Biochemistry Conception, theory, research, and application ——Logic and LIY (Learn It Yourself) Sheng Zhao (赵晟), Biochemistry and Molecular Department of Medical school in Southeast University Web: http://teaching.ewindup.info/ Email: shengzhao@seu.edu.cn or windupzs@gmail.com QQ /MSN/Skype/gChat: windupzs@gmail.com Mobile:18551669724 or 13675130010

Chapter 1: Protein Structure and Function Amino acid and peptide

As an example: A Cool Protein Magic Color Marker of the twenty-first century Nobel Prize in Chemistry 2008

Origin of GFP Jellyfish, Aequorea victoria in 1962

Functions in nature Found in the Pacific Northwest jellyfish Aquorea victoria Works as a spontaneous fluorescent protein which transduces the blue chemiluminescence of the aequorin protein into green fluorescent light Serves as a means of communication Attract prey Means of protection

Green Fluorescent Protein (GFP)

A song for GFP ——by Christopher Dinesh Raj XFPs A song for GFP ——by Christopher Dinesh Raj Oh, fluorescent one! From the time they clones you And the time, I first set my eyes upon you, Something drives us To do something new with you… …Only to make you pure. You have been glorified here, And stolen there, Still you shine like an emerald in mother nature’s crown. Allow me to make a new construct out of you, Mutate and transform you, Shine some blue light upon you, And I am certain my peers will turn green, Yes, green with envy, Because MY GFP is brighter. Labeled Endoplasmatic Reticulum Florescent Zebra

Structure of GFP Primary 236 AA residues Secondary * 1 β pleated sheet composed of 11 antiparallel strands (48 %) * 5 α helices (5%) 20 β turns (bends) 2 γ turns 12 β bulges 7 β hairpins Supersecondary β barrel Fluorophore (Chromophore) in center

Amino acids Monomeric building blocks of proteins 20 different standard protein amino acids Same general structure Differ in side chain (R group) All organisms have same set of 20 2 rare protein amino acids selenocysteine and pyrrolysine Different activities and shapes of proteins due to different amino acid sequences

Amino acids Different side-chain (R group) -Carbon atom -Amino group (N-terminal) - Carboxyl group (C-terminal) Different side-chain (R group) Different chemical and physical properties

Properties of Amino Acids Chirality Structure Acid / Base properties Chemical reactions Capacity to polymerize

1. Chirality: L-Amino Acids Since all amino Acids but glycine (R=H) are chiral molecules, it’s possible to have enantiomers/ stereoisomers. However, nature uses only one enantiomer, the L-amino acid. The two enantiomers of alanine, D-Alanine and L-Alanine

2. Structure: Amino Acid Classification Aliphatic Aromatic Sulfur containing Polar/uncharged basic/acidic Hydrophobic- water fearing. non-polar side chains Hydrophilic- water loving. polar, neutral chains negatively charged, positively charged

Non-polar Amino Acids There are 8 non-polar amino acids:

Polar, Uncharged Amino Acids There 7 polar, uncharged amino acids:

Polar, Charged Amino Acids There are 5 polar charged amino acids:

21st & 22nd AAs

Groups and Abbreviations of AA Amino Acid 3-Letter 1-Letter Side-chain polarity Side-chain charge (pH 7.4) Hydropathy index Absorbance λmax(nm) Alanine Ala A nonpolar neutral 1.8 Arginine Arg R Basic polar positive −4.5 Asparagine Asn N polar −3.5 Aspartic acid Asp D acidic polar negative Cysteine Cys C 2.5 250 Glutamic acid Glu E Glutamine Gln Q Glycine Gly G −0.4 Histidine His H positive(10%)neutral(90%) −3.2 211 Isoleucine Ile I 4.5 Leucine Leu L 3.8 Lysine Lys K −3.9 Methionine Met M 1.9 Phenylalanine Phe F 2.8 257, 206, 188 Proline Pro P −1.6 Serine Ser S −0.8 Threonine Thr T −0.7 Tryptophan Trp W −0.9 280, 219 Tyrosine Tyr Y −1.3 274, 222, 193 Valine Val V 4.2 21st and 22nd amino acids 3-Letter 1-Letter Selenocysteine Sec U Pyrrolysine Pyl O Ambiguous Amino Acids 3-Letter 1-Letter Asparagine or aspartic acid Asx B Glutamine or glutamic acid Glx Z Leucine or Isoleucine Xle J Unspecified or unknown amino acid Xaa X Unk is sometimes used instead of Xaa, but is less standard.

Maps of amino acid Amino Acid 3-Letter 1-Letter Alanine Ala A Arginine Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

Essential Amino Acids (*) Essential only in certain cases. Human body is able to synthesize only 10 of them. The other 10 must be obtained from food (essential amino acids). Essential Nonessential Histidine Alanine Isoleucine Arginine* Leucine Asparagine Lysine Aspartic acid Methionine Cysteine* Phenylalanine Glutamic acid Threonine Glutamine* Tryptophan Glycine Valine Ornithine* Proline* Selenocysteine* Serine* Tyrosine*

UV-absorbing Properties of Amino Acids Only three amino acids, Phe, Tyr, and Trp, absorb light in the near UV range (230 nm-300 nm). These amino acids dominate the UV absorption spectra of proteins. The wavelength maxima for tyrosine and tryptophan are around 280 nm. In contrast, nucleic acids (we will discuss later) have absorption maximum of 260 nm. Thus a simple UV scan can allow one to distinguish between protein and nucleic acids.

