大学精品课件：生物化学（英文版）Biochemistry-chapter 3 AA and primary structure of protein（part 1）.ppt

1、Chapter 3 Amino Acids and the Primary Structures of Proteins 3.1 Outline of Proteins 3.2 Structures of Amino Acids 3.3 Other Amino Acids and Amino Acid Derivatives 3.4 Ionization of Amino Acids 3.5 Chemical reactions of AAs: 3.6 Peptide Bonds Link Amino Acids in Proteins 3.7 Protein Purification Tec

2、hniques 3.8 Amino Acid Composition of Proteins 3.9 Determining the Sequence of Amino Acid residues 3.10 Comparisons of the Primary Structures of Proteins Reveal Evolutionary Relationships 1. Proteins is most important biological functional molecules: (1) Enzymes, the biochemical catalysts (photosynt

3、hesis,thermophilic bacteria,HIV-1 revers transcriptase) (2) Storage and transport of biochemical molecules (Hb, Mb) (3) Physical cell support and shape (tubulin, actin, collagen) 3.1 Outline of Proteins (4) Mechanical movement (flagella, mitosis, muscles) (5) Decoding information in the cell (transl

4、ation, regulation of gene expression) (6) Hormones or hormone receptors (growth hormone, insulin receptor) (7) Other specialized functions (antibodies, toxins etc) 2. Classification of proteins: (1) According to shape of protein molecules; Globular proteins Usually Usually water solublewater soluble

5、, compact, roughly spherical, compact, roughly spherical Hydrophobic interiorHydrophobic interior, , hydrophilic surfacehydrophilic surface g globular proteins include enzymes,lobular proteins include enzymes, carrier and carrier and regulatory proteinsregulatory proteins Fibrous proteins Provide me

6、chanical support Provide mechanical support not water solublenot water soluble Often assembled into large cables or threadsOften assembled into large cables or threads - -KeratinsKeratins: major components of hair and nails: major components of hair and nails CollagenCollagen: major component of ten

7、dons, skin, bones and : major component of tendons, skin, bones and teethteeth (2) According to polymerization of protein molecules; Monomeric proteins Oligomeric proteins (multimeric proteins) (3) According to conjugation of protein molecules; Simple proteins Conjugated proteins 3. Molecular size a

8、nd Mr of proteins Proteins are homogeneous. Mr of proteins vary from about 6000 Da to 1 106 Da or more。 Usually insulin (5700 Da) or RNase (126000 Da) was as the boundary of proteins and polypeptides. Mr of proteins Mr of amino acid 110. Primary structure - amino acid linear sequence Secondary struc

9、ture - regions of regularly repeating conformations of the peptide chain, such as a-helices and b-sheets Tertiary structure - describes the shape of the fully folded polypeptide chain Quaternary structure - arrangement of two or more polypeptide chains into multisubunit molecule 4. Conformation and

10、structure levels of proteins 3.2 Structures of Amino Acids 1. General structure of amino acids. More than 200 different AAs are found in living organisms,including the 20 common (standard) amino acids. 2. Structures of the 20 common (standard) amino acids. All of the common amino acids found in prot

11、eins are amino acids. C H H2N COO H R COO - R groupR group Amino group Carboxylic group a H = Glycine CH3 = Alanine H N 3 + H 3. Ionization of amino acids The R groups are different in 20 AAs. Zwitterionic form of amino acids Under normal cellular conditions amino acids are zwitterions (dipolar ions

12、): Amino group = -NH3+ (Protonated) Carboxyl group = -COO- (Ionized) Stereochemistry Stereoisomers - compounds that have the same molecular formula but differ in the arrangement of atoms in space Enantiomers - nonsuperimposable mirror images Chiral carbons - have four different groups attached 4. Co

13、nfiguration of amino acids. Stereochemistry of amino acids 19 of the 20 common amino acids have a chiral a-carbon atom (Gly does not) Threonine and isoleucine have 2 chiral carbons each (4 possible stereoisomers each) Mirror image pairs of amino acids are designated L (levo) and D (dextro) Proteins

14、are assembled from L-amino acids (few D-amino acids occur in nature) Mirror Images of Amino Acid a a Mirror image of Stereoisomers The 19 chiral amino acids used in the assembly of proteins are all of the L configuration, although a few D-amino acids occur in nature.Why ? Ball-and-stick model Perspe

