-known problem in the food industry.

3. Photoreceptors: a heritage from the very early days Light has to be turned into an electric signal that can be processed by the nervous system. This is done by rhodopsin, a molecule that has many similarities with the light-driven proton pump used by the archeabacteria Halobacterium Halobium [21] to store energy in a proton gradient. Later in evolution, photosynthesis in plants evolved as a different and much more efficient way of storing energy. In this sense the basic photodetector in vision uses a very old discovery of nature that could no longer efficiently do the job of energy transformation in more complex organisms. Because bacteriorhodopsin is available in large quantities and trimers of the protein are naturally organized in a hexagonal lattice in the membrane of Halobacterium halobium it has been far more studied than rhodopsin and is one of the most studied proteins in biophysics. Its structure is known to a resolution of about 0.5 nm [22].

pattern of human lenses as a function of age and B: correlation

between the position of the interference

maximum

and

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Rather than going through these (slow) dissipative steps one can reset the pump much more rapidly from M to BR but only at the price of another blue photon [27]. Note that unlike the case of semiconductor photovoltaic devices, light absorption by the retinal and the charge carrier transport are clearly separated. A similar approach is also used, for instance, in solar cells based on dyesensitized colloidal Ti02 films [28] or dyes trapped in sol/gel glasses [29]. The advantage of working with specific ions and small molecules as carriers is that the information pathways can be shared without danger of mixing the signals. There are also no majority and minority carriers, which require a very accurate control of purity, to worry about. Ionics has found fewer application in devices than electronics but it is perhaps more than an anecdote that ionic diodes were actually manufactured [30] and that Letaw and Bardeen even designed an ionic transistor [31]. Rapid signal processing requires electronic processes but these are confined to very small regions of space in biological systems like BR. Nearly all structural and spectroscopic techniques have been used to study BR. The contribution of time resolved SR X-ray scattering [25] was to establish that the localized conformational changes in the protein found by neutron scattering [32] are directly related to the spectroscopic

The photocycle of bacteriorhodopsin illustrates how a molecular machine, in this case a pump, works. The most important features of the present view (for a review, see Ref. [23]) are summarized in the diagram in Fig. 2. A green photon is absorbed by the all-tram retinal which forms a Schiff base with Lys216 and a transient excited state is formed which isomerizes in about 0.5 ps to the 13-cis isomer J. This is followed by a succession of spectroscopically distinct states, K, L during which the Schiff base is protonated. Asp85 accepts a proton from the Schiff base which is transferred to the extracellular space in the L to M transition. At this point the work is effectively done and all there remains to do is to dissipate the excess energy in the BR molecule. The following step (M to N, or Ml to M2 to N) in which a proton from the protonated Asp96 is transferred to the Schiff base is irreversible [24]. The conformational relaxation of the protein which is accompanied by a large scale (0.1-0.2 nm) movement especially in the vicinity of helices G and B [25] occurs in all likelihood during the N to 0 or N to BR transition. Simultaneously Asp96 is reprotonated from the cytoplasmic side and the retinal returns to its all-trans conformation. The connectivity in the system thus seems to be achieved through a proton conductance consisting of a network of hydrogen bonds [26].

work

Light

proton

transport

ZD

uptake

r

kT

I ),

proton

gradient

kJ/mOl

AG

relaxation

proton

BR* A5 = + 250 J(mol”K)-’

AS = - 300 J(mol”K)+

I1 I 1 I 1

H

Asp 65, H’

c-------_-1

r-l2

u

N

\

- BR

fH’

[Asp 96

cis

trans 4 0.5

ps

cis

3 Ps

cis

1 PS

ClS

50 ps

cis

cis 10 ms ms-s

trans IOms

*

Fig. 2. Schematic diagram of the photocycle of bacteriorhodopsin (after [24]). The energy of the J intermediate is provided

by the Ml-M2

is not known. Irreversibility

transition.

