《分子生物图谱》PPT课件.ppt

1、Obligatory course for biology students Prerequisite: General biology, Biochemistry,Molecular Biology,Prokaryotic Cell (does not have nucleus) Archaebacteria and eubacteria *Archea: Methnogens (anaerobic) Extreme halophiles (dead sea) Extreme thermophiles (hot spring, geysers) Eukaryotic Cell (contai

2、n a nucleus) Protista, fungi, animals and plants,Cells:,Cytoplasm: proteins, ribosome, metabolites and ions Plasma membrane: phospholipid bilyer, associated proteins and carbohydrates DNAs, mtDNA,Basic Cellular Components,Prokaryotic cells,Bacteria: Gram-negative- Cell wall (3 layers: Periplasmic sp

3、ace; peptidoglycan; outer membrane) Gram-positive-Cell wall (thicker peptidoglycan layer) *Quite sensitive to lysozyme and penicillin Capsule and slime: The hydrophilic gel surrounding the cell wall in most bacteria Flagella: Long, rigid protein roads, facilitating the movement of motile bacteria Fi

4、mbriae and pili: Short hair-like structure and attach other cells (essential infecting other organisms) Spores: A small, often unicelluar, reproductive unit of plants, algae, fungi, protoza, and bacteria,Organization of Eukaryotic Cells,Endoplasmic reticulum,Biomembrane,Nucleus,Nuclear envelope Nucl

5、eolus Nucleoplasm,Chromosome and karyotype Long arm: q (for queue) Short arm: p (for petit) Gimsa (Quinacrine) Dark band: G band or G + band (AT rich) Light band: R band or G band (GC rich),Organelles in the Cytoplasm,(The membrane-bound structure in a cell) Mitochondria Chloroplasts Endoplasmic ret

6、iculum Golgi apparatus Peroxisomes Lysosomes Vacuoles Glyoxisomes,Mitochondria,Size: 1.5-2.0 in length, 0.5-1.0 um in diameter Approx the same as E. coli Maternal inheritance Many copies: occupying of the cytoplasmic volume Role: produce ATP Encode: proteins and RNAs,mtDNA,Chloroplasts,Thylakoloids

7、类囊体 Chlorophylls: located on the Thylakoloid membrane to absorb light for photosynthesis,Light 2H2O-O2 + 4H + +4e- H+ +ADP 3- + Pi 2- -ATP 4- + H2O,Endoplasmic reticulum,Rough ER: process newly synthesized peptides from ribosome Smooth ER: involved in the synthesis and metabolism of lipids,Golgi app

8、aratus,Major site for sorting and modifications of proteins and lipids rough ER-transport vesicles-Golgi-Glycoproteins,Peroxisomes,Peroxisome: contain enzymes for degrading amino acids and fatty aids Catalase 2H202-2H20 +02,Lysosomes,Lysosomes : 1) nuclease for degrading DNA and RNA 2) Protease for

9、degrading proteins and other enzymes for degrading polysacchrides and lipids 3) lysosomes exist only in animal cells (plant -vacuoles for degrading macromolecules),Glyoxisomes 乙醛酸循环体,Found mainly in plant seeds Fatty acids-Acetyl CoA Microbodies: Peroxisomes +Glyoxisomes,Viruses,Baltimore Classifica

10、tion Structure Life Cycles,The life cycle of Viruses,1 ) Attachment: viral surface protein-host cell receptor e.g: HIV GP120 - CD4 glutamatic acids Basic: lysine, arginine ligand-gated ion channel Others: porins 3 - Membrane attached proteins Ras, v-Src, others: myristate, farnesyl, glycosylphosphat

11、idylinositol,Ras: One of the large family of GPT-binding proteins that help relay signals from cell-surfaces receptors to the nucleus. Named for the ras gene, first identified in viruses cause rat sarcoma V-Src: Name of the first retroviral oncogene discovered (v-src) and its precursor proto-oncogen

12、e (c-src). The product of these genes is a membrane-associated protein kinase that phosphorylates many target proteins on tyrosine residues. (From sarcoma, the type of cancer that the src virus causes; pronounced “SARK”),Miscellaneous proteins,Chaperone Interleukins Interferons Tumor Necrosis Factor

13、s Chemokins,Chaperone: Proteins that helps other proteins avoid misfolding pathways that produce inactive and aggregated states,Conjugated protein: Glycoproteins, metalloproteins,Cytokines,Regulate immunity, inflammation, apoptosis, and hematopoiesis e.g: Interleukins Interferons Tumor Necrosis Fact

14、ors Chemokins,Hormones: insulin, growth hormone, and prolactin Structural proteins : collagen, myosin Transcirptional factors: zinc fingers, leucine zipper, helix-turn-helix, helix-loop-helix Ubiquitin: the marker for protein degradation. If a protein binds to ubiquitin, it will be degraded by prote

