Novel Approaches in Protein Engineering
23-25 April 2010
Istanbul, Turkey

Organisers:

Draft Report

Summary

Protein engineering is the process of developing value added proteins that can be used in industrial, medical and environmental applications. It is a young discipline, with much research taking place into the understanding of protein structure and function relationship.

There are two general strategies for protein engineering, rational design and directed evolution. These techniques may complement each other and often researchers will apply both. More detailed knowledge of protein structure and function, as well as advancements in high-throughput technologies, may greatly expand the capabilities of protein engineering. Protein Engineering is a highly interdisciplinary field covering areas from molecular biology to computer science. It is important to bring together scientists from these fields to promote collaborations resulting in new methods and technologies that can advance the field.

The Protein Engineering Workshop was held in Istanbul, Turkey, 23-25 April, 2010 at Sabanci University Karakoy Kampus. The main objective of the meeting was to discuss new experimental methods, emerging technologies, new algorithms, software tools and their applications in protein engineering. In particular the workshop focused on:

1. computational approaches that are used in finding structural and sequence motifs in proteins,
2. computational approaches that are used in determining impact of mutations on function and stability,
3. molecular dynamics studies,
4. molecular mechanics studies,
5. understanding the structure function relationship,
6. protein engineering related databases,
7. experimental approaches in rational design,
8. experimental approaches in directed evolution,
9. emerging technologies.

Fourteen researchers from all over Europe were invited as speakers to the workshop covering all the priority topics mentioned above. Some of these researchers worked on directed evolution and experimental techniques and others were more involved in development of computational methods for the protein engineering and rational design. They presented their latest works in the field followed by question and answer sessions. Three speakers other than the ones that gave a presentation during the workshop could not attend the workshop due to medical problems and travel gridlock due to Volcanic Ash over Euro fly zone. We also lost several European participants due to uncertainity of the flights leaving from most of Europe

Over 80 researchers (mainly from Turkey) from different backgrounds attended the workshop. 14 researchers presented their works as short talks. There were two poster sessions where 12 researchers presented their poster and discussed their works with other students and the invited speakers.

One of the goals of the workshop was to discuss state of the art methods and techniques in protein engineering. The talks given covered wide range of applications ranging from drug design to industrial enzyme development which promoted collaborations amongst the scientists working in these fields. We also had very positive feedbacks from the students. They could present their works to other students and experienced researchers in the field and also got feedback from the experts on use of computational and experimental methods in their research. Overall the workshop successfully achieved its goals.

There was a section on computational methods consisted of two parts. The first part covered bioinformatics tools developed to determine functional motifs and other related information that can be used in protein engineering. The second part of the computational methods involved use of Molecular Dynamics and Molecular Mechanics methods in protein engineering applications.

There were also series of talks focusing on experimental approaches used in rational design as well as directed evolution. These talks focused on difficulties of such tasks and the benefits arising from the outcome of such experiments.

Scientific Content

The first speaker was Rita Casadio (Bologna University) who talked about Support Vector Machines based Predictor of Protein stability Changes upon Single Point Mutation from the Protein Sequence and Structure (three states) and of human Deleterious Single Nucleotide Polymorphysms after giving a brief introduction to the factors that may have an impact on protein stability.

The first talk by Rainer Merkl (University of Regensburg) was on identification of evolutionary important residues by means of an entropy based analysis of multiple sequence alignments. They espacially look at correlated mutations and use these mutations in engineering proteins for a specific function.

The first talk given by Reinhard Sterner (University of Regensburg) was on the evolution of the prototypical (beta alpha)(8)-barrel protein imidazole glycerol phosphate synthase (HisF) which was recently studied by complementary computational and experimental approaches. The 4-fold symmetry of HisF suggested that its constituting (beta alpha)(2) quarter-barrels have a common evolutionary origin. They recombinantly produced HisF-N1 protein which was properly folded and formed a tetramer being stabilized by disulfide bonds. The introduction of a disulfide bond in HisF-C1 [corresponding to (beta alpha)(5-6)] also resulted in the formation of a stable tetramer. The fusion of two identical HisF-N1 quarter-barrels yielded the stable dimeric half-barrel HisF-N1N1. Based on their findings they deduced the evolution of beta alpha(8)-barrel protein.

