1. INTRODUCTION AND WORK PROGRAMME

The basic idea of this workshop is to bring together the international expertise from the different fields of research activities, e.g. bio-physical chemistry, cell biology, pharmacology, all aiming, in one way or the other, at the development of new antimicrobial peptides for medical and veterinary use. Antimicrobial peptides, or generally defense peptides, kill the invading microbe by breaking it into pieces: the primary locus of action is the bacterial membrane, being disrupted by the antimicrobial peptide. In the search for potential antimicrobial agents that fulfill the requirements for use in medicine, the first approach is to look whether and how a given peptide interacts with a biological or model membrane. This is where Trieste comes in.

Why Trieste?

ELETTRA, near Trieste, is a synchrotron light source of the latest generation that combines extreme brilliance in the X-ray region with complete dedication to user-friendliness. Among the first beamlines to come into operation last year was the Austrian CRG (cooperating research group) beamline designed for high-flux X-ray small-angle scattering (SAXS). The prime motivation to build this excellent research tool was to investigate the nano-structural events during processes of biological interest, such as membrane transitions or protein conformational changes in solution, in real time, i.e. to enter the sub-millisecond domain with time-resolved SAXS. The interaction of membrane-active agents, and specifically of membrane-transforming peptides, with lipid model membranes is one key project area, supported by wide expertise in biochemistry and biophysics of lipids and peptides existing at the respective Italian and Austrian research institutes. This is presently one of the world’s best instruments in the field of SAXS, and we would like to see this being reflected by the work done with it, particularly in the field covered by the workshop.

The workshop should, therefore, be more than a scientific conference with an impressive list of high-level presentations. It is supposed to produce results in the following ways:

• update the current opinions in the various sectors of the field
• stimulate structural investigations
• provide a key for priorities with the X-ray structural methods, in particular with synchrotron radiation.

The present preview shall provide those who are not well familiar with the potential of X-ray structural methods in general and synchrotron radiation in particular, with a concise introduction, so that they can reach a quick apprehension what this technique can do for the field.

For further reading, a list of reviews and original papers is given in the Annex.

2. BACKGROUND ON X-RAYS AND MODEL MEMBRANES

What type of information can be gained from X-ray measurements?

X-ray diffraction is by no means limited to the availability of well-developed single crystals. Indeed, with biological membranes as with many other important supramolecular structures, the main contribution by X-ray diffraction comes from its potential to provide structural information also from random or partly ordered systems.

X-ray diffraction offers a highly convenient and informative way to study lipid polymorphism by a comparatively simple approach which does, in most cases, not require extensive procedures for data analysis. The techniques have become comparable in their ease of application to spectroscopic or thermodynamic techniques, and thus form an integrating component in the methodical arsenal of membrane biophysics.

Polar lipids, such as phospholipids, when dispersed in water, form aggregates with a certain type of local symmetry that can be crudely classified as lamellar, hexagonal, or cubic structures, depending on the nature of the lipid and the physico-chemical state of the system (T,p, concentration, salts, proteins or peptides, or other additives), and they exhibit mesomorphic phase transitions. With the exception of cubic phases, which can grow to large monocrystalline dimensions of the size of the sample container, these phases normally are present in microscopic crystalline domains, randomly oriented throughout the sample, so that the X-ray beam (its cross section normally being much larger than the individual crystallite) "sees" the situation of a crystalline powder. This has the effect, that all lattice planes, spaced at a repeat distance d, simultaneously meet the Bragg-condition

n·l = 2d·sinq

(n=1,2,3..., ‘order of the reflection’, 2q ...diffraction angle, l ...wavelength)

thus leading to a powder-pattern of concentric rings about the direction of the primary X-ray beam.

With powder patterns from phospholipid dispersions, except for some cases of complex cubic structures, only a few reflections are sufficient to describe the type of symmetry (e.g. lamellar or hexagonal) and the relevant dimensions, i.e. the repeat distances. With these characteristic distances being in the order of 10 to several 100 Å, the corresponding diffraction signals (with wavelengths l in the order of 1 Å) lie in the small-angle region, typically between 10-100 mrad.

Important structural information is also contained in the wide-angle region, between about 10 and 50°, corresponding to distances between about 9 and 2 Å, which are the characteristic dimensions of the hydrocarbon chain lattice, if they are not in a liquid disordered state. Ideally, these two distinct regions of the diffraction pattern should be measured simultaneously to characterize the structure of a given phase. Such simultaneous small-and wide-angle (SWAX) cameras are available as commercial instruments, and also the Austrian CRG-station at ELETTRA is equipped with such a modification.

The following table summarizes the type of structural information and the way, how it may be obtained from X-ray diffraction/scattering :

3. SOME EXAMPLES

In the following, a few results of representative studies performed by the Graz group on the effects of membrane active peptides shall be presented to illustrate the above described potential.

Melittin

It has been known for many years from calorimetric and dilatometric data, that melittin has drastic effects, already at peptide-to-lipid ratios of less than 1:1000, on the phase transition behavior of phosphatidylcholine liposomes. SWAX experiments have dramatically verified this notion (see Fig.1). Qualitatively, the most relevant effects are the following: in the liquid crystalline state of the lipids, which is closest to the one which the peptide acts on in real life, a considerable change in solvation and/or induction of curvature is observed. Melittin seems to force the bilayers to curve. This may destroy the proper contiguity between lipids and proteins of cells, e.g. erythrocytes and thus lead to lysis.

WAX data have confirmed the calorimetric data by showing that already at very low doses of melittin (1:1000), the hydrocarbon chain packing is changed throughout the system.

Fig 1.(next page): SAX patterns of 20% aqueous dispersions of DPPC, with 0.1 and 1 mol-% melittin (top to bottom), in the temperature range between 4 and 50 °C.

d -Lysin

The system d-lysin, a bacteriotoxin from S. aureus, and dimyristoyl-phosphatidylcholine (DMPC) has been studied. Pure DMPC liposomes exhibited small-angle X-ray scattering profiles with sharp Bragg peaks characteristic for multilamellar vesicles (Panel A). In the presence of peptide the intensity of the Bragg peaks decreased significantly and were superimposed upon a diffuse scattering background (Panel B). Additionally, a strong increase of intensity of the innermost part the small-angle scattering range suggests the presence of smaller lipidic particles. X-ray diffraction patterns of the pellet obtained after centrifugation of the lipid-peptide mixture were essentially similar to those of pure DMPC liposomes demonstrating the multilamellar nature of the pellet (Panel D). In the case of the supernatant the scattering curve shows a broad reflection typical of a single bilayer transform (Panel C).

Fig. 2 : SAX data of DMPC/d -Lysin

The presence of single bilayer particles in these samples was further verified by indirect Fourier transformation of the experimental small-angle X-ray scattering curve of the supernatant. This analysis was consistent with large unilamellar vesicles with a lamellar thickness of 5.2 nm. These data clearly show that d-lysin above a certain treshhold concentration induces disruption of multilamellar vesicles which rearrange in unilamellar liposomes.

PGLa

Wide-angle X-ray diffraction (Fig.3) experiments give information on the hydrocarbon chain packing arrangements. Such type of experiments are described exemplary for the antimicrobial peptide PGLa, isolated from the skin sekretion of the South African clawed frog, and liposomes composed of the main phospholipid components of bacteria, namely phosphatidylglycerol (PG) and -ethanolamine (PE). Wide-angle X-ray diffraction patterns of liposomes consisting of pure PG or PG rich mixtures were characterized by an asymmetric reflection with a peak at 0.412 nm and a shoulder around 0.402 nm. This is typical for phospholipids with the hydrocarbon chains tilted relative to the normal of the bilayer plane. However, as shown in Figure 3, a rather sharp and symmeric peak centered around 0.413 nm was observed in the presence of PGLa, indicating that the chains become oriented normal to the bilayer plane. These results demonstrate that PGLa penetrates into the hydrophobic core of the bilayer inducing an untilting of the lipid chains.

Fig.3: WAX data of PE/PG lipid mixtures with and without PGLa

4. WHY SYNCHROTRON RADIATION ?

The main advantages of using synchrotron radiation SAX and SWAX techniques lies in the drastically reduced time to measure a diffractogram. This can be exploited in two fundamentally different ways.

Mass Screening

In the search for a systematic pattern of interaction types between different antimicrobial peptides and multiple combinations of lipid model membrane substrates, it is necessary to screen large numbers of samples and conditions. With classical laboratory X-ray equipment one would be able to perform about a dozen measurements per 8 hours shift. This number is limited simply by the low intensity of the laboratory X-ray source (even with a rotating anode).

With the Austrian SAXS beamline at ELETTRA a one-second exposure is sufficient per typical multilamellar liposome sample. Given the development of fast sample changing devices, several thousand patterns could be screened during one shift. This could be turned into an enormous advantage for the systematic investigation on membrane modulation. There the limits are essentially set by the speed of sample preparation and changing.

Real Time X-Ray Diffraction

While the above method is static, giving a structural picture at equilibrium, synchrotron radiation with its high X-ray flux can also be used for fast time-resolved studies on structural transitions. With lipid systems, as in the present case, exposure times of milliseconds and below have been reached. This could certainly be useful in the field of antimicrobial peptides, where it is interesting to not only find out which peptides destroy membranes, but also how they do it. This could be done e. g. in a rapid mixing / stopped-flow experiment.

