Protein-lipid interaction

Objetivo

A.GENERAL BACKGROUND

A.1.Why a COST action on this topic?

The need of a multidisciplinary and collaborative approach to protein-lipid interactions and exchange of scientific knowledge for a better understanding of the complex phenomena investigated are principal reasons for carrying out the proposed project of research and cooperation in the framework of a COST Action. The organisation of workshops, round table discussions, training courses, and exchange visits which are guaranteed under the COST Action network will provide necessary ways and means for an effective coordination of research activities.

Protein-lipid interactions are essential features of biological membranes, nevertheless many questions related to Chemistry and physics of lipids and proteins are still not understood nowadays. The lack of proper understanding of molecular mechanisms important for the functioning of the biological membranes also hinders practical applications in the industries. For instance, the application of drug encapsulation in lipid vesicles is hampered by short lifetime of the drug in the blood circulation caused by lipid bilayer unstability. In view of the scope of the field, international contacts are thus of vital importance, not only between academic groups, but also with appropriate R&D groups of (i.e. biotechnology, pharmaceutical) industries.

Due to a broad range of activities and multidisciplinary character, the field of Protein-Lipid Interactions would gain substantially from a European cooperation, since in one country scientists could not cover all relevant topics essential for adequate understanding of complex phenomena underlying functioning of biological membranes.

A.2.Status of research in the field

The basic structure of the biological membrane is provided by a lipid bilayer. The lipid bilayer serves as a relatively impermeable barrier to the passage of most water-soluble molecules. The protein molecules, on the other hand, are dissolved in the lipid bilayer and mediate transport of specific molecules, function as enzymes, form structural links of plasma membrane to the cytoskeleton or other extracellular matrices, mediate energy conversion in the cell, and are important receptors for receiving and transducing chemical signals.

Protein-lipid interactions are critical for the above mentioned functions of the membrane. The protein-lipid interface has been extensively characterised, and it is well documented that the configuration of the lipid chains, interactions of the lipid head groups with protein residues, structure of the protein, overall conformation of the protein and protein oligomeric state are important for a proper function of the membrane. Some of these effects arise from general physiochemical properties of the lipid environment, such as lipid composition, hydrophobic and electrostatic interactions, lipid bilayer fluidity, lipid bilayer curvature, protein to lipid ratio, while others may be induced by protein-lipid selectivity, or protein-lipid hydrophobic mismatch. There is, however, relatively little detailed experimental information how proteins and lipids are interacting on a molecular level.

The study of protein and lipid structures has revealed some important consequences for the functioning of the biological membrane. For instance, the study of membrane domain formation suggests that metabolic processes in the membrane generally are made kinetically more efficient in membrane domains by a restricted diffusion of lipids and proteins in the plane of the membrane. Recently, new and fascinating possibilities have appeared, which enables one to measure the size and dynamics of lipid domain formation. Also, related to the domain formation is the assembly of proteins in the membrane, which includes different phenomena like; membrane channel formation, assembly of bacteriophages in the membrane, enzymatic reactions and assembly of protein responsible for electron transport. All these biological processes demand a concerted action of more than one membrane protein. However, relatively little is known about the specificity of these interactions, and the molecular forces involved. A great deal of biophysical research has been focused on the structural studies of lipids and proteins. Lipid structures in an aqueous environment display a variety of morphological structures, which can be classified in general as bilayer and non-bilayer structures. While the role of the lipid bilayer as a fluid matrix for proteins is commonly accepted, the functional role of non-bilayer lipid structures is not well understood. As high-resolution structural data for membrane proteins are becoming available (~ 10) their number is still strikingly lower compared with the number of known high-resolution structures of water-soluble proteins (> 3000). This is mainly due to difficulties with the crystallisation of membrane protein for the purpose of X-ray crystallography, or relatively large protein-lipid complexes for high-resolution NMR structural determinations. Despite these difficulties there has been recently progress in the structural characterisation, due to introduction of 2D electron density maps, and site-directed labelling of membrane proteins. Also, with the progress in membrane molecular biology relatively versatile schemes are now available for the attachment of spectroscopic labels to membrane proteins. In conjunction with selected spectroscopic techniques like FTIR, ESR, CD and fluorescence this will provide a powerful structural tool. It is expected that with the new technology more detailed membrane protein structures will be available in the near future.

