ododododododo dodododododod ododododododo dodododododod o □ o □ o ^ ~ o □ o □ o Kód předmětu: BÍ8980 1 MASARYKOVA UNIVERZITA □ o d^MNA ° D ODODOu^uODODO dodododododod onODODODOnono Protein expression and purification I. The molecular principles for understanding proteins Lubomír Janda, Blanka Pekarové and Radka Dopitová Tento projekt je spolufinancován Evropským sociálním fondem a státním rozpočtem České republiky. ministerstvo školství, mládeže a tělovýchovy EVROPSKÁ UNIE « NVESTICE DO ROZVOJE VZDĚLÁVÁNÍ OP Vzdělávání pro konkurenceschopnost Název prezentace v zápatí 1 Dteins-Lubomír Janda This course aims to develop students' confidence in their ability to understand the way proteins function and the basis of the methods used to distinguish, identify, and characterize them. •This lecture is intended to provide a concise summary of the principles with which you should be familiar in order to understand the structures and functions of proteins. Literature: Exploring proteins - Nicholas C. Price and Jacqueline Nairn Basic Methods in Protein Purification and Analysis -Richard J. Simpson, Peter D. Adams and Erica A. Golemis High Throughput Protein Expression and Purification - Sharon A. Doyle Cloning, Gene Expression and Protein Purification - Charles Hardin, Jennifer Edwards etal. Protein Purification, Principles and Practise -Robert K. Scopes -standing proteins - Lubomír Janda 1.2. The amino acids H The constituents of proteins, the amino acids H NH2 — C-C02H R H +NH3- -c-R -CO; General structure of an amino acid The zwitterionic form of an amino acid 1.2. The amino acids Dteins- Lubomír Janda 1.2.1. The variety of amino acids 1.2.2. Clasification of the amino acids in terms of polarity Non-polor side chain Ala, Gly, Ile, Leu, Met, Phe, Pro, Trp, Val Polar, uncharged side chain Asn, Cys, Gln, Ser, Thr, Tyr Polar charged side chain Arg, Asp, Glu, His, Lys lUC-NPOLAR. HYDROPHOBIC PCLAR. UNCHARGED Alanine Ma A MW = *9 > H M R GR OUPS H - ^ COO" NH-+ * Gl* C MW»7S Vbln-B VbI V mw- 17 " OOC > H M I lO-CI 2 - CM N HL t 3 Leuon* Llu L MW= J31 " OOC > H M -^H,-Clf OH^C Tlirecnlnt Tir T MUV= 119 I v.n ri i li r II? 1 MVJ = 31 "OOC H M xCH2-GHj Hi - CH2 ■CHSNH, Í~:jpi1í>in-p c MW= 121 Plienylalsniie Ptie F MW-31 HJM -CH, O ,coo~ -»CH«, Tyrosine TTT Y Mw- iai Trp W MW - Í04 OOC u > ** H NH 'CH^NH, AjiMnaojnt N Methionine Met M MW= 49 " OOC -CHE-CHE-S-CHa NH^ ^C-CH,-CH, -COO" Gl Uta man? Ulm Q P*din* p MW ■ 15 NHa-CHj-(CH POLAR BASIC ^ coo" r J K MW- m partis aod D MW ■ - 33 POLAí ACIDIÍ " OOC >H H_N 0 Ml I NH2^ Auntie R MW- 174 GklH rrin ě acid Gh E MW= I47 > HNVNH + "H-NH. t 3 Hi&tidne Hf, H MW= 155 http://www.cem.msu.edu/~reusch/VirtualText/sterism3.htm Dteins-Lubomír Janda Clockwise Counterclockwise (R) (S) 1.2. The amino acids 1.2.3. General properties of the amino acids 1.2.3.1. Stereochemistry The Sequence Rule for Assignment of Configurations to Chiral Centers. Assign sequence priorities to the four substituents by looking at the atoms attached directly to the chiral center. 1. The higher the atomic number of the immediate substituent atom, the higher the priority. For example, H- < C- < N- < O- < Cl-. 2. If two substituents have the same immediate substituent atom, evaluate atoms progressively further away from the chiral center until a difference is found. For example, CH3- < C2H5- < ClCH2- < BrCH2- < CH3O-. 