Chapter 24 Amino Acids and Proteins Created By Prof. Gary F. Porter, Ph.D. Copyright © 2014 by John...

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Chapter 24 Amino Acids and Amino Acids and Proteins Proteins Created By Prof. Gary F. Porter, Ph.D. Copyright © 2014 by John Wiley & Sons, Inc. All rights reserved.

Transcript of Chapter 24 Amino Acids and Proteins Created By Prof. Gary F. Porter, Ph.D. Copyright © 2014 by John...

Chapter 24Amino Acids and ProteinsAmino Acids and Proteins

Created By Prof. Gary F. Porter, Ph.D.

Copyright © 2014 by John Wiley & Sons, Inc. All rights reserved.

Table of Contents

1. Introduction2. Amino Acids3. Synthesis of α–Amino Acids4. Polypeptides and Proteins5. Primary Structure of Polypeptides and Proteins6. Examples of Polypeptide and Protein Primary Structure7. Polypeptide and Protein Synthesis8. Secondary, Tertiary, and Quaternary Structures of Proteins9. Introduction to Enzymes10. Purification and Analysis of Polypeptides and Proteins11. Proteomics

© 2014 by John Wiley & Sons, Inc. All rights reserved.

• Amino acids are the monomeric unit for proteins.• Proteins are polyamides.

• There are:– 20 different common α-amino acids, and – 4 levels of protein structure.

1. Introduction

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• In the 20 common amino acids, the α carbon has an L configuration.

1. Introduction

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2A. Structures and Names:

The four classes of amino acids are:Neutral, NonpolarNeutral, PolarCharged Positive, Polar Acidic (at pH 7)Charged Negative, Polar Basic (at pH 7)

2. Amino Acids

© 2014 by John Wiley & Sons, Inc. All rights reserved.

2. Amino Acids

© 2014 by John Wiley & Sons, Inc. All rights reserved.

2. Amino Acids

© 2014 by John Wiley & Sons, Inc. All rights reserved.

2. Amino Acids

© 2014 by John Wiley & Sons, Inc. All rights reserved.

2. Amino Acids

© 2014 by John Wiley & Sons, Inc. All rights reserved.

2. Amino Acids

© 2014 by John Wiley & Sons, Inc. All rights reserved.

2B. Essential Amino Acids:– Are not synthesized by higher animals.– Also, Histidine and arginine are essential in infants.

The Essential Eight

Valine Tryptophan

Leucine Threonine

Isoleucine Methionine

Phenylalanine Lysine

2. Amino Acids

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2C. Amino Acids as Dipolar Ions:– Contain a basic group (-NH2) and an acidic group

(-CO2H), and each of these has a pKa.

– Exist as dipolar ions at physiological pH.

• Dipolar ions are also called zwitterions.• Isoelectric point (pI) is the pH at which the net

charge on an amino acid is 0.

2. Amino Acids

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Alanine has two ionization equilibria:

2. Amino Acids

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• Note the titration curve for Alanine below.– As base is added and pH is increased, the net charge

on Alanine goes from positive to negative.

2. Amino Acids

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3A. Method 1: From Potassium Phthalimide.• Methionine Synthesis

3. Synthesis of α–Amino Acids

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3B. Method 2: The Strecker Synthesis• treat aldehyde with NH3 and HCN, and

• produce an α amino acid.

3. Synthesis of α–Amino Acids

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• Peptides – are amino acids joined together to form a molecule.

• Peptide Bonds – are amide linkages between the amino acids.

N terminus on left C terminus on right

No. of Amino Acids linked together

Name

2 Dipeptide

3 Tripeptide

3-10 Oligopeptide

Multiple polypeptides Protein

4. Polypeptides and Proteins

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4A. Hydrolysis• Peptide bond cleavage occurs in 6 M HCl for 24 h.• AAs separate on cation-exchange resin.– AAs will bind at low pH. Amino

Acids (AA)– AAs release as pH increases.

Resin

4. Polypeptides and Proteins

© 2014 by John Wiley & Sons, Inc. All rights reserved.

Detection of AAs:– Ninhydrin Reagent– λmax = 570nm

Formation of blue complex occurs with this reagent.

4. Polypeptides and Proteins

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Primary Structure is defined by: – amino acid sequence,– molecular weight,– amino acid composition.

Edman Degradation:– identification of N-Terminus,– sequential degradation of AAs from N- terminus,– Automated sequencing is good up to ~60 AAs.

5. Primary Structure of Polypeptides and Proteins

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5A. Edman Degradation:uses phenylisothiocyanate,cleaves the N-terminal AA,as a phenylthiohydantion,The R group identifies the AA.

5. Primary Structure of Polypeptides and Proteins

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5B. Sanger N-Terminus Analysis: an N-terminal AA analysis, uses 2,4-dinitrofluorobenzene.

