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Centre of the University and School of Milan for Bioscience Educa3on
www.cusmibio.unimi.it
INVISIBLE FORMS: PROTEINS IN 3-­‐D 16-­‐19 April 2011 Copenhagen Denmark
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INVISIBLE FORMS: PROTEINS IN 3-­‐D Biological macromolecules measure only a few nanometres and cannot be observed even with the strongest light microscope. One of the major technologies available to make protein structures ‘visible’ is X-­‐ray crystallography.
The 3D coordinates of each single atom are collected in a file and can be viewed using dedicated soFware.
GeHng regular crystals is the first step of 3D protein structure determina3on. X-­‐ray irradia3on of crystals allows magnifica3on at atomic resolu3on and determina3on of the 3D structure of the molecule. The spa3al structure of a protein is very important for its func3on. These structures are collected in specialized databases and can be viewed using dedicated soFware to compare structures of normal and mutated proteins, design new vaccines and therapeu3c molecules.
This workshop provides an interdisciplinary exercise for biology, chemistry and bioinforma3cs. Main techniques for 3D structure determinaNon of proteins
Accurate 3D macromolecular structures are obtained by two main techniques: Nuclear MagneNc Resonance (NMR) and X-­‐ray crystallography (83%). When a protein with unknown structure shows a good level of sequence iden3ty with a protein with known structure, a rough structure prevision can also be obtained by compara3ve modelling. This kind of analysis, although less accurate, can also provide important insights on the structure/func3on of a protein, but at present templates are available for only 20% of protein domains. Whatever method is used to determine the structure of a macromolecule, the 3D coordinates of each single atom are collected in a file, deposited in the PDB (Protein Data Bank) database and can be viewed using dedicated soFware. Determinazione
3D structuredella
struttura in 3D
Interpreta3on of electron density
Crystal characteriza3on
Crystal diffrac3on spectrum
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Nuclear MagneNc Resonance is a type of spectrometry in which proteins are hit with radio waves while they're in a strong magne3c field. Since atomic nuclei selec3vely absorb electromagne3c radia3ons, the peaks you see represent individual atoms in the protein -­‐ actually the interac3ons between specific atoms. The posi3ons of the peaks and their sizes are used to determine the distances between the atoms, and to put together a model of the molecule that fits those constraints using complex soFware. One of the major limits of NMR is that only structures of rela3vely small proteins (no more than 200 aminoacids) can be resolved.
X-­‐ray crystallography is a method of determining the arrangement of atoms within a crystal in which a beam of X-­‐rays strikes a crystal and diffracts into many specific direc3ons. From the angles and intensi3es of these diffracted beams, a crystallographer can produce a three-­‐dimensional picture of the density of electrons within the crystal. From this electron density, the mean posi3ons of the atoms in the crystal can be determined, as well as their chemical bonds and various other informa3on.
To perform X-­‐ray crystallography, it is necessary to grow crystals with edges around 0.1-­‐0.3 mm. Finding the op3mal condi3ons of crystal growth is not an easy task. Nevertheless crystals of complex and large macromolecules have been obtained. In 1954 Max Perutz's group developed X-­‐ray cristallography methods to solve the molecular structure of globular proteins and define the structure of haemoglobin. In 1953 Watson and Crick published their model of the DNA double helix based on crystallography data provided by Rosalind Franklin. More recently, the 2009 Nobel Prize for chemistry was assigned for the defini3on of the 3D structure of a complex organelle such a ribosome. Obtaining the crystal of the first macromolecular complex took almost 30 years! Which are the main applications of biocrystallography?
study protein structure and functions relationship
study protein-­‐ligand interactions
drug design
Fig. 1 The vapour diffusion method is the most frequently used technique to grow protein crystals. A. A small amount of a crystallisa3on solu3on is put into the chamber reservoir. A drop of protein solu3on and a drop of crystallisa3on solu3on are placed onto the siHng drop post. B. The chamber is sealed to start the crystallisa3on process.
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Among the various methods of growing protein crystals, the most common is Vapour Diffusion (Hanging Drop Method).