3. If double or triple bonded groups are encountered as substituents, they are treated as an equivalent set of single-bonded atoms. For example, C2H5- < CH2=CH- < HC=C- http://www.cem.msu.edu/~reusch/VirtualText/sterism3.htm Dteins-Lubomír Janda 1.2. The amino acids 1.2.3. General properties of the amino acids Amino Acid 1.2.3.2. Ionization 12 pH Alanine Titration Curve / CHj j / H^N-CH-COO" j *" " — H3N-CH-COO": pK\ / i _______ HsN-ch-cqoh; i i 0.5 10 1.5 2,0 OH" equivalents Symbol pKa (COOH) pK2 (NH2) pK R Group Glycine Gly 2,4 9,8 Alanine Ala 2,4 9,9 Valine Val 2,2 9,7 Leucine Leu 2,3 9,7 Isoleucine lie 2,3 9,8 Serine Ser 2,2 9,2 Threonine Thr 2,1 9,1 Cysteine Cys 1,9 10,8 8,3 Methionine Met 2,1 9,3 Aspartic Acid Asp 2 9,9 3,9 Glutamic Acid Glu 2,1 9,5 4,1 Asparagine Asn 2,1 8,8 Glutamine Gln 2,2 9,1 Arginine 1,8 9 12,5 Lysine Lys 2,2 9,2 10,8 Histidine His 1,8 9,2 6 Phenylalanine Phe 2,2 9,2 Tyrosine Tyr 2,2 9,1 10,1 Tryptophan Trp 2,4 9,4 Proline Pro 2 10,6 Dteins-Lubomír Janda 1.2. The amino acids 1.2.3. General properties of the amino acids 1.2.3.3. Hydrophobicity (Engelman et al 1986) Amino acid Transfer free energy kJ/mol Phe F 15,5 Met M 14,2 lie 1 13 Leu L 11,7 v Cys C 8,4 Trp W 7,9 Ala A 6,7 Thr T 5 Gly G 4,2 Ser S 2,5 Pro P -0,8 Tyr Y -2,9 His H -12,5 Gln Q -17,1 Asn N -20,1 Glu E -34,3 Lys K -36,8 Asp D -38,5 Arg R -51,4 Kyte and Rase, et al Wollenden Janin Boolittle , etal (1979) (1) m (3) KJ He Cys GlyjLeuJIe Cys Vél Valwig lie PheJIe Val Leu Val Phe leu,Phe Leu, Met, Trp Met Phe Met A^GlyjTrp Oys MetjAla Hb Th^Ser TVr Trpjyr Hi&Ser Gly Ala Thr Thi-jSer Gly Prp Trpjyr Thr Tyr Pro Asn AspjLys,Gln Asp Hb Ser GlUjHis Gl^Glu AsrijGln Prc^Arg Asp AspjGlu Asn GlMspjGlu Arg Arg Arg Lvs Dteins-Lubomír Janda 1.2. The amino acids 1.2.4. Chemical characteristic of the amino acids 1.2.4.1. Aliphatic side chains Ala, Gly, lie, Leu Vol 1.2.4.2. Aromatic side chains Phe, Tyr, Trp 1.2.4.3. Basic side chains Arg, Lys 1.2.4.4. Acidic side chains Asp, Glu 1.2.4.5. Hydroxy! side chains Ser, Thr 1.2.4.7. Sulphur-containing side chains Met, Cys 1.2.4.6. Amide side chains Asn, Gln 1.2.4.8. Proline Pro 1.2.4.9. Amid side chains Histidine 1.2. The amino acids 1.2.4. Chemical characteristic of the amino acids Dteins- Lubomír Janda Amino Acid Symbol One-letter Mass pKaR Group Frequency of occurence wo H20 % Alanine Ala A 71,08 Arginine Arg R 15649 12,5 5,35 Asparagine Asn N 114,1 4,18 Aspartic Acid Asp D 115,09 3,9 5,32 Cysteine Cys c 103,14 8,3 Glutamic Acid Glu E 128,13 4,1 3,95 Glutamine Gin Q 129,12 6,64 Glycine Gly G 57,05 6,93 Histidine His H 137,14 6 2,29 Isoleucine lie l 113,16 5,91 Leucine Leu L 113,16 9,64 Lysine Lys K 128,17 10,8 5,93 Methionine Met M 131,2 2,38 Phenylalanine Phe F 147,18 4 Proline Pro P 97,12 4,83 Serine Ser S 87,08 6,86 Threonine Thr T 101,11 5,42 Tryptophan Trp W 186,21 1,15 Tyrosine Tyr Y 163,18 10,1 3,06 Valine Val V 99,13 6,71 )teins-Lubomír Janda Resonance stabilization of the peptide bond mding proteins - Lubomír Janda The cis and trans forms of the peptide bond o >7 \ An C-N (b) 0 H \ / C-N ^c7 / ^ (d) C - terminus NH,—CH—CO—NH—CH—CO—NH—CH—CO—NH—CH—CO,H Side chains 1.3.2. Information available from the amino acid sequence of a protein 1.3.2.2. Isoelectric point Dteins- Lubomír Janda COO" NH, cocr Dteins-Lubomír Janda 1.3. The primary structure of proteins 1.3.2. Information available from the amino acid sequence of a protein 1.3.2.3. Absorption coefficient 1.3.2.4. Hydrofobicity Type of modification N-terminal acylation 1.3.2.5. Post-translational modifications Possible effect on function Removal of targeting