5. Primary Structure of Polypeptides and Proteins

© 2014 by John Wiley & Sons, Inc. All rights reserved.

5C. C-Terminus Analysis– uses carboxypeptidases,– continually cleaves carboxyl AAs, and– can only be used with smaller peptide chains.

5. Primary Structure of Polypeptides and Proteins

© 2014 by John Wiley & Sons, Inc. All rights reserved.

6B. Insulin– is secreted by pancreas, and – regulates (lowers) blood glucose.

Thousands of other polypeptides and protein structures are known.

6. Examples of Polypeptide and Protein Primary Structure

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7D. Automated Peptide Synthesis– Involves solid

phase addition,– addition of AA, – followed by wash. – High yield at each

step is realized.

FMOC =Fluorenylmethyloxycarbonyl

7. Polypeptide and Protein Synthesis

© 2014 by John Wiley & Sons, Inc. All rights reserved.

Secondary Structure• is defined by conformations of polypeptides.• Specific types are

– α helix,– β sheets, and– coil or loop.

• Rotation about the α-carbon atom in each residue.– AA residues have limited or no rotation.– The side groups have free rotation.– There is free rotation about the α-carbon but not the peptide bond.

8. Secondary, Tertiary, and Quaternary Structures of Proteins

© 2014 by John Wiley & Sons, Inc. All rights reserved.

Secondary Structure• β pleated sheet (below) has

– high nonpolar AA content, and– predominates in silk.

• α helix (right) has– 3.6 AA’s per right handed turn, and – is in fibrous proteins.

8. Secondary, Tertiary, and Quaternary Structures of Proteins

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8B. Tertiary Structure– has a three dimensional shape that arises from

secondary structures orientation to each other, and

– results in two classes of proteins.• Fibrous Proteins have mainly α helices.• Globular Proteins

– have all types of secondary structure.

– AA classes have specific locations:• nonpolar are favored in the interior and • polar aare favored on the exterior.

8. Secondary, Tertiary, and Quaternary Structures of Proteins

© 2014 by John Wiley & Sons, Inc. All rights reserved.

Carbonic Anhydrase Myoglobin

8. Secondary, Tertiary, and Quaternary Structures of Proteins

© 2014 by John Wiley & Sons, Inc. All rights reserved.

8C. Quaternary Structures Three dimensional shape that arises from polypeptide chain orientation to each other.

• See the four polypeptide chains of hemoglobin to the right.

8. Secondary, Tertiary, and Quaternary Structures of Proteins

© 2014 by John Wiley & Sons, Inc. All rights reserved.

• Enzymes– catalyze all chemical reactions found in cellular

metabolism,– have highly specific reactants called substrates,– form an enzyme-substrate complex, and– are stereospecific.

• Enzyme-substrate complex– induces conformational changes in the enzyme,– is the place where the reaction is catalyzed, and– resides within the active site.

9. Introduction to Enzymes

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• Competitive Inhibitors– have a similar structure to substrate and – binds to the active site instead of the substrate.

• Cofactors– is an extra molecule required for enzyme activity.– i.e. metal ions

• Coenzyme – Organic cofactor

9. Introduction to Enzymes

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• Lysozymes cleave the cell wall of gram positive bacteria.• Lysozymes hydrolyze the glycosidic linkages in peptoglycans.• Blue AAs are found at the active site.

10. Lysozyme: Mode of Action of an Enzyme

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• Pancreatic digestive enzymes• Chymotrypsin– is activated from Chymotrypsinogen– is a peptidase– Catalytic triad - Asp 102, His 57, Ser 195– The triad only forms in Chymotrypsin– The inactive form exists in the pancreas to avoid

damage

11. Serine Proteases

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• Hemoglobin is a conjugated protein with a nonprotein prosthetic group.

• Heme is the prosthetic group.• Fe2+

12. Hemoglobin: A Conjugated Protein

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13A. Purification– Involves• chemical and• chromatographic means.

– Depends upon• molecular weight• pI, and• stability.

13. Purification and Analysis ofPolypeptides and Proteins

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13B. Analysis Methods– Gel Electrophoresis (based upon pI)– Mass Spectroscopy

• Obtains molecular weight

– Electrospray Ionization (ESI) uses• peak corresponds to m/z ratio and • mixtures separated by HPLC

– Matrix-assisted Laser Desorption Ionization (MALDI)• Used for ionization of nonvolatile molecules

13. Purification and Analysis of Polypeptides and Proteins

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Involves the identification, structure, and function of proteins

14. Proteomics

© 2014 by John Wiley & Sons, Inc. All rights reserved.

Ch. 21 - 39

END OF CHAPTER 24