15、asome,Nucleic Acids, Genomics and Proteomics,1: Building blocks-nucleotides2: DNA structure 3: RNA structure 4: Chromatin Structure 5: The genetic code6: Genes 7: Repetitive DNA sequences8: Genomics and proteomics,Section B Protein Structure,Molecular Biology Course,B1 Amino acids- basic structure,4

16、.A few proteins contain nonstandard amino acids that are formed by post-translational modification of proline and lysine.,proline,Common structure of 19 AAs,a-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 amphot

17、eric (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 reactivity,Protein structure,B1Amino acids: structure, side chains (charged, polar uncharged, nonpolar aliphatic, aromatic) B2Protein structure and function: Structure: size and shapes, primarysecondary tertiary

18、quaternary, prosthetic groups Domains, motif, and family Function B3Protein analysis Purification Determine sequence, mass, and structure (X-ray crystallography and NMR),1.“Acidic” amino acids (2): containing additional carboxyl groups which are usually ionized,Form salt bridges, are hydrophilic (亲水

19、),aspartic acid (Asp, D，天冬氨酸),glutamic acid (Glu, E，谷氨酸),Protein structure,B1 Amino acids- charged (5),2. “Basic” amino acids (3): containing positively charged groups,Lysine (Lys, K, 赖氨酸),The imidazole group (咪唑基) has a pKa near neutrality. This group can be reversibly protonated under physiologica

20、l conditions, which contribute to the catalytic mechanism of many enzymes.,Arginine (arg, R,精氨酸),d,e,a guanidino group (胍基）,Histidine (His, H，组氨酸),Protein structure,Contain groups that form hydrogen bonds with water, hydrophilic,Contain hydroxyl groups.,Asparagine (Asn, N，天冬酰氨),B1 Amino acids- polar

21、 uncharged (5),Serine (Ser, S，丝氨酸),Threonine (Thr, T，苏氨酸),Glutamine (Gln, Q，谷氨酰氨),Protein structure,Cysteine (Cys, C，半胱氨酸) has a thiol (巯醇) group， which is often oxidizes to cystine (胱氨酸),x-S-S-x,Protein structure,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):

22、 contains a sulfur atom,Protein structure,B1 Amino acids- nonpolar aliphatic (7),(hydrophobic 疏水),Alkyl (烷基) side chains,Alanine (Ala, A，丙氨酸),Valine (Val, V，缬氨酸),Leucine (Leu, L，亮氨酸),Isoleucine (Ile, I，异亮氨酸),Protein structure,Phenylalanine (Phe, F，苯丙氨酸),Tyrosine (Tyr, Y，酪氨酸),Tryptophan (Trp, W，色氨酸),

23、Protein structure,B1 Amino acids- aromatic (3),Accounts for most of UV absorbance of proteins at 280 nm hydrophobic （疏水的),B2Protein structure and function,Molecular Biology Course,Structure: size and shapes, primarysecondary tertiary quaternary, prosthetic groups (辅基，nonprotein molecules of conjugat

24、ed proteins (结合蛋白) Domains, motif, and family Protein function,B2Protein 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 fun

25、ctionalities of the protein complex.,Protein structure,Globular proteins: enzymes,Complementary fit of a substrate molecule to the catalytic site on an enzyme molecule.,Protein structure,B2Protein structure Shapes,Fibrous proteins: important structural proteins (silk fibroin, keratin in hair and woo

26、ls ),keratin in hair,Keratin (角蛋白),Protofibril (初原纤维),microfibril (微管),Protein structure,Polypeptides contain N- and C- termini and are directional, usually ranging from 100-1500 aa,Formation of a peptide bond (shaded in gray) in a dipeptide.,Protein structure,B2Protein structure Primary,Structure o

27、f the pentapeptide Ser-Gly-Tyr-Ala-Leu.,N terminus,C terminus,Protein structure,a-helix right-handed 3.6 aa per turn hydrogen bond N-HO=C,Collagen triple helix: three polypeptide intertwined,A stereo, space-filling representation,Protein structure,B2Protein structure Secondary,b-sheet: hydrogen bond

28、ing of the pepetide bond N-H and C=O groups to the complementary groups of another section of the polypeptide chain,A stereo, space-filling representation of the six-stranded antiparallel b sheet.,Parallel b sheet: sections run in the same direction Antiparallel b sheet: sections run in the opposite

29、 direction,Protein structure,Protein structure,B2Protein structure Tertiary,The different sections of a-helix, b-sheet, other minor secondary structure and connecting loops of a polypeptide fold in three dimensions,Noncovalent interaction between side chains that hold the tertiary structure together

30、: van der Waals forces, hydrogen bonds, electrostatic salt bridges, hydrophobic interactions Covalent interaction: disulfide bonds,Denaturation of protein by disruption of its 2o and 3o structure will lead to a random coil conformation,Protein structure,Many proteins are composed of two or more poly

31、peptide 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;

32、heme-red,back,Protein structure,B2Protein structure Quaternary,Advantages 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 betwee

33、n the subunits can often be modified by binding of small molecules and lead to the allosteric effects seen in enzyme regulation.,Protein structure,Covalently or noncovalently attached to many conjugated proteins, and give the proteins chemical functionality. Many are co-factors in enzyme reactions.