Giorgio Colombo (CNR Milan) talked about molecular modeling and molecular dynamic simulations and how they can be used to understand receptor ligand interactions. He also described how these simulations can reveal insights about the mechanism of proteins functionality by giving several examples on proteins. He talked about identification of hot spots and functional sites as well.

Canan Atilgan (Sabanci University) talked about a new tool called perturbation-response scanning which relies on the systematic use of computational perturbation/response techniques based on linear response theory, by sequentially applying directed forces on single-residues along the chain and recording the resulting relative changes in the residue coordinates. She also talked about application of this tool to explain ferric transport dilemma of of the bacterial ferric binding protein: it uptakes iron from the host with remarkable affinity, yet releases it with ease in the cytoplasm for subsequent use.

Ali Rana Atilgan (Sabanci University) started by giving brief overview of the concepts and the philosophy behind then he talked on hierarchy of timescales in protein dynamics is linked to enzyme catalysis. He continued by describing how coase grained methods can give us information on time scales of coupled protein dynamics and enzyme catalysis.

These talks were followed by six short talks given by graduate students which was followed by the first poster session.

The second day started by a talk by Francisco Valero (Autonomous University of Barcelona) who approached the protein engineering from a different perspective. What are the aspects one should consider in production of these engineered proteins. He presented Pichia pastoris as a suitable cell factory to produce heterologous proteins. He also talked about means of optimizing the production and the parameters involved in this process.

During the coffee break we also had the second poster sessions where the attendees went over the posters presented by the designated researchers.

After the coffee break Xavier Daura of Autonomous University of Barcelona gave a talk on assessing the structural conservation of protein pockets to study functional and allosteric sites. He talked about the use of Molecular dynamics in drug discovery based on the information gained on conserved sites of ligand binding regions.

Piotr Zielenkiewicz, of Polish Academy of Sciences talked on Protein-protein interaction inhibitors - blocking the 'unhealthy' interactions in Cystic Fibrosis. He started his talk by describing protein interaction networks and their network properties. He described in detail how they found key interactions from their analysis. Then he gave a detailed drug design procedure aimed at inhibiting this key protein-protein interaction. This talk inspired many researchers since it went through all the processes involved in drug development.

Afternoon session started with a second talk by Reinhard Sterner was on activation of a thermostable enzyme by directed evolution. This talk went over the exerimental methods used in directed evolution as well as the highthroughput selection mechanism of the enzyme with the desired property. He discussed the impact of several mutations generated by directed evolution the the function and the stability of the enzyme.

Rainer Merkl's second talk was on computational evidence for the evolution of a (ß / a) 8-barrel protein from an ancestral quarter-barrel stabilised by disulfide bonds following Prof. Sterners first talk. He talked on computational methods they developed to determine the conservation of disulfide bonds in this family.

Cenk Selçuki of Ege University gave a talk on Molecular Modeling of Metal Binding Properties in Proteins. He discussed potential uses of metal binding proteins and gave a review of molecular modelling methods and how they are used in finding the motifs used by proteins in binging different types of metals. He specially focused on MXCXXC Motif of Atx1 in Saccharomyces Cerevisiae that they have determined using the methods they developed in their group.

Pier Luigi Martelli of University of Bologna gave a talk on prediction of membrane protein topology and localization. He discussed the state of the art in this topic then gave a detailed description of the Machine Learning approaches they deveoped in their group. He also talked about the predictors and the servers they established in their group regarding membrane bound proteins.

This talk was followed by a coffee break and the third poster session was held during the break.

Mehmet Ali Kilinc (Akdeniz University) talked about a Naturally Engineered Protein for Biotechnological Applications. His talk was focussed on an Iron Storage Protein. He talked about how they identified the functional sites of this protein especially resiudes involved in iron binding process. He discussed the experimental approaches they followed to verify their predicitions. He also gave examples of potential biotechnological uses of this protein.