15、ctive Fischer projections Fischer projections - horizontal bonds from a chiral center extend toward the viewer, vertical bonds extend away from the viewer (1).The three-letter and one-letter abbreviations. 5.Classification of the 20 common amino acids (2). Classification of amino acids by chemical c

16、onstruction. A.Aliphatic R groups: B.Aromatic R groups: C.Sulfur-containing R groups: D.Side chains with alcohol groups: E.Basic R groups: F.Acidic R groups and their amide derivatives: A. Aliphatic (hydrophobic) R Groups Glycine (Gly, G) - the a-carbon is not chiral since there are two Hs attached

17、(R=H) = smallest and fits easiest into small nitches Four amino acids have saturated side chains: Alanine (Ala, A) Valine (Val, V) Leucine (Leu, L, The occurrence in proteins is the highest.) Isoleucine (Ile, I) Proline (Pro, P) 3-carbon chain connects a-C and N = ring structure Four aliphatic amino

18、 acid structures Important in protein structure and folding since their R groups cluster away from water Proline has a nitrogen in the aliphatic ring system Proline (Pro, P) - has a three carbon side chain bonded to the a-amino nitrogen The heterocyclic pyrrolidine ring restricts the geometry of pol

19、ypeptides = causes abrupt changes in the direction of the polypeptide chain B. Aromatic R Groups (benzene ring properties) Side chains have aromatic groups Phenylalanine (Phe, F) - benzene ring Tyrosine (Tyr, Y) - phenol ring Tryptophan (Trp, W) - bicyclic indole group The occurrence in proteins is

20、the lowest. Aromatic amino acid structures they absorb UV light at 280 nm C. Sulfur-Containing R Groups Methionine (Met, M) - (-CH2CH2SCH3) Cysteine (Cys, C) - (-CH2SH) Methionine usually first amino acid in a protein Two cysteine side chains can be cross-linked by forming a disulfide bridge (-CH2-S

21、-S-CH2-) Disulfide bridges may stabilize the three- dimensional structures of proteins Methionine and cysteine Many extracellular proteins contain disulfide bridges that stabilize the 3D structure of proteins by joining adjacent peptide chains. D. Side Chains with Alcohol Groups Serine (Ser, S) and

22、Threonine (Thr, T) have uncharged polar side chains = hydrophilic E. Basic R Groups Hydrophilic with nitrogenous side chains Histidine (His, R) - imidazole Lysine (Lys, K) - alkylamino group Arginine (Arg, R) - guanidino group Side chains are nitrogenous bases which are substantially positively char

23、ged (proton acceptors) at pH 7 = gives a positive charge to proteins Structures of histidine, lysine and arginine F. Acidic R Groups and Amide Derivatives Confer negative charges to proteins because their side chains are ionized at neutral pH Aspartate (Asp, D) and Glutamate (Glu, E) are dicarboxyli

24、c acids, and are negatively charged at pH 7 Asparagine (Asn, N) and Glutamine (Gln, Q) are uncharged but highly polar and are amides of Asp and Glu whose R groups can form H bonds Structures of aspartate, glutamate, asparagine and glutamine Le u L -C-C-CONH2 -C-CONH2 -C-COOH -C-C-COOH -H -CH3 -C-OH

25、-C-SH -C-C-S-C P Pro -C-C C N N+ -C-C-C-C-NH3+ -C- -C- -OH -C- N Aliphatic Amide Acidic Imino, Circular Basic Sulfur Alcohol Aromatic -C-C-C-N-C-N N+ = C -C-C-C C -C-C-C C C -C C C C HN C-COOH a a -C-C OH Gln Q Asn N Asp D Glu E Phe F Arg R Lys K His H Gly G A A Ala V Val I Ile Y Tyr Ser S Thr T Met

26、 M Cys C Trp W (3). Classification of amino acids by polarity. Highly hydrophobic: Highly hydrophilic: Val Ile Leu Met Phe His Asp Glu Gln Asn Lys Arg Less hydrophobic: Tyr Gly Cys Ser Thr Ala Trp Pro The Hydrophobicity of Amino Acid Side Chains Hydropathy: the relative hydrophobicity of each amino

27、acid (5 are highly hydrophobic and 7 are highly hydrophylic) The larger the hydropathy, the greater the tendency of an amino acid to prefer a hydrophobic environment Hydropathy affects protein folding: hydrophobic side chains tend to be in the interior hydrophilic residues tend to be on the surface