An additional advantage offered by synchrotron radiation SAX facilities as the one at ELETTRA is the wide freedom for choice of sample environment. This means that, unlike with most available laboratory X-ray cameras, the configuration of the sample, e. g. application of an electric or magnetic field, elevated pressure or other exotic conditions, can be installed without great difficulties.

5.THE MOST FREQUENTLY ASKED QUESTIONS

- How is a peptide inserted into a membrane?

- What is its local structure and that of its immediate surroundings?

- Is it inserted as a monomer or as an oligomer?

- Is it located in the polar/apolar interface or rather penetrating into the

hydrocarbon interior of the lipid bilayer?

This sort of questions cannot be answered in a straightforward manner by X-ray diffraction techniques. In the limit of low peptide abundance at peptide-to-lipid ratios of 1:1000 and less, the scattering power of the peptide and its immediate surroundings is greatly outweighted by the excess of lipid material. At higher doses, where the interaction already leads to massive membrane destruction and to the production of small micellar fragments, the question could be answered by solution X-ray scattering techniques. It would remain to be established, however, whether the structures so obtained are also representative for the situation, where one peptide molecule transforms thousands of lipids.

On the basis of the initially stated questions is certainly the desire to understand the local structural features of peptide folding, self-association and interaction with neighbouring lipid molecules, which are responsible for membrane destruction. It appears that these questions can be satisfactorily solved by an indirect approach: The systematic search for the minimum essential components of a peptide structure aided by computer modelling and screening of the effects on target model membranes, is certainly the method of choice, where X-ray techniques can play a most helpful role.

6. SUGGESTED READING

Review Literature:

P. Laggner. 1994.X-Ray Diffraction on Biomembranes with Emphasis on Lipid Moiety.
In: "Subcellular Biochemistry" Vol. 23, Physico-chemical Methods in the Study of Biomembranes (eds. H. J. Hilderson and G. B. Ralston), Plenum Press, pp. 451-491

P. Laggner and M. Kriechbaum. 1995.
Time-Resolved X-Ray Small-Angle Diffraction with Synchrotron Radiation on Phospholipid Phase Transitions. Pathways, Intermediates and Kinetics.
In: "Modern Aspects of Small-Angle Scattering" (ed. H. Brumberger), Kluwer Academic Publ., pp. 387-407.

P.Laggner and M. Kriechbaum. 1997.

Small-Angle X-Ray Scattering under Extreme Conditions of Temperature, Pressure and Time.

In: "X-Ray Investigations of Polymer Structures" (ed. A.Wlochowicz, J.Janicki, and Cz.Slusarczyk), SPIE Proceedings 29XR, pp.17-34

K. Lohner and R.M. Epand. 1997.

Membrane Interactions of Hemolytic and Antibacterial Peptides.

Adv. Biophys. Chem., Vol. 6, pp. 53-66, JAI Press Inc.

Original Papers:

P. Laggner and H. Mio. 1992.
SWAX - A Dual-Detector Camera for Simultaneous Small- and Wide-Angle X-Ray Diffraction in Polymer and Liquid Crystal Research.
Nucl. Instr. Meth. in Phys. Res. A323, 86-90.

P. Laggner and H. Mio. 1992.
SWAX - A Dual-Detector Camera for Simultaneous Small- and Wide-Angle X-Ray Diffraction in Polymer and Liquid Crystal Research.
Nucl. Instr. Meth. in Phys. Res. A323, 86-90.

A. Colotto, K. Lohner and P. Laggner. 1991.
Small-Angle X-Ray Diffraction Studies on the Effects of Melittin on Lipid Bilayer Assemblies.
J. Appl. Cryst. 24, 847-851

A. Colotto, D. P. Kharakoz, K. Lohner and P. Laggner. 1993.
Ultrasonic Study of Melittin Effects on the Phase-Transitions of Phospholipid Multibilayer Systems.
Biophys. J. 65, 2360-2367.

H.Amenitsch, S.Bernstorff and P.Laggner. 1995.

High Flux Beamline for Small-Angle X-Ray Scattering at ELETTRA.

Rev. Sci. Instrum. 66(2), 1624-1626

K. Lohner, A. Latal, R.I. Lehrer and T. Ganz. 1997.
Differential Scanning Microcalorimetry Indicates that Human Defensin, HNP-2, Interacts Specifically with Biomembrane Mimetic Systems.
Biochemistry 6, 1525-1531.

A. Latal, G. Degovics, R.F. Epand, R.M. Epand and K. Lohner. 1997.
Structural Aspects of the Interaction of PGLa, a Highly Potent Antimicrobial Peptide from Frog Skin, with Lipids.
Europ. J. Biochem. 248, 938-946

G. Degovics, A. Latal, E. Prenner, M. Kriechbaum and K. Lohner. 1997.
Structure and Thermotropic Behavior of Mixed Choline Phospholipid Model Membranes.
J. Appl. Cryst., in press