Amino Acids not found in Proteins -Amino group (N-terminal) - Carboxyl group (C-terminal) -Carbon atom

3. Acid / Base properties: Zwitterions A Zwitterion is a dipolar ion. Since amino acids contain both an acid and a base, an internal acid-base reaction forms a zwitterion. amino acid zwitterion Amino acids exist primarily as zwitterions.

Zwitterions Amino acid zwitterions are amphoteric. They can react as either acids or bases. In acid solution zwitterion protonated In base solution zwitterion deprotonated

Isoelectric Points The isoelectric point of an amino acid occurs at the pH where the amino acid exists as the zwitterion. protonated acid solution low pH zwitterion isoelectric point deprotonated base solution high pH

Isoelectric Points No net charge Minimum solubility in water Protein will precipitate out at its isoelectric point Principle for separating amino acids and peptides based in electrophoresis: + charged amino acids move to – electrode - charged amino acids move to + electrode 0 amino acids at their isoelectric points do not move

Electrophoresis A mixture of histidine, serine, and glutamic acid can be separated by electrophoresis at pH = 5.68. - + positively charged (protonated) histidine negatively charged (deprotonated) glutamic acid serine at its isoelectric point at pH = 5.68

Electrophoresis

Amino Acid separation by Ion Exchange Unfortunately, amino acids are not colored as described in this overhead. Therefore, what methods would you use to first check if an amino acid is indeed present?

Ion Exchange

4. Chemical reactions Different side-chain (R group) -Carbon atom -Amino group (N-terminal) - Carboxyl group (C-terminal) Different side-chain (R group) Different chemical and physical properties

Reaction with Ninhydrin Amino group Reaction with Ninhydrin Used to visualize spots or bands of amino acids separated by chromatography or electrophoresis. (Qualitatively ) Deep purple color formed with traces of any amino acid except Pro (yellow) Reaction with Nitrous acid (HNO2 ) and Van Slyke Determination Nitrogen gas which can be observed qualitatively or measured quantitatively.

Reaction with formaldehyde and formol titration Amino group

Amino group and Carboxyl group A Peptide Bond is an amide bond linking together amino acids forming peptides and proteins. Amino group and Carboxyl group peptide bond

R-Groups In solution it is the nature of the amino acid R-groups that dictate structure-function relationships of peptides and proteins. The hydrophobic amino acids will generally be encountered in the interior of proteins shielded from direct contact with water. Conversely, the hydrophilic amino acids are generally found on the exterior of proteins as well as in the active centers of enzymatically active proteins. The imidazole ring of histidine allows it to act as either a proton donor or acceptor at physiological pH. Hence, it is frequently found in the reactive center of enzymes. The primary alcohol of serine and threonine as well as the thiol (-SH) of cysteine allow these amino acids to act as nucleophiles during enzymatic catalysis. Additionally, the thiol of cysteine is able to form a disulfide bond with another cysteine. Disulfide bonding between cysteines in different polypeptide chains of oligomeric proteins plays a crucial role in ordering the structure of complex proteins, e.g. the insulin and its receptor. Cysteine-SH + HS-Cysteine <=> Cysteine-S-S- Cysteine (Cystine)

+ OOC CH SH NH HS - CH COO NH OOC CH S NH COO R group reaction -HH  2 Cysteine become 1 Cystine + - OOC CH 2 SH + NH 3 HS - CH 2 COO + NH 3 -HH - OOC CH 2 S + NH 3 COO Disulfid bond Cystine

Structure of GFP Primary 236 AA residues Secondary * 1 β pleated sheet composed of 11 antiparallel strands (48 %) * 5 α helices (5%) 20 β turns (bends) 2 γ turns 12 β bulges 7 β hairpins Supersecondary β barrel Ligand * Fluorophore (Chromophore)

Peptides Dipeptides: two amino acids linked by peptide bond (amide linkage) Peptides are written so that the free NH2 group is on the left and the free COOH group is on the right. N-terminus: alanine C-terminus: aspartic acid Ala-Asp alanylaspartic acid

Peptides Tripeptides Ser-Cys-Asn Serinylcysteinylasparagine

Properties of Peptides Acid-base properties——also have pI Biuret reaction (react with copper sulfate in highly alkaline solutions to produce a violet color. First step of Lowry assay) Degradation Biological function

Naturally-occurring peptides Peptides synthesized on the ribosome (1) coded by their own genes (2) synthesized in the form of precursor (3) never contain D-AA (4) an example: oxytocin Peptides not synthesized on the ribosome (1) synthesized by a series of enzymes outside the ribosome (2) may contain D-AA (3)an example: glutathione

Glutathione GSH Glutamate Cysteine Glycine -Carbon atom Antioxidant in vivo to protect DNA

glutathione reductase (GSR) Two status of GSH: GSH (reduced status) vs. GSSG (oxidized status) H2O2 2GSH NADP+ GSH peroxidase glutathione reductase (GSR) 2H2O GSSG NADPH+H+ Antioxidant in vivo to protect DNA

Peptide hormone Many hormones (e.g. neuropeptide) are oligopeptide or polypeptide Thyrotropin-releasing hormone (pyro)Glu His Pro-NH2

Polypeptides Proinsulin

Peptides & Proteins Peptides contain 50 or fewer amino acids Dipeptides contain 2 amino acids. Tripeptides contain 3 amino acids. Oligopeptides contain 4~10 amino acids Polypeptides contain 10~50 amino acids. Proteins contain greater than 50 amino acids. An amino acid in the peptides or proteins is called amino acid residue Peptide chain and Polypeptide chain