28、Hydropathy scale for amino acid residues (Free-energy change for transfer of an amino acid from interior of a lipid bilayer to water) Free-energy change for transfer (kjmol-1) Amino acid The relative hydrophobicity or hydrophilicity of each amino acid is called its hydropathy. - -0.290.29 - -0.750.7

29、5 - -1.11.1 - -1.71.7 - -2.62.6 - -2.72.7 - -2.92.9 - -3.03.0 - -4.64.6 - -7.57.5 Nutritional requirement of human on amino acids: Nonessential amino acid Essential amino acid Gly Ala Ser Tyr Cys Pro Asn Asp Glu Gln Arg Met Trp Lys Val Ile Leu Phe Thr (His) 3.3 Other Amino Acids and Amino Acid Deriv

30、atives More than 200 different AAs are found in living organisms. Most species contain a variety of L-AAs that are either precursors of the common AAs or intermediates in other biochemical pathways. For examples: Homocysteine, homoserine, ornithine, citrulline (AAs precursors or intermediates); SAM

31、(S-Adenosyl Metionine,CH3 donor), -aminobutyrate (neurotransmitter), Histamine (blood pressure,HCl secretion), Epinephrine, Thyroxine (metablism regulation), N-formylmethionine (bacteria), Selenocystein (enzyme). Precursor AA: (a) glutamate (b) histidine (c) tyrosine (d) tyrosine 13 of AAs have 2 io

32、nizable groups; 7 of AAs have ionizable side chains with additional,measurable pKa values aspartate, glutamate, cysteine, tyrosine, lysine, histidine and arginine 3.4 Ionization of Amino Acids Each ionizable group is associated with a specific pKa value. When the pH of the solution is below the pKa,

33、 the protonated form predominates; When the pH of the solution is above the pKa, the unprotonated form predominates; High Low H+ lone pair electrons H H H+ N H H N Amino H+ Ampholyte contains both positive and negative groups on its molecule Carboxylic C O O H C O O Proton Is Adsorbed or Desorbed pK

34、a Low High pH pKa pK1 pK2 COOH N H3+ COO- R - C - H N H3+ R - C - H COO- NH2 R - C - H Acidic environment Neutral environment Alkaline environment A+ A- Ao pK1 2 pK2 9 pHpH = = pKpKa a + log+ logproton acceptorproton acceptor/proton donorproton donor Henderson-Hasselbalch equation The pKa values of

35、AAs are determined from titration curves. (Ala) pK1 = 2.4 pK2 = 9.9 pH pI ， toward anode pH pI ， toward cathode pH = pI ， no migrate The net charge on Ala at pH2.4 averages +0.5 and the net charge at pH9.9 averages -0.5. At pH6.15 , the avergae net charge of Ala is zero. The pH is referred to as the

36、 isoelectric point (pI). Ala would not migrate in a electric field at its pI. Ionization and the titration curve of Glu. Ionization and the titration curve of His. At pH7.0, the ratio of imidazole to imidazolium ion is 10:1. Thus, the 2 forms of His are both present in significant concentrations nea

37、r physiological pH. This property makes the side chain of His ideal for the transfer of protons within the catalytic sites of enzymes. Amino acids -COOH -NH2 -R Gly G 2.34 9.60 Ala A 2.34 9.69 Val V 2.32 9.62 Leu L 2.36 9.68 Ile I 2.36 9.68 Ser S 2.21 9.15 Thr T 2.63 10.4 Met M 2.28 9.21 Phe F 1.83

38、9.13 Trp W 2.38 9.39 Asn N 2.02 8.80 Gln Q 2.17 9.13 Pro P 1.99 10.6 Asp D 2.09 9.82 3.86 Glu E 2.19 9.67 4.25 His H 1.82 9.17 6.0 Cys C 1.71 10.8 8.33 Tyr Y 2.20 9.11 10.07 Lys K 2.18 8.95 10.53 Arg R 2.17 9.04 12.48 pK1 pK1 pK2 pK2 pK3 OH- pH pI pI ? pK1 + pK2 2 three pKa two pKa ? ? How to calcul

39、ate pI of AAsHow to calculate pI of AAs HOOC-CH2-C-COOH NH3+ H HOOC-CH2-C-COO- NH3+ H -OOC-CH2-C-COO- NH3+ H -OOC-CH2-C-COO- NH2 H A+ Ao A- A2- pK1 = 2.1 pK2 = 3.9 pK3 = 9.8 2.1 + 3.9 2 = 3.0 first second third Isoelectric point is the average of the two pKa flanking the zero net-charged form pK1 pK