A drop of protein solu3on is suspended over a reservoir containing buffer and crystalliza3on solu3on. Water diffuses from the drop to the solu3on leaving the drop with op3mal crystal growth condi3ons (Fig. 1). The protein we will crystallize in this lab ac3vity is chicken lysozyme (129 aminoacids, from hen egg white).
Lysozyme is a potent bactericide agent. It is a small enzyme that aiacks the protec3ve cell walls of bacteria. Bacteria build a tough skin of carbohydrate chains, interlocked by short pep3de strands, that braces their delicate membrane against the cell's high osmo3c pressure. Lysozyme breaks these carbohydrate chains, destroying the structural integrity of the cell wall. The bacteria burst under their own internal pressure. Substrates of lysozyme i n c l u d e p e p 3 d o g l y c a n ( F i g . 2 ) , t h e polysaccharide component of the cell walls of certain bacteria; lysozyme hydrolyzes the ß(1-­‐4) glycosidic bond between residues of N-­‐acetylmuramic acid (NAM) and N-­‐
acetylglucosamine (NAG), the two monomer sugars of pep3doglycan chains. Lysozyme is one of the natural immunity weapons against infec3ons. It is abundant in all vertebrate secre3ons to resist infec3on of body exposed surfaces; in humans lysozyme is present in tears and mucus. Unfortunately, lysozyme is a large molecule that is not par3cularly Fig. 2 Peptidoglycan structure
useful as a drug.
PROTOCOL
Equipment, materials and chemicals
•crystallisa3on plates •sealing tape (5 cm) •1 ml and 1 μl micropipeie
•A microscope to observe the crystals
•Storage space at 20 °C
•Hen egg white lysozyme (a single chain of 129 aa residues) 40 mg/ml in H2O
•crystalliza3on solu3on NaCl 3.4 M (20% w/vol)
•Na-­‐acetate buffer pH 4 1M
•bidis3lled H2O
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A small amount of a crystallisa3on solu3on is added to the reservoir of the crystallisa3on chamber. A drop of protein solu3on and a drop of crystallisa3on solu3on (1:1 vol) are pipeied onto the siHng drop post, located in the centre of the chamber.
well 1
well 2
NaCl (200 μl)
NaCl (225 μl)
Final crystallisaNon condiNons:
Buffer pH 4.0 (25 μl)
Buffer pH 4.0 (25 μl)
H2O (275 μl) H2O (250 μl)
well 1: NaCl 8%, Acetate Buffer pH4, 50 mM
well 2: NaCl 9%, Acetate Buffer pH4, 50 mM
In details:
Pipeie 0.5 ml of crystallisa3on solu3on (prepared by mixing precipita3on solu3on NaCl 20% + Na-­‐Acetate Buffer 1M + H2O) in 2 wells of the crystallisa3on plate (according to the volumes of each solu3on given in the table above).
Pipeie 2 μl of iysozyme solu3on ( 40mg/ml) into the crystallisa3on cup on the siHng drop post in each well. Pipeie 2 μl of the reservoir solu3on into the crystallisa3on cup on the siHng drop post in each well. Mix gently. The final volume of the drop is 4 μl.
Immediately seal the chamber to avoid evapora3on and to guarantee the correct vapour diffusion equilibrium in the chamber. Leave the plates at 21 °C. WARNING: crystal forma3on requires absolute s3llness
Crystal growth can be monitored directly under the microscope aFer 1-­‐2 hours. The plates may be stored un3l the next day for final analysis. AFer about 1-­‐2 weeks, crystals will have grown to their final size. (A sealed plate will keep up to a year, some3mes even longer).
By comparing the results from the 2 chambers, determine the op3mal condi3ons for crystallisa3on.
