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Workshop Programme

for period 21 - 23 June 2004

Protein Interactions in Vitro and in Vivo

21 - 23 June 2004

Timetable

Monday 21 June
Session: Protein-Protein Interactions in Vitro and in Vivo
13:30-14:30 Dobson, CM (Cambridge)
  Protein interactions in the context of folding, misfolding \& aggregation Sem 1
 

Protein folding is perhaps the most fundamental process associated with the generation of functional structures in biology. There has been considerable progress in the last few years in understanding the underlying principles and dominant interactions that govern this highly complex process. Recently, much research has also focused on the realisation that proteins can misfold in vivo and that this phenomenon is linked with a wide range of highly debilitating diseases that are becoming increasingly prevalent in the modern world. We have been investigating in particular the nature of the amyloidogenic conditions, that include Alzheimer's disease, type 2 diabetes and the spongiform encephalopathies, e.g. BSE and CJD, in which protein misfolding leads to the aggregation of proteins, often into thread-like amyloid structures. Our studies have led us to put forward new ideas concerning the fundamental origins of the various diseases associated with their formation and the various strategies that can be used for their prevention and treatment. We have also speculated more generally that the need to avoid aggregation could be a significant driving force in the evolution of protein sequences and structures.

References:

M. Vendruscolo, J. Zurdo, C.E. MacPhee and C.M. Dobson, “Protein Folding and Misfolding: A Paradigm of Self-Assembly and Regulation in Complex Biological Systems”, Phil. Trans. R. Soc. Lond. A 361, 1205-1222 (2003).

M. Dumoulin, A.M. Last, A. Desmyter, K. Decanniere, D. Canet, G. Larsson, A. Spencer, D.B. Archer, J. Sasse, S. Muyldermans, L. Wyns, C. Redfield, A. Matagne, C.V. Robinson and C.M. Dobson, “A Camelid Antibody Fragment Inhibits the Formation of Amyloid Fibrils by Human Lysozyme”, Nature 424, 783-788 (2003).

F. Chiti, M. Stefani, N. Taddei, G. Ramponi and C.M. Dobson, “Rationalisation of Mutational Effects on Protein Aggregation Rates Using Simple Physical Principles”, Nature 424, 805-808 (2003).

C.M. Dobson, “Protein Folding and Misfolding”, Nature 426, 884-890 (2003).

C.M. Dobson, “In the Footsteps of Alchemists”, Science 304, 1259-1262 (2004).

 
14:30-15:30 Fink, AL (California)
  Factors affecting the aggregation of the natively unfolded protein alpha-synuclein Sem 1
 

The etiology of Parkinson’s disease is unknown; however, substantial evidence implicates the aggregation of alpha-synuclein as playing a critical role in the disease. We have found that a variety of endogenous and exogenous factors induce a conformational change in alpha-synuclein and directly accelerate the rate of formation of alpha-synuclein fibrils in vitro; other factors inhibit the fibrillation. The mechanism of alpha-synuclein aggregation involves at last three competing kinetic pathways, leading to fibrils, amorphous aggregates, and soluble oligomers. Thus, many factors may cause acceleration of alpha-synuclein fibrillation, and some of these factors are likely to be important in the pathophysiology of alpha-synuclein and Parkinson’s disease. Various aspects of how the self-assembly of alpha-synuclein occurs will be discussed.

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16:00-17:00 Minton, AP (NIH)
  Mesoscopic models for the effect of macromolecular crowding, macromolecular confinement \& surface adsorption upon protein assoc. Sem 1
 

A large fraction of the total volume of all biological fluid media is either occupied by soluble macromolecules, or lies within a distance of macromolecular dimension from the surface of an extended structural element such as a cytoskeletal fiber or a membrane. I will describe mesoscopic statistical-thermodynamic models for the excluded volume interaction of a globular protein with inert "background" macromolecules and model boundaries, and for nonspecific weakly attractive interaction between a globular protein and a planar surface. The results of calculations based upon these models will be presented, describing how nonspecific interactions of soluble proteins with features of the local environment are likely to affect specific associations between the soluble proteins. Model predictions will be compared with the result of experiment where relevant data are available.

 
Tuesday 22 June
Session: Protein-Protein Interactions in Vitro and in Vivo
09:00-10:00 Thornton, JM (European Bioinformatics Inst.)
  Protein-protein interactions from a structural perspective Sem 1
 

An update of the structural features of multimeric proteins will be presented. We have compiled low redundancy sets of homomeric proteins with different symmetry and subunit composition as well as sets of heteromeric proteins for comparison. We find significant variations between monomers and multimers and with the additional data we compare dimers, trimers, tetramers and hexamers. The variations we observe can all be seen as consequences of the hydrophobic effect, which has long been noted as a major driving force in protein folding and association. A comparison with transient complexes will also be presented.

Ponstingl, H., Kabir, T. & Thornton, J.M. (2003) Automatic Inference of Protein Quaternary Structure from Crystals. J. Appl. Cryst. 36, 1116-1122.

Nooren, I.M.A. & Thornton, J.M. (2003a) Structural characterisation and functional significance of transient protein-protein interactions J. Mol. Biol. 325, 991-1018. PMID: 12527304

Nooren, I. & Thornton, J.M. (2003b) Diversity of protein-protein interactions. EMBO Journal. 22, 3486-3492. PMID: 12853464

 
10:00-11:00 Deem, MW (Rice)
  Random energy models for interactions and dynamics in the immune response to viruses, vaccines, and cancer Sem 1
 

The adaptive vertebrate immune system is a wonder of modern evolution. Under most circumstances, the dynamics of the immune system is well-matched to the dynamics of pathogen growth during a typical infection. Some pathogens, however, have evolved escape mechanisms that interact in subtle ways with the immune system dynamics. In addition, negative interactions the immune system, which has evolved over 400 000 000 years, and vaccination, which has been practiced for only 200 years, are possible. For example, vaccination against the flu can actually increase susceptibility to the flu in the next year. As another example, vaccination against one of the four strains of dengue fever typically increases susceptibility against the other three strains. Immunodominance also arises in the immune system control of nascent tumors--the immune system recognizes only a small subset of the tumor specific antigens, and the rest are free to grow and cause tumor growth.

In this talk, I present a physical theory of original antigenic sin and immunodominance. How localization in the immune system leads to the observed phenomena is discussed.

1) M. W. Deem and H. Y. Lee, ``Sequence Space Localization in the Immune System Response to Vaccination and Disease,'' Phys. Rev. Lett. 91 (2003) 068101.

2) J.-M. Park and M. W. Deem, ``Correlations in the T Cell Response to Altered Peptide Ligands,'' Physica A, to appear.

 
11:30-12:30 Teichmann, SA (Cambridge)
  Domain interactions in multi-domain proteins Sem 1
 

Two thirds of all prokaryote proteins, and eighty percent of eukaryote proteins are multi-domain proteins. The composition and interaction of the domains within a multi- domain protein determine its function. Using structural assignments to the proteins in completely sequenced genomes, we have insight into the domain architectures of a large fraction of all multi-domain proteins. Thus we can investigate the patterns of pairwise domain combinations, as well as the existence of evolutionary units larger than individual protein domains. Structural assignments provide us with the sequential arrangement of domains along a polypeptide chain. In order to fully understand the structure and function of a multi-domain protein, we also need to know the geometry of the domains relative to each other in three dimensions. By studying multi-domain proteins of known three- dimensional structure, we can gain insight into the conservation of domain geometry, and the prediction of the structures of domain assemblies.

 
14:00-15:00 Janin, J (CNRS)
  A structural basis for the specificity of protein-protein recognition Sem 1
 

We compare the geometric and physical chemical properties of interfaces involved in specific and non-specific protein-protein interactions in crystal structures reported in the Protein Data Bank. Specific interactions are illustrated by 70 protein-protein complexes and by subunit contacts in 122 homodimeric proteins; non-specific interactions, by 188 pairs of monomeric proteins making crystal packing contacts selected to bury more than 800 Å2 of protein surface. A majority of these pairs have two-fold symmetry and form ‘crystal dimers’ that cannot be distinguished from real dimers on the basis of the interface size or symmetry. Their chemical and amino acid compositions resemble the protein solvent accessible surface, they are less hydrophobic than in homodimers and contain much fewer fully buried atoms. We develop a residue propensity score to assess preferences for the different types of interfaces, and we derive indexes to evaluate the atomic packing, which is less compact at non-specific than at specific interfaces.

These differences can be interpreted in terms of geometric and chemical complementarity in cases where conformation changes are small and recognition takes place between preformed surfaces. In contrast, large changes at an interface imply that recognition first occurs between surfaces that are not complementary. A basic question in molecular assembly is how this process takes place, and whether we can reproduce it. Molecular docking algorithms that generate protein-protein complexes based on the component structures have been tested in a blind prediction experiment called CAPRI (Critical Assessment of PRedicted Interactions). Results obtained on 13 target complexes indicate that prediction procedures often succeed when the conformation changes are small, although they fail to reproduce large changes.