40、2 pK3 Aspartic acid +1 0 -1 -2 OH The pI of 20 common AAsThe pI of 20 common AAs AAAA pIpI AAAA pIpI AAAA pIpI AAAA pIpI AlaAla 6.026.02 ProPro 6.306.30 ThrThr 6.536.53 AspAsp 2.772.77 ValVal 5.975.97 PhePhe 5.485.48 CysCys 5.025.02 GluGlu 3.223.22 LeuLeu 5.985.98 TrpTrp 5.895.89 TyrTyr 5.665.66 Lys

41、Lys 9.749.74 IleIle 6.026.02 GlyGly 5.975.97 AsnAsn 5.415.41 ArgArg 10.7610.76 MetMet 5.745.74 SerSer 5.685.68 GlnGln 5.655.65 HisHis 7.597.59 The pKa of COOH of free AAs are lower than those of typical carboxylic acids. Because of the effect of NH2 and R group. According to Henderson-Hasselbalch eq

42、uation, free AAs exist predominantly as zwitterions at neutral pH. -COOH of Glu is further removed from the influence of the NH2 , and behaves as a weak acid with a pKa of 4.1 similar to acetic acid. The pKa of ionizable side chains in proteins can differ from those of the free AAs. Because: (1) NH2

43、 and COOH lose their charges in peptide chain; (2) the position of an ionizable side chain within the 3D structure of a protein can affect its pKa. The measurement of AAs concentration The measurement of AAs concentration Whether can AAs concentration been Whether can AAs concentration been measured

44、 by acidic or basic titration ?measured by acidic or basic titration ? pH = pKpH = pKa2 a2 + log + logHH2 2NCHNCH2 2COOCOO- -/H/H3 3N N+ +CHCH2 2COOCOO- - 1. Reactions of -NH2： 2. Reactions of -COOH： 3. Reactions of -NH2 and -COOH： 4. Reactions of R groups： 3.5 Chemical reactions of AAs: 1. Reaction

45、s of NH2： (1) Reaction with HNO2（deamination）: Van Slyke methods to measure content of AAs (2) Reaction with acylating agent（for protection of NH2 in peptides chemical synthesis): NH2 R-CH-COOH + HNO2 OH R-CH-COOH + N2 + H2O Acylating reactionAcylating reaction R1CX O + H2NCH COO- R2 X=-Cl, OH,-OCOR

46、 OH- CH COO- R2 R1CHN O N(CH3)2 SO2Cl H2NCHC RO HNCHC RO SO2 N(CH3)2 + 水解水解 N(CH3)2 SO2 HNCHC RO OH + 氨基酸氨基酸 丹磺酰氯丹磺酰氯 多肽多肽N-端端 丹磺酰丹磺酰N-端氨基酸端氨基酸 丹磺酰氨基酸丹磺酰氨基酸 Reaction with DNS-Cl: DNSDNS- -氨基酸氨基酸 For the measurement of N-terminus of proteins. DNFB O2N NO2 F H2N CH COOH R DNP - AA O2N NO2 HN CH COOH R

47、 HF (3) Hydrocarbylation Reaction:(3) Hydrocarbylation Reaction: For the measurement of N-terminus of proteins by Sanger. Edman reaction N C S 40，弱碱，弱碱 N C N H R CH C O S 40， H+ 、硝基甲烷、硝基甲烷 COOH H2N CH R PITC 异硫氰酸苯酯异硫氰酸苯酯 PTC- AA 苯氨基硫甲酰氨基酸苯氨基硫甲酰氨基酸 PTH- 氨基酸氨基酸 苯乙内酰硫脲氨基酸苯乙内酰硫脲氨基酸 H N C N H R CH C O OH

48、 S 可用层析法鉴可用层析法鉴 定出定出aa的种类的种类 ( (4 4) ) FormingForming ShifeShife basebase: : (5) Deamination reaction by enzyme catalyse(5) Deamination reaction by enzyme catalyse： RCHCOO - NH3+ RCHCOO - O + NH4+ 氨基酸氧化酶氨基酸氧化酶 ( (6 6) ) ReactionReaction withwith fluorescentfluorescent agentsagents: : For the labelling and modification of For the labelling and modification of proteins; measurement of free amine of Lys. proteins; measurement of free amine of Lys. EX=390nm=390nm，EM=475nm=475nm