Since the concentra3on of salt ions is higher in the crystallisa3on solu3on than in the mixture on the siHng drop post, solvent molecules will move from the protein drop to the reservoir by vapour diffusion in the gas phase. During this process, the solubility of the protein in the drop decreases. The protein solu3on in the drop eventually becomes supersaturated, which is a thermodynamically unstable state. This causes some of the protein in the drop either to form crystal nuclei that finally grow into larger protein crystals, or to precipitate as amorphous protein which is useless for X-­‐ray analysis. Crystallisa3on and precipita3on are compe3ng processes, so it is extremely important to find the op3mal condi3ons favouring crystallisa3on.
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FROM CRYSTALS TO 3D STRUCTURES
The steps necessary to analyze the crystals and reconstruct the 3D structure of a protein are depicted in Fig 3 and 4. Fig.3. X-­‐ray diffrac3on. The x-­‐rays are diffracted in a predictable paiern based on the regular crystal laHce structure formed by the protein. The diffracted X-­‐rays p ro d u c e a p a i e r n o f s p o t s o n a photographic plate.
Fig.4 From the angles and intensi3es of the diffracted beams, crystallographers produce a three-­‐dimensional picture of the electron density within the crystal and reconstruct the polypeptide aminoacid chain.
All files containing molecular coordinates of the molecules whose 3D structure has been determined are collected (in different formats) in the PDB (“Protein Database) hip://www.rcsb.org/pdb/home/home.do and can be viewed with dedicated soFwares. We will use YASARA soFware.
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In this scenario, the molecular analysis is cri3cal to the iden3fica3on of toxic substances which may have caused harm to a person’s health.
A young woman is admiied to the emergency room of a local hospital with severe symptoms of anaphylac3c reac3on. She was enjoying a dinner at an italian restaurant with a friend when the first symptoms occurred. She refers to be allergic to egg proteins and soya and therefore to have chosen food which should not contain any of the substances she knows to be allergic to.
Her friend, a lawyer, wants to suit the restaurant for food fraud.
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SCENARIO
You will be given 3 protein par3al sequences: your task is to find to which protein >Protein 1
CCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR
HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEK
LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS
>Protein 2
DQAMEDIKQM EAESISSSEE IVPNSVEQKH IQKEDVPSER YLGYLEQLLR LKKYKVPQLE
IVPNSAEERL HSMKEGIHAQ QKEPMIGVNQ ELAYFYPELF RQFYQLDAYP SGAWYYVPLG
TQYTDAPSFS DIPNPIGSEN SEKTTMPLW
>Protein 3
KVFGRCELAAAMKRHGLDNYRGYSLGNWVCAAKFESNFNTQATNRNTDGSTDYGILQINSRWWCN
DGRTPGSRNLCNIPCSALLSS
they belong to and which of them can be responsible for the severe anaphylac3c reac3on in the woman. !
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BLAST (Basic Local Alignment Search Tool) is a collection of searching programs for biological sequence databases. The program uses the BLAST algorithm to compare protein or DNA sequence queries to protein or DNA sequence databases. The Basic Local Alignment Search Tool (BLAST) Dinds regions of local similarity between sequences. The program compares nucleotide or protein sequences to sequence databases and calculates the statistical signiDicance of matches. BLAST can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families.
To find the protein to which each par3al sequence belongs to open hip://
mrs.cmbi.ru.nl/mrs-­‐web/, the MRS homepage, a search engine for biological
and medical databanks. Click the link Blast, on the top page grey bar.
Paste the aa sequence of protein 1 in the white box. Don’t forget the > symbol in front of the protein name and sequence, to conform to the correct FASTA format. Check that the op3on Filter sequence (low complexity) is selected. ClicK Run Blast, top right in the page.
The new page shows a table giving the number of hits, i.e. similar to the query sequence and the degree of similarity of each alignment; Blast has iden3fied 48 proteins whose sequence has a certain degree of similarity to your query. Click the first row in the table to obtain the complete hit list. The first hit has the highest BitScore and the lowest E-­‐value, i.e. the match has the highest sta3s3cal significance. The first hit corresponds to albu_bovin, i.e. to bovine serum albumin. Look carefully at the hit list. For each protein different codes and values are reported:
•-­‐ the ID column with the iden3fica3on code of the protein sequence in the database (the protein name followed by the name of the organism);
-­‐ the Coverage
column with the color code degree of correspondence between •
the query sequence and the homologous sequence in the database; a red line means that the alignment of the query and albu_bovin is complete; blue and light blue lines mean a par3al correspondence;
-­‐ the Descrip3on column with a short descrip3on of the hit protein;
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•-­‐ the Hsps, Bitscore and E-­‐value columns with the values giving the sta3s3cal significance of each match.