The computer simulation of biomembranes by molecular dynamics, stochastic dynamics, and Monte Carlo is another area of rapid growth in protein-lipid research. The results of simulations were used in a number of ways: to interpret details that experiments cannot resolve; to refine intuition and develop models; to predict responses to changes in composition and environment; and finally to determine from first principles the parameters of biological membrane behaviour. Ultimately, however, the results of simulations should be put to experimental trial. In this respect, more research effort is needed to devise suitable model systems for experimental verification of computer simulations.

The highlights of the current development of protein-lipid interactions are summarised in the following topics:

1.Relationships between structure and function of biological membranes. Studying relevant membrane model systems with a variety of techniques, including X-ray crystallography, NMR, ESR, CD, FTIR, Raman spectroscopy, fluorescence spectroscopy and molecular dynamics is deepening the understanding.

2.Protein-protein assembly in the biological membrane. The amino acids and their patterns that are responsible for protein recognition and aggregation in the membrane are being recognised. Some of the molecular details of protein insertion in the membrane were determined.

3.Membrane domain formation. The role of hydrophobic mismatch, lipid and protein lateral diffusion and induction of membrane domain formation in response to different environmental factors is now well established. The importance of lipid domain formation for electron transport in mitochondria and chloroplasts was demonstrated.

A.3.Relations with other European Programmes

There is no particular specific European programme devoted at present to Protein-Lipid Interactions.

The significant interest in the field of protein-lipid interactions is exemplified by recent European research conference on molecular basis of biological membrane protein structure and function, Albufeira, 1997.

B.OBJECTIVES OF THE ACTION AND SCIENTIFIC CONTENT

B.1.Main objectives

The main objective of the Action is an increase of the knowledge of protein-lipid interactions on molecular level and time scales, based on interdisciplinary approach by chemists, physicists and biologists.

Strong links between academics and relevant industries will reinforce these efforts. The expected results are of high scientific, economic and educational value. Understanding the Chemistry and physics of biological membranes is central of this Action.

The scientific goals can be summarised as follows:

-Integration of chemical and physical knowledge on biological membrane systems.
-Better understanding of molecular processes and time scales important for the functioning of biological membranes, with emphasis on structural aspects of lipids and proteins, protein aggregation and membrane domain formation by using different membrane model systems.
-Reinforcement of research efforts by strong links between academic institutions and industries.

B.2.Sub topics

1.Structure-Function Relationship

In biological systems function is ultimately related to structure, and protein-lipid systems are no exception. There exists a great deal of information concerning lipid structures, but a limited amount of direct structural information is available on membrane proteins. Consequently, relatively little is known about the detailed structure-function relationship of protein-lipid systems. In particular, the effects of bilayer physicochemical properties on protein structure and function are anticipated to give further insight in membrane functioning. With respect to the bilayer properties the focus is expected to lie on lipid physical states, lateral pressure profiles, bilayer thickness, and comparison of protein structural parameters obtained from various spectroscopic techniques (NMR, FTIR, CD, ESR, fluorescence) in different bilayer model systems.

It is proposed to study:

-Effects of symmetrical hydrophobic peptides on the structure and dynamics of lipid bilayers.
-The regulatory effect of hydrophobic thickens of lipid bilayer on protein functioning.
-The effect of protein secondary structures on the ion channel activity.
-The influence of bilayer lateral pressure on the activity of membrane enzymes or conducting properties of membrane ion channels.
-The effect of protein-lipid topology on membrane functioning.
-Structure and function of water soluble protein-lipid assemblies.

2.Protein-Protein Assembly

Most biological processes demand a concerted action of more than one protein. In the biological membrane there are several processes where protein-protein recognition plays a very important role. For instance, making a channel, insertion of proteins in the membrane, assembly of enzymes, assembly of electron transport chains, and phage assembly. Recent theoretical and experimental studies have started to address the fundamental problem of how proteins recognise each other, and which molecular forces are involved. However, more effort should be directed towards amino acid motifs that are important for dimer or oligomer formation, and the importance of hydrophobic and electrostatic interactions between proteins. An important contribution to the understanding of processes that lead to the aggregation of the proteins in the membrane came from simulation work using Monte Carlo simulations on model membrane systems. In such simulations the effects of Van der Waals and electrostatic forces, local curvature, lipid-mediated attraction, and perturbations due to inclusions was studied in detail. However, better model systems are needed to experimentally verify the conclusions from computer simulations.