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changes during the photocycle. These conformational changes, which were not expected to be of such magnitude [33], were later confirmed by a variety of techniques [34,35] and similar changes were also found by EPR in rhodopsin, the pigment of the eye [36]. Rhodopsin has, however, a different mechanism than BR, with a retinal conformational change from 11-cis to trans, which does not involve proton transport. That conformational changes of this magnitude take place also severely limits what one can reasonably hope to achieve in the foreseeable future in the understanding of molecular machines using computer simulations. There is thus still no alternative to experiments. The material scientist would, of course, like to know whether one can modify or even improve BR. The effect of replacing single amino acids by point mutation has been extensively studied [21]. If these mutations have any effect at all on the activity of the protein it is to reduce it. This is perhaps not astonishing if we believe that nature has already found an optimum by mutation and natural selection. Note that the steps of the cycle which can be modified are the slow (> lo-’ s) dissipative steps, not the fast ones (reactions) where the work gets done. As illustrated in the energy diagram in Fig. 2 most of the steps take place near equilibrium (i.e. AC < a few kT). This is characteristic of biological macromolecules. As already pointed out by McClare [37] when dealing with molecular machines the wording of the second law of thermodynamics has to explicitly include time. Indeed, work is only done when one form of stored energy is converted into another and this must happen in times which do not allow exchange with thermal energies, at the molecular level this means less than lo-’ s. At a more general level, the time resolved X-ray experiments on BR [25] were useful because they provided the first direct (nonspectroscopic) observation of a proteinquake [38]. This concept is part of current theories about the dynamics of biomolecules which are very closely related to those about glasses or spin glasses [39]. Up to here, we could still refer to groups of atoms (e.g. individual side chains, helices), which hopefully we shall some time see in detail in a crystal structure. The protein is, however, not the only part of the photoelectric system in BR and the membrane plays at least two very important roles in the photocycle. Without the vectorial transport across the membrane no energy could be stored. Although the conformational change takes place in membrane patches [25,32] or even in crystals [40] it is not associated with any work since it is equivalent to pumping water from a lake into itself - somewhere one needs a dam. The second role is to provide a soft medium in which the large anharmonic low frequency motions associated with the relaxation of the protein in the second part of the photocycle can take place. At low temperatures or in dried (solid) membranes these movements are no longer possible and the photocycle is interrupted [41].

By changing the environment (i.e. by altering the Gouy-Chapman layer) one can also alter the rate of some of the steps in the cycle [42] or alter the optical properties of BR producing so-called blue membranes. This gives an extra level of control over the process. Due to its potential applications in optoelectronics BR must also be the only protein that is sold by an electronics company (Wacker, Munich). The applications of BR as a material for information processing have been recently reviewed [43] and a differential light sensor was produced at Fuji Corp. [44]. With oriented samples, the light-induced electrostatic field can be visualized by spreading toner from a standard copier on the membrane [45]. BR also has interesting nonlinear optical properties [46]. Before leaving the topic of photoreceptors it is perhaps useful to analyze the reasons for which others, using considerably more sophisticated equipment than ours, failed to detect the conformational changes in BR. As there is no reason to assume that there are major differences in the quality of the X-ray sources used or the skills of the experimentalists, one is inevitably led to the conclusion that the difference lies in the detectors. Whereas others used a phosphor-image intensifier or an imaging plate drum, we used a (linear) proportional gas detector with delay line readout [47]. Such an efficient, noise free, photon counting device is a prerequisite to detect rapid (l-100 ms) intensity changes that represent 6% overall in the integrated intensities after background subtraction.

4. Nonlinear properties of protein assemblies If we can find photoelectric devices in nature what about other devices, such rectifiers and amplifiers or components like resistors and capacitors needed to build a dynamic analogue network which is exactly what organisms are? To give an answer to this one has to consider both the spatial and temporal organization of these systems. The role of a diode can be played by a cascade of reactions, for instance, where irreversibility results from different rate constants in successive steps as shown recently for the enzyme succinate dehydrogenase [48]. Proteins often assemble into specific aggregates or oligomers and this gives them the potential for nonlinear behaviour as in allosteric effects and other control mechanisms in catalysis. I am not so sure that this key feature of biological materials has yet been fully recognized by supramolecular synthethic chemists (see e.g. Ref. [49]). The catalytic activity of many enzymes can be influenced by substrates, products or other effecters, making the enzyme an n-port device. The effect of substrate and effecters on the switching between the T and the R states in the allosteric enzyme aspartate transcarbamylase (ATCase) has been studied in detail using SR [50]. The results support the model involving an equilibrium between only

MHJ.

two states [51] with rapid switching between the T and the R state. In this case the nonlinearity is built into a single assembly. Catalytic activity, however, sometimes depends on equilibria between oligomers, dimers and tetramers, for instance, in the case of the enzyme pyruvate decarboxylase (PDC) where there is a slow pH-dependent equilibrium. Such equilibria and the structural influence of effecters can be studied by X-ray solution scattering. The shape of the oligomers of PDC in solution was determined and it could be shown that binding of the substrate analogue pyruvamide induces a change in the quatemary structure of the tetramer which is the catalytically active species [52]. In the case of oligomeric enzymes the potential for nonlinearity is usually built into a reaction implying autocatalysis (e.g. 2X + Y -+ 3X) or gain or, if you prefer,

amplification

(for an abstract model, see the Brusselator

[531). One of the more extensively studied nonlinear systems is the tubulin-microtubule system and SR has made a very significant impact on the study of its temporal oscillations [54]. The proposed mechanism for these oscillations is shown in Fig. 3. The nonlinearity in this system is built in mainly through the exponential dependence of the rate constant k,, of the so-called catastrophe (breakdown of microtubules), on the inverse of the concentration of the complex between the tubulin dimer and GTF’ ([Tu-GTF]). Under appropriate conditions self-similar spatial banding patterns on the scale of Frn to mm can also be produced [55] as illustrated in Fig. 4. The formation of these patterns is facilitated by gravitational or magnetic fields, for instance.