34、Examples : heme groups in hemogobin (Figure),Protein structure,B2Protein structure Prosthetic groups,Protein structure,B2Protein structure Domains, motifs and families,Domains: structurally independent units (tertiary) of a protein, being connected by sections with limited higher order structure wit

35、hin the same polypeptide. (Figure) They can also have specific function such as substrate binding,Protein structure,Structural motifs: Groupings of secondary structural elements that frequently occur in globular proteins Often have functional significance and represent the essential parts of binding

36、 or catalytic sites conserved among a protein family,bab motif,Protein families: structurally and functionally related proteins from different sources,The primary structures of c-type cytochromes from different organisms,Protein structure,Motif,The tertiary structures of the above c-type cytochromes

37、,Cys, Met and His side chains covalently linked to the heme to the protein,Protein structure,Domain,Back,Enzymes: substrate binding, side chain in catalysis Signaling: cell membrne Transport and storage: hemoglobin transports oxygen Structure and movement: collagen, keratin, tubulin in cytoskeleton,

38、 actin and myosin for muscle contraction Nutrition: casein (酪蛋白) and ovalbumin(卵清蛋白) Immunity: antibodies Regulation: transcription factors,Protein structure,B2Protein functions,B3Protein analysis,1. Purification: to obtain enough pure sample for study,2. Sequencing: determine the primary structure

39、of a pure protein sample,3. 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.,Protein structure,Protein purification,To purify the interested protein from other proteins and nonprote

40、in molecules existing in the cells,An essential experimental step prior to study of any individual protein,Protein structure,The principal properties of proteins used for purification,Size: gel filtration chromatography,2. Charge: ion-exchange chromatography, isoelectric focusing, electrophoresis,3.

41、 Hydrophobicity: hydrophobic interaction chromatography,4. Affinity: affinity chromatography,5. Recombinant techniques: involving DNA manipulation and making protein purification so easy,Protein structure,Gel filtration chromatography,Protein structure,Bead: the matrix determine the size of the pore

42、,Protein structure,Ultracentrifugation: can be used to separate proteins according to their size and shape that determine their sedimentation rate. Very large protein complex.,Protein structure,2. Charge: ion-exchange chromatography, isoelectric focusing, electrophoresis,Isoelectric point (pI): the

43、pH at which the net surface charge of a protein is zero,-,-,-,-,-,-,-,-,+,+,+,+,+,+,+,+,pH=pI,pH pI,pH pI,Protein structure,Charge 1: Ion-exchange chromatography,Column + anions,+,+,+,Sample mixture,Protein binding,Column + proteins,Column + anions,Ion displacing,Purified protein,Protein structure,+

44、,Charge 2: Electrophoresis,Protein migrate at different position depending on their net charge,Protein structure,Charge 3: Isoelectric focusing,A protein will stop moving at position corresponding to its isoelectric point (pI) in a pH gradient gel.,Protein structure,3. Hydrophobicity: hydrophobic in

45、teraction chromatography,Similar to ion-exchange chromatography except that column material contains aromatic or aliphatic alkyl groups,Protein structure,4. Affinity chromatography,Enzyme-substrate binding,Receptor-ligand binding,Antibody-antigen binding,Protein structure,5. Recombinant techniques:,

46、Clone the interested protein encoding gene in an expression vector with a purification tag added at the 5- or 3 end of the gene,Protein overexpression in a cell,Protein purification with affinity chromatography.,Protein structure,Mass Determination,Gel filtration chromatography and SDS-PAGE,Comparin

47、g of the unknown protein with a proper standard,Popular SDS-PAGE: cheap and easy with a 5-10% error,SDS: sodium dodecyl sulfate, makes the proteins negatively charged and the overall charge of a protein is dependent on its mass.,Protein structure,Mass spectrometry:,Molecules are vaporized and ionize

48、d, and the degree of deflection (mass-dependent) of the ions in an electromagnetic field is measured,extremely accurate, but expensive,MALDI can measure the mass of proteins smaller than 100 KDa,Helpful to detect post-translational modification,Protein sequencing: relying on the protein data base,Protein sequencing : Determine the primary structure of a protein,Specific enzyme/chemical cleavage: Trypsin cleaves after lysine (K) or arginine (R) V8 proteease cleaves after glutamic acid (E) Cyanogen bromide cleaves after methionine (M),Edaman degradation: