Presentation on theme: "Section B Protein Structure"— Presentation transcript:
1Section B Protein Structure Molecular Biology CourseSection BProtein Structure
2B2 Protein structure and function: Cross with BiochemistryMolecular Biology CourseB1 Amino acids: structure, side chains (charged, polar uncharged, nonpolar aliphatic, aromatic)B2 Protein structure and function:Structure: size and shapes, primary secondary tertiary quaternary (prosthetic groups) ; Biological functionsStructure & function: Domains, motif, and familyB3 Protein analysisPurification Determine sequence, mass, andstructure (X-ray crystallography and NMR)
3B1 Amino acids- basic structure Protein structureB1 Amino acids- basic structureH3NCOO-RHCaCommon structure of 19 AAsprolinea-carbon is chiral (asymmetric) except in glycine (R is H)2. Amino acids are dipolar ions (zwitterions [兼性离子]) in aqueous solution and are amphoteric (兼性的同时有酸碱性或正负电荷的)3. The side chains (R) differ in size, shape, charge and chemical reactivity4. A few proteins contain nonstandard amino acids that are formed by post-translational modification of proline and lysine.
4B1 Amino acids- charged (5) Protein structureB1 Amino acids- charged (5)Form salt bridges, are hydrophilic (亲水)1. “Acidic” amino acids (2): containing additional carboxyl groups which are usually ionizedaspartic acid (Asp, D，天冬氨酸)glutamic acid (Glu, E，谷氨酸)
5Lysine (Lys, K, 赖氨酸) Arginine (arg, R,精氨酸) Histidine (His, H，组氨酸) Protein structure2. “Basic” amino acids (3): containing positively charged groupseLysine (Lys, K, 赖氨酸)Arginine (arg, R,精氨酸)da guanidino group (胍基）Histidine (His, H，组氨酸)The imidazole group (咪唑基) has a pKanear neutrality. This group can be reversiblyprotonated under physiological conditions,which contribute to the catalytic mechanism of many enzymes.
6Contain groups that form hydrogen bonds with water, Protein structureB1 Amino acids- polar uncharged (5)Contain groups that form hydrogen bonds with water,hydrophilicSerine (Ser, S，丝氨酸)Contain hydroxyl groups.Threonine (Thr, T，苏氨酸)Asparagine (Asn, N，天冬酰氨)Glutamine (Gln, Q，谷氨酰氨)
7Protein structureCysteine (Cys, C，半胱氨酸) has a thiol (巯醇) group， which is often oxidizes to cystinex-S-S-x
8B1 Amino acids- nonpolar aliphatic (7) Protein structureB1 Amino acids- nonpolar aliphatic (7)(hydrophobic 疏水)Glycine (Gly, G，甘氨酸)Proline (Pro, P，脯氨酸): imino acid (亚氨基酸)Methionine (Met, M，甲硫氨酸): contains a sulfur atom
9Isoleucine (Ile, I，异亮氨酸) Protein structureAlkyl (烷基) side chainsAlanine (Ala, A，丙氨酸)Leucine (Leu, L，亮氨酸)Valine (Val, V，缬氨酸)Isoleucine (Ile, I，异亮氨酸)
10B1 Amino acids- aromatic (3) Protein structureB1 Amino acids- aromatic (3)Accounts for most of UV absorbance of proteins at 280 nm hydrophobic （疏水的)Phenylalanine (Phe, F，苯丙氨酸)Tyrosine (Tyr, Y，酪氨酸)Tryptophan (Trp, W，色氨酸)
11B2 Protein structure and function Molecular Biology CourseB2 Protein structure and functionStructure: size and shapes, primarysecondary tertiary quaternary, prosthetic groups (辅基，nonprotein molecules of conjugated proteins (结合蛋白)Domains, motif, and familyProtein function
12B2 Protein structure －Sizes A few thousands Daltons (x 103) to more than 5 million Daltons (x 106)Some proteins contain bound nonprotein materials (prosthetic groups or other macromolecules), which accounts for the increased sizes and functionalities of the protein complexs.
13Complementary fit of a substrate molecule to the catalytic site Protein structureB2 Protein structure －ShapesGlobular proteins: enzymesComplementary fit of a substrate molecule to the catalytic siteon an enzyme molecule.