References:

The structural basis of macromolecular recognition. S.W. Wodak & J. Janin (2002) Adv. Prot. Chem. 61 9-68. Dissecting protein-protein recognition sites. P. Chakrabarti & J. Janin (2002) Proteins 47, 334-343 Dissecting protein-protein interfaces in homodimeric proteins. R.P. Bahadur, P. Chakrabarti, F. Rodier & J. Janin (2003) Proteins 53, 708-719 A dissection of specific and non-specific protein-protein interfaces. R P Bahadur, P Chakrabarti, F Rodier & J Janin (2004) J. Mol. Biol. 336, 943-955

 
16:00-17:00 Truskett, TM (Texas, Austin)
  Towards a simple coarse-grained strategy for modelling unfolding, phase separation, and aggregation in protein solutions Sem 1
 

Protein stability, aggregation, and crystallization are of fundamental scientific and technological importance. However, molecularly-detailed models that can account for both the proteins and the solvent are computationally prohibitive to study under relevant solution conditions. As a result, the relations between misfolding/aggregation events in solution and protein sequence (mutations), solvent conditions, solution composition, and the presence of interfaces are still poorly understood. Here, we introduce a strategy for investigating these phenomena through use of a new coarse-grained model that combines an analytical theory for heteropolymer collapse with a recently introduced model for solvation in aqeuous solution. This approach can qualitatively reproduce the basic effects of temperature, pressure, and sequence on protein stability. We have used the model to derive effective center-to-center interactions for native and denatured proteins. We are currently using these effective interactions as inputs to liquid-state theory and simulation to gain new insights into the global experimental behavior of protein solutions.

 
Wednesday 23 June
Session: Protein-Protein Interactions in Vitro and in Vivo
09:00-10:00 Harden, JL (Johns Hopkins)
  Sequence patterning in simple de novo proteins with tailored association behavior Sem 1
 

Patterning of charged or hydrophobic residues in repetitive amino acid sequences is often associated with the formation of well-defined assemblies such as helix bundles and beta sheets. In recent years, de novo protein designs have extensively utilized such patterned motifs to construct minimalist sequences that fold or aggregate in a predetermined manner via the specific association of secondary structures. This talk will discuss examples of de novo designs based on associating amphiphilic secondary structures. In particular, we will focus on a series of modular de novo proteins that preferentially form homo- and hetero-trimeric assemblies. The stability of these assemblies and the secondary structure of the associating elements in various solution conditions will be presented and discussed in the context of opportunities for theoretical models of these simple systems

 
10:00-11:00 Vekilov, PG (Houston)
  Phase transitions in protein solutions Sem 1
 

Dense liquid, gel-like and solid, ordered in three, two, or one dimension, or completely disordered phases form in protein solutions and underlie physiological and patho-physiological, laboratory, and technological processes. The loss of phase stability of the protein solution represents the ultimate form of intermolecular interactions at high solution concentrations. The loss of phase stability can be accompanied by loss of conformational stability, as in the formation of the amyloid fibrils, or occur with preservation of the protein conformation.

Two aspects of the phase transitions will be discussed.

The first one is the role of water, structured at the hydrophobic and hydrophilic patches on the surface of the protein molecules. Examples will be provided illustrating that this structuring often determines the entropy and enthalpy balance of the phase transition, leads to unusual intermolecular interaction potentials with one or more outlying maxima, which severely affect the phase diagrams, and that the dynamics of destruction of the water shell is the major determinant of the kinetics of association of molecules into solid phases. Because of the water structuring, the fastest pathway of nucleation of ordered solid phases is not the one with the lowest free-energy barriers.

The second aspect is the interaction between the phases. Examples from the nucleation of two types of ordered solid phases: three-dimensional crystals and the polymers of sickle cell hemoglobin, which have one-dimensional translational symmetry, show that nucleation proceeds via a disordered liquid-like intermediate. In crystal nucleation, the structuring of the intermediate is the rate determining step in the nucleation process, while in the nucleation of the HbS polymers, the formation of the intermediate determines the overall nucleation rate.

Phys. Rev. Lett. 84, 1339 (2000); Nature 406, 494 (2000); Proc. Natl. Acad. Sci. USA 97, 6277 (2000); Solid State Physics 57, 1 (2002); Proc. Natl. Acad. Sci. USA 100, 792 (2003); Methods in Enzymology 368, 84 (2003); Biophys. J. 85, 3935 (2003); J. Am. Chem. Soc. 125, 11684 (2003); J. Mol. Biol. 336, 43–59 (2004); Crystal Growth and Design 5, in print (2004).

 
11:15-12:15 Pearl, L (Institute of Cancer Research)
  Achieving specificity in regulated protein-protein interactions Sem 1
 

The crowded environment of a cell presents an individual protein with a considerable evolutionary challenge if it is to achieve specific 'signal' interactions with authentic macromolecular partners and minimise non-specific 'noise'. Simply maximising the interaction energy of the specific complexes improves the signal-to-noise ratio, but at the price of reversibility. Proteins involved in regulated rather than constitutive complexes, must achieve specificity by more subtle mechanisms, which will be illustrated by examples of macromolecular complexes determined in our laboratory.

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