5. Reaction-diffusion

,

300

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and morphogenesis

I

200

100

0

0

300

Tu-GTP knuc Tu-GTP + UG(n) -=+=

t [al

MC

nucleation

MG(n+l)

elongation

kcat nG i%F

ns

MS kdepol

oligomer

( oligomer oligomer

koli

Tu-GDP + GTP -

600

catastrophe / rescue depolymerization

ring ) T”_GDp

Tu-GTP + GDP

nucleotide exchange

Fig. 3. Proposed mechanism for the assembly of the complex of tubulin dimer (Tu) with guanosine triphosphate (GTE’) or guanosine diphosphate (GDP) into microtubules. Under appropriate conditions the numbers of growing (MG) and shortening (MS) microtubules oscillate, as illustrated by the time course of cP,, the concentration of tubulin incorporated in microtubules (MG and MS). The model calculations [54] (full lines) reproduce all the features of the experimental data in a variety of conditions.

Although both the temporal and spatial oscillations can be cast in the formalism of reaction-diffusion, an approach that is usually traced back to the seminal paper of A.M. Turing [56], or dissipative chemical systems [53], one should be cautious in the interpretation of such formal models unless all proposed intermediate species and steps can be detected. Reaction-diffusion models can be applied at very different size scales of morphogenesis in biology [57] but there is a great need to experimentally document the specific processes which they represent if these models are to have an operational value. The dissipative (diffusion) processes come into the picture as soon as things become too complex (for us). Dissipative phenomena are a subject which therefore also usually deserves less attention in physics textbooks. This is because, as Feynmann [58] put it, “ You know, however, that on a microscopic level - on the deepest level of physics - there are no nonconservative forces. Nonconservative forces, like friction, appear only because we neglect complications - there are too many particles to analyze”. The size and complexity of biological macromolecules put them in a region of transition between a description in terms of (fast) fundamental phenomena, (e.g the first part of the BR photocycle), and (slower) dissipation phenomena (e.g. the second part of the photocycle). In recent years there has been a number of papers concerning patterns formed in non-equilibrium conditions [59-611, but the potential of these methods for the fabrication of patterned materials does not seem to have yet been fully realized. That it can be done is well illustrated by the patterns of seashells, which can be fully accounted for by reaction-diffusion models [62]. When admiring their beauty we should, however, keep in mind that they are only the fossil signature of the processes from which they result.

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Fig. 4. Self-similar organization of microtubules at different distance scales. The individual stripes ( - OS mm) which form part of a la]rger structure with a repeat distance of about 3 mm, consist of pseudohelical bands of about 100 pm separation. A: Elliptically polarized 1ight view of a sample prepared from tubulin, guanosine ~phosphate (GTP), acetyl phosphate and acetate kinase. The latter two substances are needed to rephospho~late the GDP into GTP, thus keeping the GTP ~ncentration constant (i.e. the system is kept far from equilib~um) . B: A solution observed through cross polars with a wavelength retardation plate. Different orientations of the microtubules (acute and obh3se) give rise to blue and yellow patches respectively. (From Ref. [.5.5]with permission (OAAAS)).

6. Conciusion

The task of illustrating that biological systems have devices that are formally similar to those of electronics is done and one may ask whether it is more than a smart linguistic trick to replace autocatalysis by amplification, directionality by rectification or diffusion by resistance. I believe that the advantage is to provide a more familiar frame of thinking in which, for instance, thermodynamic networks 1631could be used for the description of metabolic pathways. It is interesting in this respect that some of the standard modelling software for analogue circuit design is also used to study complex reaction networks [64]. I have taken most of the references from recent papers in so-called high impact journals to illustrate that the connection between materials sciences and biology is a

very lively one, although many seem not to have realized this yet (so much for high impact!). I guess that it will take some time before material scientists learn how to implement as subtle nonlinear control mechanisms as those of biological systems. In the meantime, however, their progress will increase our understanding of biological materials. What are then realistic expectations for the (nonmediCal) use of biological materials ? The fact that they are not very good at dealing with large energy densities and hence usually also not for very fast signal processing - a property they tend to share with synthetic polymer devices severely limits their range of applications beyond what our ancestors already knew, except perhaps as sensors [65]. As a source of (technological) inspiration and beauty they will, however, remain incomparable.

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Acknowledgements I thank Professor Neville Greaves without whose gentle but continuous pressure this article would never have seen the light, Drs. A. Marx and J. Tabony for kindly providing Figs. 3 and 4, respectively, and Miss Patricia Brouillon for her help in preparing the manuscript.

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