14Protein structureFibrous proteins: important structural proteins (silk fibroin, keratin in hair and wools )Protofibril (初原纤维)microfibril (微管)Keratin (角蛋白)keratin in hair
15Formation of a peptide bond (shaded in gray) in a dipeptide. Protein structureB2 Protein structure －PrimaryPolypeptides contain N- and C- termini and are directional, usually ranging from aaFormation of a peptide bond (shaded in gray) in a dipeptide.
16Protein structureN terminusC terminusStructure of the pentapeptide Ser-Gly-Tyr-Ala-Leu.
17B2 Protein structure －Secondary a-helixright-handed3.6 aa per turnhydrogen bondN-H···O=CA stereo, space-filling representationCollagen triple helix:three polypeptide intertwined
18Protein structureb-sheet: hydrogen bonding of the pepetide bond N-H and C=O groups to the complementary groups of another section of the polypeptide chainParallel b sheet: sections run in the same directionAntiparallel b sheet: sections run in the opposite directionA stereo, space-filling representation of the six-stranded antiparallel b sheet.
19B2 Protein structure －Tertiary The different sections of a-helix, b-sheet, other minor secondary structure and connecting loops of a polypeptide fold in three dimensions
20Covalent interaction: disulfide bonds Protein structureNoncovalent interaction between side chains that hold the tertiary structure together: van der Waals forces, hydrogen bonds, electrostatic salt bridges, hydrophobic interactionsCovalent interaction: disulfide bondsDenaturation of protein by disruption of its 2o and 3o structure by heat and extremes of pH will lead to a random coil conformation
21B2 Protein structure －Quaternary Many proteins are composed of two or more polypeptide chains (subunits). These subunits may be identical or different. The same forces which stabilize tertiary structure hold these subunits together. This level of organization called quaternary structure.The quaternary structure of hemoglobin。 a1-yellow; b1-light blue; a2-green; b2-dark blue; heme-redback
22Advantages of the quaternary structure: Protein structureAdvantages of the quaternary structure:It allows very large protein molecules to be made, such as tubulin。It can provide greater functionality to a protein by combining different activities into a single entity.The interactions between the subunits can often be modified by binding of small molecules and lead to the allosteric effects seen in enzyme regulation
23B2 Protein structure － Prosthetic groups Covalently or noncovalently attached to many conjugated proteins, and give the proteins chemical functionality. Many are co-factors in enzyme reactions.Examples : heme groups in hemogobin (Figure)
24B2 Protein structure － Domains, motifs and families Domains: structurally independent units (tertiary) of a protein, being connected by sections with limited higher order structure within the same polypeptide. (Figure)They can also have specific function such as substrate binding
25Protein structureStructural motifs:Groupings of secondary structural elements that frequently occur in globular proteinsOften have functional significance and represent the essential parts of binding or catalytic sites conserved among a protein familybab motif
26The primary structures of c-type cytochromes from different organisms Protein structureProtein families: structurally and functionally related proteins from different sourcesMotifThe primary structures of c-type cytochromes from different organisms
27The tertiary structures of the above c-type cytochromes Protein structureDomainThe tertiary structures of the above c-type cytochromesCys, Met and His side chains covalently linked to the heme to the proteinBack
28Protein structureB2 Protein functionsEnzymes: substrate binding, side chain in catalysisSignaling: cell membrneTransport and storage: hemoglobin transports oxygenStructure and movement: collagen, keratin, tubulin in cytoskeleton, actin and myosin for muscle contractionNutrition: casein (酪蛋白) and ovalbumin(卵清蛋白)Immunity: antibodiesRegulation: transcription factors
29Protein structureB3 Protein analysis1. Purification: to obtain enough pure sample for study2. Sequencing: determine the primary structure of a pure protein sample3. Mass determination: determine the molecular weight (MW) of an interested protein.4. X-ray crystallography and NMR: determine the tertiary structure of a given sample.