sequences. Generation of several new products (hormones). Activation of proteins (enzymes). Stabilization of structure of secreted proteins. Formation of hydroxy -Lys or -Pro increases the stability of the triple helix of collagen. Many cell surface proteins are involved in cell - cell recognition. Attachment of glycosyl-phosphatidyinositol groups anchors proteins to membrane. The polar nature of proteins can be enhanced. Phoshorylation of Ser, Thr, Tyr, His, Asp side chains can regulate the activity of proteins, especially in signalling pathways. Attachment of C14 (myristoylation) or C16 (palmitoylation) chains will enhance the association of the protein with membranes. 1.3. The primary structure of proteins Dteins- Lubomír Janda 1.3.2. Information available from the amino acid sequence of a protein 1.3.2.6. Structural and functional motifs >AHK4/CRE1 Receptor histidine kinase MNWALNNHQEEEEEPRRIEISDSESLENLKSSDFYQLGGGGALNSSEKPRKID FWRSGLMGFAKJMQQQQQLQHSVAVKMNNNNNNDLMGNKKGSTFIQEHRALLPK ALILWIIIVGFISSGIYQWMDDANKIRREEVLVSMCDQRARMLQDQFSVSVNH VHALAILVSTFHYHKNPSAIDQETFAEYTARTAFERPLLSGVAYAEKWNFER EMFERQHNWVIKTMDRGEPSPVRDEYAPVIFSQDSVSYLESLDMMSGEEDREN ILRARETGKAVLTSPFRLLETHHLGWLTFPVYKSSLPENPTVEERIAATAGY LGGAFDVESLVENLLGQLAGNQAIVVHVYDITNASDPLVMYGNQDEEADRSLS HESKLDFGDPFRKHKMICRYHQKAPIPLNVLTTVPLFFAIGFLVGYILYGAAM HIVKVEDDFHEMQEL KVRAEAADVAK •Transmembrane domains •Targeting sequences -S-K-L peroxisomes K-D-E-L endoplasmic reticulum •Metal binding -C-X4-C-X2-C- Fe binding •Glycosylation sites -N-X-S/T- •Phosphorylation sites -R-X1-2-S/T- Protein kinase A -R-R-X-S/T- •Nitrosylation -(G,S,T,C,Y,N,Q)-(K,R,H,D,E)-C-(D,E) www.expasy.org/prosite 1.3.2. Information available from the amino acid sequence of a protein Dteins- Lubomír Janda different species, same function (beta-glucosidase, Zea mays and Brassica napus) same species, different function histidine kinase - ETR1 (ethylene signalling pathway) histidine kinase - AHK4 (cytokinin signalling pathway) independent folded and functional unit AHK4 - extracelular CHASE domain histidine kinase domain receiver domain 1. The molecular principles for understanding proteins - Lubomír Jar Please solve a problem. Question 1: 1 am an amino acid. My name in Greek means „sweet". 5 points 1 belong to the group of amino acids with non-polar side chains. 3 points 1 am frequently found in secondary structures called loops. 2 points 1 am the smallest amino acid. 1 point Glycin 1. The molecular principles for understanding proteins - Lubomír Janda Please solve a problem. Question 3: 1 am an amino acid. My name in Greek means "silk". 5 points 1 belong to the group of polar, uncharged side chain amino acids. 1 am not an aromatic amino acid. 1 contain a hydroxyl group and my MW is less than 100 Da. 3 points I am very often phosphorylated 2 points I am related to threonine. 1 points Serine Dteins-Lubomír Janda ■standing proteins - Lubomír Janda )teins-Lubomír Janda Beta Sheet Structure HydrophiEic side edge view Ribbon Representation top view Dteins-Lubomír Janda Met, Glu, Leu,Ala + Pro, Gly, Tyr - Structural preferences of the different amino acids alpha - helices alpha helices beta sheets beta sheets beta turn beta turn + + Dteins-Lubomír Janda 1.5. The tertiary structure of proteins 1.5.1. General principles Close packing Elements of secondary structure Distribution of side chains Pairing of polar group Formation of domains Average Conformational Parameters of Helical Elements Conformation Alpha helix 3-10 helix Pi-helix Polyproline I Poly proline II Polyproline III Phi Psi Omega Residues per turn Translation per residw -57 -47 180 Ü -49 -26 180 3.0 2.0 57 -70 1&0 4.4 1.15 -83 +158 0 3.33 1.9 -78 +149 180 3.0 3.12 -80 +150 180 3.0 31 1.5. The tertiary structure of proteins 1.5.2. Classification of protein structures 1.5.3. Forces involved in stabilizing tertiary structure SCOP Classification Statistics SCOP: Structural Classification of Proteins. 