This talk was followed by 8 short talks given by graduate students describing their work on protein engineering.

The last day started day by a talk by Ugur Sezerman (Sabanci University) on Automatic Domain Identification in Proteins. He talked on how they represented protein structures as graphs and the algorithm they developed in identifying common domains in proteins using graph matchingwhich is based on network properties of the graphs. They could also find out functional sites of the domains using their algorithm. He gave several examples on potential uses of their algorithm in protein engineering.

The last talk of the workshop was given by Turkan Haliloglu of Bogazici University on Prediction of Functionally Important Residues in proteins and protein protein interaction as well. She talked about Gaussian Network Models they had developed and how these ideas can be used to identify functional residues. She also talked about use of Molecular Dynamics in functional site identification.

The meeting ended with a short round table discussion about the computational and experimentals needs of the field. We also discussed on how to find means of collaborations between the interested partners and COST calls were pointed out as the best solution for such collaborations.

We also had a number of young researchers who were invited to present their cutting edge algorithmic work on protein engineering they were mostly focused on molecular dynamics studies but few were on novel algorithm developments for functional residue identification. There were several experimental application talks on protein engineering by young researchers. Some of the work presented employed both experimental and computational work.

Assessment of the results & impact of the event on the future direction of the field

The main objective of the meeting were to discuss new algorithms, software tools and their applications as well as new experimental methods developed for directed evolution and rational design approaches. In particular we aimed to bring in some of the world's leading scientists who have made significant recent progress in (i) computationally identifying the functional residues in protein-protein inetraction (ii) predicting the impact of mutations to the function and the stability of the protein, (iii) understanding the exact functionality of structural and sequence motifs, (iv) applications of Molecular Dynamics and Molecular Mechanics approaches to Protein Engineering (v) experimental methods involved in directed evolution especially high throughput selection mechanisms. Once the functionality and the structure-function relatipnship of proteins are well understood, one can hope to develop (vi) novel proteins for specific purposes especially in industrial processes. In principle, these approaches can be used for designing molecules that could be employed as drugs for the treatment of a variety of human diseases such as several types of cancer, rheumatoid arthritis, brain diseases and viral infections.

Overall the meeting inspired several interesting discussions carried out during question sessions, coffee breaks and lunch breaks. PROTIST'10 brought together 82 prominent scientists (71 from Turkey and 11 from Europe) from 4 different countries (Italy,Spain,Germany and Poland) and promoted to set up a forum for discussing some of the existing and future challenges related to protein engineering and emerging technologies and related computational tools that can help to address these challenges. Unfortunately we lost several participants to travel gridlock due to volcanic ash over Euro fly zone which limited the international impact we aimed to achieve in this workshop. Still we had many particpants from Turkey and had several fruitful discussions prompting collaborations between the participants.

Programme

Friday 23 April 2010

08.30 – 08.50 Registration

Session 1
08.50 – 09.00 Opening Remarks
09.00 – 10.00 Rita Casadio, University of Bologna
The effect of mutations on protein stability
10.00 – 10.30 Tea Break
10.30 – 11.30 Rainer Merkl, University of Regensburg
Protein Design Directed by Correlated Mutations
11.30 – 12.30 Reinhard Sterner, University of Regensburg
Evolution and design of (ß / a) 8-barrel proteins
12.30 – 14.00 Lunch Break

Session 2
14.00 – 15.00 Giorgio Colombo, CNR
Investigating protein function and interactions through molecular simulations
15.00 – 16.00 Canan Atilgan, Sabanci University
Perturbation-Response Scanning Techniques Reveal Stability, Function and Allosteric Control in Proteins
16.00 – 16.30 Tea Break
16.30 – 17.30 Ali Rana Atilgan, Sabanci University
Coarse-grained models for coupled protein dynamics and enzyme catalysis