It is proposed to study:

-Assembly of membrane channels.
-Helix-helix interactions in monotopic and polytopic membrane proteins.
-Transmembrane helix stability.
-Protein attraction induced by lipid fluctuations.
-Integration of membrane proteins through channels into the lipid bilayer.
-Monte Carlo simulations of protein aggregation in the lipid bilayer.

3.Membrane Domains

The reason for the great variety of lipids found in biological membranes, and the relations between lipid compositions and membrane function pose one of major unsolved problems in membrane biology. If a single role of lipids were to maintain the fluidity and assembly of the membrane, such a variety of lipids would not be required. In addition, there are certain conceptual problems involved in assuming a constant bilayer structure. It is becoming increasingly clear that such basic processes as cell fusion, exo- and enodcytosis, facilitated transport, and the rate of reactions are difficult to reconcile with the classical model of a membrane mosaic proposed in 1972 by Singer and Nicolson. The recent development in the field provides strong evidence that phase separation occurs in biological membranes. Phase co-existence of microscopic domains provides a basis for understanding long-range translational diffusion, lateral segregation of membrane components and efficiency of bimolecular reactions that occur in membranes.

Now, that we are in the position to challenge mosaic model of the biological membrane, the research should focus on processes that are important in formation of membrane domains. Study of origin of phase separation and domain formation in membranes may reveal relevant thermodynamical parameters important for domain formation. The extent of hydrophobic matching between proteins and lipids in membranes, which can play an important role in the lateral segregation of proteins in biological membranes, should be studied in different membrane model systems. To get more insight in the dynamics of membrane domains, percolation limit, domain topology and bimolecular reactions in phase-separated domains, a further theoretical and experimental

development is needed. On the more practical side, the role of various membrane proteins regulating lipid asymmetry (flippases, scramblases and MDR proteins) in the control of enzyme activity, apoptosis and possibly endo-exocytic activities should be tested. Generally speaking, all physico-chemical studies concerning membrane domain formation will have a direct impact on our perception and understanding of biological membrane functioning.

B.3.Benefits coming from the implementation of this Action

-Knowledge gained from multidisciplinary research using different membrane model systems.
-Significantly increased personal contacts and collaborations between scientists.
-Practical application of knowledge on protein-lipid interactions.
-Efficient co-ordination of European research activities on protein-lipid interactions.
-Improved chances of employment for PhD students and post-docs in other COST countries.

B.4.Some specific examples of research

Many studies using different techniques have shown that the activity of membrane proteins is sensitive to mismatch between proteins and lipids. For instance, Ca2+-ATPase and (Na++K+)-ATPase from sarcoplasmic reticulum reconstituted in mono-unsaturated PC (phosphatidylcholine) bilayers of varying length showed an optimum activity at a lipid chain length of 18 carbon atoms. Longer or shorter lipid chain length markedly decreased activity. A similar chain length dependence of enzyme activity was found for mitochondrial cytochrome c oxidase or leucine transport system of Lactobacilus lactis. On the other hand, the 2H-NMR studies on gramicidin in a bilayer of PC with different thickness showed that in di-C14:0-PC the average lipid chain order was significantly increased, but less so in di-C 6:0-PC, while a small decrease was observed in di-C 8:0-PC.

Eukaryotic membrane proteins are normally transported through the secretory pathway from the endoplasmic reticulum to Golgi and then to the plasma membrane. Due to different membrane thickness in this cell structures different protein-lipid interactions are responsible for protein anchoring in the membrane. It was demonstrated, using genetically modified protein UBC6, that lengthening of the tail anchor from 17 hydrophobic amino acids to 21 amino acids resulted in targeting protein to the Golgi, while a further increase to 26 hydrophobic amino acids resulted in localization at the plasma membrane. This clearly demonstrates the importance of physico-chemical processes in protein trafficking in the cell.