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Click the code albu_bovin. In the new page you will find many data about the protein, its func3on and its effects on human health. 8
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The page is ar3culated in different sec3ons. Consult the sec3ons Entry informa3on, Name and origin, Comments, Features key and Sequence informa3on to answer the following ques3ons about the protein albu_bovin.
Return to the hit list page. Click the red line corrresponding to albu_bovin. You will find that the 150 aminoacids of your query completely correspond (100% Iden3ty) to 150 residues of albu_bovin; click the red line again to find a sec3on where the alignment of protein1 and albu_bovin is shown.
The sequence of protein 1 is iden3fied by the leier Q (query) and the sequence of albu_bovin is iden3fied by the leier S (sequence). Between these two sequences is the sequence of the shared aminoacids. Numbers on the leF and right correspond to aminoacid posi3ons. Note that the 150 residues of the query sequence correspond to residues 301-­‐ 450 of bovin albumin. Following the same procedure, iden3fy protein 2 and protein 3, find the requested info and answer the 6 ques3ons in the box. Draw your conclusions about the case of the allergic woman.
Among the 3 proteins, only lysozyme, abundant in hen egg white, can be responsible for the allergic reac3on shown by the woman. 1. Protein name
2. Species of origin
3. Func3on
4. Lenght in aminoacids
5. Is this protein present in the food ingested by the woman?
6. Could this protein be responsible for the allergic reac3on suffered by the woman? Explain your answer
Which entrée in the Menu chosen by the woman may contain egg white lysozyme? Egg white lysozyme is largely used in cheese produc3on for its ly3c effect on bacteria cell wall. Lysozyme is efficient in countering “late blowing” during the ripening of cheese, such as parmesan, provolone and the like. “Late blowing” is caused by the outgrowth of clostridial spores (origina3ng mainly from the use of silage in the feeding of dairy caile) present in raw milk.
While the use of lysozyme as cheese preserva3ve is allowed in the produc3on of many cheese types, its use (and that of ensilaged forages) is forbidden in the 9
disciplinary of produc3on of PDO (Protected DesignaNon of Origin) Parmigiano-­‐
Reggiano. Lysozyme (not exceeding 300 mg/kg) can instead be added to Grana Padano, a cheese similar to Parmigiano Reggiano and oFen used as a Reggiano surrogate, but whose organolec3c quali3es (and price) are far below those of Reggiano. Strong suspect exists that the Carpaccio served to the woman, differently from what declared on the Restaurant Menu, was accompanied by slivers of Grana Padano (and not of the more expensive Parmigiano Reggiano).
The woman and her lawyer have good chance of winning the case for fraud brought to the restaurant.
WORKING WITH YASARA
Yasara is a graphics program used to visualise and manipulate protein models. It was developed by Elmar Krieger, mainly at the CMB. If Yasara is not installed on your computer yet, go to the the soFware sec3on hip://www.bioinforma3csatschool.eu/soFware.html to see how to do that.
