1. Folding@home and SWISS-MODEL
Two Different Approaches to Protein Structure Modeling
Taru Tukiainen ja Sini Sipponen S

2. Outline Introduction to protein modelling
Modelling the folding process with
Comparative modelling with SWISS-MODEL

3. Introduction Proteins are formed from a sequence of amino acids
Primary structure = polypeptide chain
Secondary structure
alpha helix
beta sheet
Tertiary structure is 3D
Quaternary structure is comprised of several tertiary structures
Native state is the functional form
General recombination, also known as homologous recombination, takes place between a pair of homologous DNA sequences (meaning DNA sequences that share a large degree of identity with one one another, similarity), Usually this is two copies of the same chromosome.
General recombination is needed especially in meiosis. Genetic variation is crucial to allow organisms to evolve in response to changing enviroment. General recombination is a simple method for generating genetic diversity in a population.
GR is also used in gene manipulation, f.ex. in the generation of gene knockout mice.
The general recombination mechanism is also essential for every proliferating cell, because accidents occur during nearly every round of DNA replication that interrupt the replication fork and require general recombination mechanisms to repair. (as was discussed in the previous presentation)
In this presentation we will mostly concentrate on general recombination and its mechanism in meiosis.

4. Hydrophobic effect on Folding
Important force affecting the forming of the tertiary structure
Many residues of amino acids are hydrophobic
leads to the formation of a hydrophobic core
After folding, entropy of protein decreases, but the entropy of system decreases
Entropy promotes the folding process
The GR in meiosis has the following characteristics:
Two homologous DNA molecules that were originally part of different chromosomes ”cross over”. In this process their double helices break and the two broken ends join to their opposite partners to re-form two intact double helices, each composed of parts of the two initial helices
The site of exchange, the place where the double helices cross over, can occur anywhere in the homologous nucleotide sequences of the two participating DNA molecules
A heteroduoplex join that links the two double helices is formed when one DNA molecule becomes base paired with another DNA molecule at the site of exchange (picture). This heteroduplex joint can be thousands of base pairs long.
The re-joining occurs so precisely that no nucleotide sequences are altered at the site of exchange, though some DNA replication can take place

5. Misfolding Prions are result of faulty folding
Little known about how they form
Can convert normal protein molecules to prions
General recombination is a complex process and requires many proteins. One of these proteins is RecA, which has a central role in the recombination between chromosomes.
RecA has two DNA-binding sites, one for single stranded DNA and one for a double helix. This allows it to hold a single strand and a double helix together and this way catalyze a multistep DNA synapsis reaction between a DNA double helix and a homologous region of single-stranded DNA.
This picture shows how the RecA protein catalyzes the reaction.
First a non-base-paired complex is formed. As soon as a region of homologous sequence is found, this non-base-paired complex is converted to a threestranded structure through transient base-flipping. This complex is unstable because it involves an unusual form of DNA, and as a result it spins out a DNA heteroduplex (one strand green and the other strand red) plus a displaced single strand from the original helix (green). Thus the structure shown in this diagram (the proteins) migrates to the left, reeling in the “input DNAs” while producing the “output DNAs.” The net result is a DNA strand exchange identical to that diagrammed earlier in the previous slide.

6. The Problems
Impossible to predict 3D structures from polypeptide chains
Folding processes and mechanisms are mostly unknown
the 3D, native state is very expensive and time consuming to solve

7. Modeling the Folding Process
Proteins fold in about 10 µs
Simulation would take dozens of years
Proteins are formed of thousands of atoms
Presented often as force fields
E.g. temperature, pH, covalent bonds between residues and hydrophobic effect must be taken in consideration
Proposed that the native state = minimum of potential energy curve

8.
Objective to study the dynamics of protein folding and misfolding and the ensuing diseases
Uses distributed computing, volunteers let their PC to be utilized when they aren’t needed
Program is a screen saver
Uses packages
AMBER, TINKER and GROMAS

9. Ensemble Dynamics Method
Polypeptide chain is considered as a system waiting for enough free energy to overcome the free energy barrier (= the folding)
Group of several molecules M is simulated at the same time
simulation rate is then M times faster than a single simulation
The simulations are completed in hours not in years
Wait until the first one of the simulations overcomes the energy barrier
All the simulations are restarted from the new energy level

10. Comparative modelling
aims at building a 3D model for a protein with unknown structure
relies on detectable similarities between the protein sequence being modelled (the target) and at least one empirically determined protein structure (the template)
a small change in the protein sequence usually results only in a small change in its 3D structure

11. SWISS-MODEL fully automated web-server for protein structure modelling
developed in 1993
nowadays the most widely-used free web-based automated facility

12. Using SWISS-MODEL User-friendly
User only submits the amino acid sequnce on a web form
optionally templates can be submitted as well
Results in min by

14. How SWISS-MODEL works? Five steps that can be repated iteratively
1 Search for suitable templates
2 Check sequence identity with target
3 Create ProModII jobs
4 Generate models with ProModII
5 Energy minimisation with Gromos96
First Approach Mode (regular)
First Approach Mode (with user-defined templates)
Optimise Mode

15. How SWISS-MODEL works? Step 1 Step 2
search for suitable templates from ExNRL3D
program used: BLASTP2
Step 2
find sequences with good degree of similarity (>25%)
aling target and template sequences
program used: SIM

16. How SWISS-MODEL works? Step 3 Step 4 Step 5
create ProModII input files
Step 4
generate models
program used: ProModII
Step 5
minimize energy
program used: Gromos96

17. Are there problems with SWISS-MODEL?
Results must be concidered with care
procedure is non-experimental
no human intervention during model building
Chosen template affects the results
the more the template and the target sequence share identity the more accurate the results will be

18. Accuracy of SWISS-MODEL