Short Talk Presentations
17.30 – 17.40 Devrim Gümral, Yeditepe University
Helix Propensity and ß2-Microglobulin Fibrillogenesis
17.40 – 17.50 Emel Durmaz, Sabanci University
High Throughput Protein Expression in Pichia pastoris using Non-repressing Carbon Source
17.50 – 18.00 Güzin Tunca, Universitat Autonoma de Barcelona
Structure-Based Drug Desig for Identification of Pharmacological Chaperones for Glucocerebrosidase  
18.00 – 18.10 Markus Gall, Institut für Biochemie, Universität Greifswald
The Role of the GGG(A)X-motif on Enantioselectivity of Pig Liver Esterase towards Tertiary Alcohols
18.10 – 18.20 Aydin Albayrak, Sabanci University
Clustering of Protein Families into Functional Subtypes Using Relative Complexity Measure
18.20 – 18.30 Cem Meydan, Sabanci University
Prediction of Heterologous Protein Expression in Pichia pastoris

18.30 – 19.30 Poster Session-I

Saturday 24 April 2010

Session 3
09.00 – 10.00 Francisco Valero, Autonomous University of Barcelona
Pichia pastoris, an exciting cell factory to produce heterologous protein
10.00 – 10.30 Tea Break+ Poster Session II
10.30 – 11.30 Xavier Daura, Autonomous University of Barcelona
Assessing the structural conservation of protein pockets to study functional and allosteric sites: implications for drug discovery
11.30 – 12.30 Piotr Zielenkiewicz, Polish Academy of Sciences
Protein-protein interaction inhibitors - blocking the 'unhealthy' interactions in Cystic Fibrosis
12.30 – 14.00 Lunch

Session 4
14.00 – 15.00 Cenk Selçuki, Ege University
Molecular Modeling of Metal Binding Properties in Proteins
15.00 – 16.00 Pier Luigi Martelli, University of Bologna
Prediction of membrane protein topology and localization
16.00 – 16.30 Tea Break + Poster Session III
16.30 – 17.00 Mehmet Ali Kilinç, Akdeniz University
A Naturally Engineered Protein for Biotechnological Applications: A Story of an Iron Storage Protein

Short Talk Presentations
17.00 – 17.10 Günseli Bayram, Sabanci University
Impact of Loop Mutations on Endoglucanase1 of T. reesei cloned in P. pastoris
17.10 – 17.20 Gülgez Gökçe Yildiz, Abant Izzet Baysal University
Characterization of three conserved amino acid mutations in cbb3-type oxidase enzyme of Rhodobacter capsulatus
17.20 – 17.30 Mehmet Öztürk, Abant Izzet Baysal University
Characterization of three conserved aminoacid mutations in cbb3-type oxidase enzyme of Rhodobacter capsulatus
17.30 – 17.40 Seyhan TÜRKKAN, Marmara University
The Effect of Active Site Tyrosine Orientation on Binding of New Pyrazoline Derivatives to MAO-B: Molecular Docking and Dynamic Simulations
17.40 – 17.50 Vildan Enisoglu Atalay, Marmara University
Effect of Tyr407 and Tyr444 Mutations on Substrate Nucleophilicity in Monoamine Oxidase A
17.50 – 18.00 Fatma Uzbas, Sabanci University
Trichoderma reesei as an Expression System
18.00 – 18.10 Yasin Bakis, Abant Izzet Baysal University
A New Approach in Prediction of Functional Classification of Proteins
18.10 – 18.20 Mithat Çelebi, Yalova University
Kinetic study on enhanced dye decolorization efficiency by horseradish peroxidase

Sunday 25 April 2010

Session 5
09.00 – 09.30 Reinhard Sterner, University of Regensburg
Activation of a thermostable enzyme by directed evolution
09.30 – 10.10 Rainer Merkl, University of Regensburg
Computational evidence for the evolution of a (ß / a) 8-barrel protein from an ancestral quarter-barrel stabilised by disulfide bonds
10.15 – 10.45 Tea Break
10.45 – 11.30 Turkan Haliloglu, Bogazici University
Prediction of Functionally Important Residues through Dynamics
11.30 – 12.30 Ugur Sezerman, Sabanci University
Automatic Domain Identification in Proteins
12.30 – 13.00 Round Table Discussion and Closing Remarks