AFer the prac3cum, you will have a chance to look at several other interes3ng proteins. You can also use the program to make nice pictures for reports and science projects. Exercise 1:
Start Yasar. In Yasara, you can load a polypep3de using the menu on the top leF of the window: File > Load > Complete scene. Choose the file introduc3on.sce and click OK. You can now see the structure of the pep3de with sequence Asp-­‐His-­‐Arg-­‐Gly-­‐Gly-­‐Met-­‐Lys-­‐Tyr in the so-­‐
called ball and s3ck representa3on. Individual atoms are shown as balls, connected by s3cks represen3ng the atomic bonds. Note: the hydrogen atoms are not represented because that would make the model much more complicated. Below, you can see a 2D representa3on of the same pep3de. You will no3ce a "cloud" around the pep3de. This represents the Van der Waals surface. This surface shows how much space the atoms actually occupy: there is barely any empty space between adjacent atoms. 10
If you move your mouse cursor along the boiom of the Yasara screen, the sequence bar appears. To keep this bar visible, click on the blue pushpin at the leF end of the bar. When you click on a residue in the sequence bar, the Cα-­‐atom of this residue will flash. If you push Ctrl while you click on the residue, the protein will rotate and zoom so that you can clearly see the Cα-­‐atom. You can manipulate the protein by holding the mouse buions and moving your mouse. Try the following: •LeF: Rotate
•Middle: Translate
•Right: Zoom
The atoms are coloured by atom type. If you click on an atom, extra informa3on about this atom will appear on the leF side of the window. Exercise 2:
Look up which types of atoms (elements) can be found in proteins, using this list of amino acids. Look at different atoms in Yasara and find out which elements are coloured red, dark blue, green, and light blue.
Exercise 3:
In the representa3on that you see, covalent bonds are shown as short s3cks of a specific colour. Explain the difference between the yellow and white bonds using the list of amino acid structures. The 3D structure shows three of the four different types of interac3ons that are important for protein folding. These different interac3ons are represented by green, blue and orange s3cks. Exercise 4:
Which interac3on belongs to each colour? Locate these interac3ons in the 2D representa3on of the pep3de. Exercise 5:
Reset Yasara by clicking File > New and then Yes. Now, load the file 1JYV.pdb (you can download lysozyme’s structure in .pdb format, at the site: hip://www.rcsb.org/
pdb/results/results.do?ou•ormat=&qrid=932BF699&tabtoshow=Current ) via File > Load > PDB file .
You now see lysozyme in Ball representa3on: you can see all atoms (except the hydrogens) as large balls. This is not a very clear representa3on. Fortunately, a number of alterna3ve representa3ons exist. You can browse through them by pushing the F1 through F8 keys on your keyboard:
F1: Ball representa3on (individual atoms)
F2: Ball-­‐and-­‐s3ck representa3on (individual atoms + bonds)
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F3: S3ck representa3on (the atoms look like s3cks) F4: Cα-­‐trace (only the Cα are represented, connected with s3cks)
F5: Backbone-­‐trace (showing only the backbone, no side chains)
F6: Cartoon representa3on (secondary structure-­‐elements)
F7: Alterna3ve cartoon representa3on
F8: Add/Remove the side chains of the residues (the "R" groups) in any other representa3on
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Exercise 6:
Using F5, F6 and F7 you can easily recognise secondary structure elements. Which secondary structures match the red and blue parts or the protein? Exercise 7: Find the following five structure-­‐elements: a double bonded oxygen atom, a pep3de bond, a his3dine side chain, an alpha helix and a disulfide bond. Use the appropriate representa3on of the protein for each element and write those down.
The acNve site
We will now look for the ac3ve site of the protein. That is where the most important amino acids are located and where the actual chemical reac3on takes place. !
A characteris3c feature of the ac3ve site of enzymes is that they (almost) always lye in a cavity or cleF on the surface of the protein. Therefore, a quick-­‐and-­‐dirty way to find the ac3ve site is to look for the largest cavity on the surface of the protein. 12
Exercise 8:
Why is the ac3ve site usually located in a cavity? Exercise 9:
Find the ac3ve site by rota3ng the protein and zooming. Choose the representa3on that you think is most appropriate. The Cus-­‐Mi-­‐Bio staff, composed of both University Professors and High School teachers, are the scien3fic editors and authors of the contents of this Handbook.
Workshop Leaders
Cinzia Grazioli and Cris3na GriH
High school teacher fully working at CusMiBio, via Celoria 20 Milan, Italy
ScienNfic supervisor
Giovanna Viale
Professor of Biology and GeneDcs, Dept. of Biology and GeneDcs for Medical Sciences, University of Milan, via VioJ 3/5, Milan, Italy
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