The protein-conducting channels in the plasma membrane of bacteria and membrane of endoplasmic reticulum in eukaryotic cells are built from the same basic components. Three integral membrane proteins Sec61?, ?, ? in eukaryots and SecA, B, E, G and J proteins in prokaryots together form the central translocation complex through which the protein is threaded. The most interesting aspect of the chemistry of protein insertion is, no doubt, the mechanism by which sufficiently hydrophobic segments are first recognized by the translocation complex, are arrested during translocation, and finally escape laterally into the lipid bilayer. Recent method developments, with site-specific generation of cross-links between membrane lipids and polypeptide chains in transit through the translocation complex, have revealed that at least three distinct environments exist as a transmembrane segment first enters and than leaves the translocation channel. Upon entering the translocation channel the transmembrane segment is first found close to Sec6?, upon further elongation it moves into a hydrophobic canyons between Sec61?, ?, ? trimers, from where it can be released into the bilayer. It was also shown that membrane proteins could be transported in reverse, i.e. from the membrane through the translocation channel to the cytoplasm.

C.SCIENTIFIC PROGRAMME

The scientific programme will depend on the projects submitted by individual research teams. The working group projects will be selected according to the objectives outlined above. Summarising these topics we can formulate three broad lines of research; structure-function relationship, protein-protein assembly and membrane domain formation. At this stage there is no specific scientific programme suggested for this action in order to place no limitation on the invited proposals. The selection, however, will strictly occur according to the outlined objectives.

D.ORGANISATION AND TIME-TABLE

D.1.Organisation

Research projects fitting in the sub-topics described in section B2 will be submitted by scientists to the Management Committee members. This Committee will establish contacts between scientists.

The Management Committee has responsibilities for:

-Drawing up an inventory of research groups working in the field in the participating COST countries during the first year.
-Organising workshops.
-Coordinating activities with other COST Actions; joint meetings are likely to result from this activity.
-Exploiting wider participation and exchange of information with other EC specific programmes as well as contacts to relevant industries.
-The planning of the intermediate report, the final report and the concluding symposium.

D.2.Timetable

Stage 1: After the first meeting of the Management Committee, a detailed inventory of ongoing research and existing plans of the participating groups to begin joint projects will be made. This will result in a discussion document, which will allow further planning to occur.

Stage 2: From the inventory it will be evident which projects are closely related and would benefit from joint activities. Researchers will set up joint collaborative projects and exchange their recent research results. It may be appropriate to explore wider collaboration with other European countries during this stage.

Stage 3: An intermediate progress report will be prepared after 2 years for review by the COST Technical Committee for Chemistry and for information to the COST Senior Officials Committee.

Stage 4: The final phase will begin after 4 years and will involve the evaluation of the results obtained. It may include the organisation of a symposium for all the participants and co-workers. The final report will be submitted to the COST Technical Committee for Chemistry for scientific assessment and after to the COST Senior Officials Committee.

E.DURATION OF THE ACTION:

The Action will last for five years.

F.ECONOMIC DIMENSION

On the basis of national estimates provided by the representatives of the COST countries that have actively participated in the preparation of the Action or have otherwise indicated their interest the overall cost of the activities to be carried out under the Action has been estimated, in 1999 prices, at roughly EUR 4,7 million/year.

This estimate is valid under the assumption that all the COST countries mentioned above would participate in the Action. Any departure from this will change the total cost accordingly.

Scientific staff:38 man x EUR 60 000 = EUR 2,28 million
Technical staff:14 man x EUR 40 000 = EUR 0,56 million
Doctoral students:27 man x EUR 25 000 = EUR 0,67 million
Total staff:79 mantotal EUR 3,51 million
Laboratory equipment and consumables EUR 0,8 million
Overhead costs EUR 0,4 million
Total estimated costs covered from national sources EUR 4,71 million/year.

G.DISSEMINATION OF SCIENTIFIC RESULTS

All publications arising from research carried out under COST Action will credit COST support and the Management Committee will encourage and promote all co-authored papers. Results of research carried out by the working groups under COST Action will be submitted to international scientific journals and reviews.

The Management Committee in conjunction with the working groups will invite potential users and interested parties to annual Management Committee meetings.

When appropriate The Management Committee will organise joint meetings with relevant COST Actions in such a way to best promote inter-disciplinary communication.

Programa(s)

IC-COST - European cooperation in the field of scientific and technical research (COST), 1971-

Tema(s)

13 - Chemistry

Convocatoria de propuestas

Data not available

Régimen de financiación

Data not available

Coordinador

N/A

Aportación de la UE

Sin datos

Dirección

Coste total