1.75 release 38221 PDB Entries (23 Feb 2009). 110800 Domains. 1 Literature Reference (excluding nucleic acids and theoretical models) Number of Number of Class Number of folds superfamilies families All alpha proteins 284 507 871 All beta proteins 174 354 742 Alpha and beta proteins (a/b) 147 244 803 Alpha and beta proteins (a+b) 376 552 1055 Multi-domain proteins 66 66 89 Membrane and cell surface 58 110 123 proteins Small proteins 90 129 219 Total 1195 1962 3902 (b) 1.6. The quaternary structure of proteins >teins- Lubomír Janda )teins-Lubomír Janda 1.7. Forces contributing to the structures and interactions of proteins hydrogen hydrogen bond bond acceptor donor hydrogen bond acceptor Dteins-Lubomír Janda 1.7. Forces contributing to the structures and interactions of proteins 1.7.3. Van der Waals' interactions Many molecules have such dipole moments due to non-uniform distributions of positive and negative charges on the various atoms. Such is the case with polar compounds like hydroxide (OH-), where electron density is shared unequally between atoms. 1.7.4. Hydrophobic interactions H ■4 v--5~,H / 4 Dteins-Lubomír Janda 1.7. Forces contributing to the structures and interactions of proteins 1.7.5. Balance of energy contributions Balance between an unfavourable enthalpy term but a favourable enthropy. In proteins, the folded state is in the range 20-60 kJ/mol. Entropy and enthalpy are in the range of several hundred kJ/mol. GnHCI and urea weaken hydrophobic interaction and promote the unfolding of proteins. http://fikus.omska.cz/~bojkovsm/termodynamika/vdws.html 1.7.6. The range of energies involved in protein interactions -AG°=RTlnK eq Interaction Avidin-biotin Protein-protein <—i Antibody-antigen Receptor-hormon-1 Enzyme-substrate Typical dissociation constant (Kd) (M) 10-15 10-10 10-9 10-7 10-5 AG° (kJ/mol) 89 59 53 42 30 I. The molecular pri Lubomír Janda Please solve a problem. Question 1: I am a secondary structure. The average length of the structure is 11-12 amino acids. 5 points 3 points In general I need 3.6 amino acids to be in the same position 2 points 1 point I. The molecular principles for understa Please solve a problem. Question 2: I am a weak forces. I need to have two atoms - one donor and one acceptor. This interaction is found between particular side chain and main chain atoms (N-H or C=0 groups of the peptide bond). I very often interact with water. This interaction is also found between water molecules, where five atoms are arranged in a tetrahedral structure. Hydrogen bond 1. The molecular principles for understanding proteins - Lubomír Janda Please solve a problem. Question 3: 1 am a 3D structure protein. According to structural classification of proteins, 1 belong to the smallest family with a homogenous secondary structure. 5 points „Greek key" structure represents a typical structure. 3 points My structure most frequently contains Val, Ile and Phe. 2 points The words "sheet" and "strand" are used with this Greek character. 1 point Beta proteins