30Protein structureProtein purificationTo purify the interested protein from other proteins and nonprotein molecules existing in the cellsAn essential experimental step prior to study of any individual protein
31The principal properties of proteins used for purification Protein structureThe principal properties of proteins used for purificationSize: gel filtration chromatography2. Charge: ion-exchange chromatography, isoelectric focusing, electrophoresis3. Hydrophobicity: hydrophobic interaction chromatography4. Affinity: affinity chromatography5. Recombinant techniques: involving DNA manipulation and making protein purification so easy
32Gel filtration chromatography Protein structureGel filtration chromatography
33Protein structureBead: the matrix determine the size of the pore
34Protein structureUltracentrifugation: can be used to separate proteins according to their size and shape that determine their sedimentation rate. Very large protein complex.
35Protein structure2. Charge: ion-exchange chromatography, isoelectric focusing, electrophoresisIsoelectric point (pI): the pH at which the net surface charge of a protein is zero-+--++-+-+--++-+pH < pIpH=pIpH > pI
36Charge 1: Ion-exchange chromatography Protein structureCharge 1: Ion-exchange chromatographySample mixture+++ProteinbindingIondisplacingColumn + anionsColumn + anionsColumn + proteinsPurified protein
37Charge 2: Electrophoresis Protein structureCharge 2: ElectrophoresisProtein migrate at different position depending on their net charge+
38Charge 3: Isoelectric focusing Protein structureCharge 3: Isoelectric focusingA protein will stop moving at position corresponding to its isoelectric point (pI) in a pH gradient gel.
39Protein structure3. Hydrophobicity:hydrophobic interaction chromatographySimilar to ion-exchange chromatography except that column material contains aromatic or aliphatic alkyl groups
404. Affinity chromatography Protein structure4. Affinity chromatographyEnzyme-substrate bindingReceptor-ligand bindingAntibody-antigen binding
415. Recombinant techniques: Protein structure5. Recombinant techniques:Clone the interested protein encoding gene in an expression vector with a purification tag added at the 5’- or 3’ end of the geneProtein overexpression in a cellProtein purification with affinity chromatography.
42Mass Determination Gel filtration chromatography and SDS-PAGE Protein structureMass DeterminationGel filtration chromatography and SDS-PAGEComparing of the unknown protein with a proper standardPopular SDS-PAGE: cheap and easy with a 5-10% errorSDS: sodium dodecyl sulfate, makes the proteins negatively charged and the overall charge of a protein is dependent on its mass.
43Mass spectrometry:Molecules are vaporized and ionized, and the degree of deflection (mass-dependent) of the ions in an electromagnetic field is measuredextremely accurate, but expensiveMALDI can measure the mass of proteins smaller than 100 KDaHelpful to detect post-translational modificationProtein sequencing: relying on the protein data base
44Protein sequencing : Determine the primary structure of a protein Protein structureProtein sequencing : Determine the primary structure of a proteinSpecific enzyme/chemical cleavage:Trypsin cleaves after lusine(K) or arginine (R)V8 proteease cleaves after glutamic acid (E)Cyanogen bromide cleaves after methionine (M)Edaman degradation:Performed in an automated protein sequencerDetermine the sequence of a polypeptide from N-terminal amino acid one by one.Expensive and laborious
45Most protein sequences are deduced from the DNA/cDNA sequence Protein structureMost protein sequences are deduced from the DNA/cDNA sequenceDirect sequencing: determine the N-terminal sequences or some limited internal sequence construction of an oligonulceotide or antibody probe fishing the gene or cDNA
46X-ray crystallography and NMR Protein structureX-ray crystallography and NMRDeterming the tertiary structure (3-D) of a proteinX-ray crystallography:Measuring the pattern of diffraction of a beam of X-rays as it pass through a crystal. The first hand data obtained is electron density map, the crystal structure is then deduced.A very powerful tool in understanding protein tertiary structureMany proteins have been crystallized and analyzed
47NMRMeasuring the relaxation of protons after they have been excited by radio frequencies in a strong magnetic fieldMeasure protein structure in liquid but not in crystalProtein measured can not be larger than 30 KDa
48Protein structure Protein Folding: back chaperones are involved in vivoThe rapidly reversible formation of local secondary structureFormation of domains through the cooperative aggregation of folding nulceiAssembly of domains into a “molten” globuleConformational adjustment of the monomerFinal conformational adjustment of the dimeric protein to form the native structure.back