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BIOCHEMISTRY I

Midterm exam schedule (Chap 2-7)
4/24 (Fri) 8pm
No withdrawal allowed!

• Key term report submission:
3/4
3/9.
3/16.
3/23.
4/1.
4/8.

Chap 2. aqueous system.
Chap 3. amino acids, peptides, and proteins
Chap 4. tertiary and quaternary structure of proteins
Chap 5. protein functions
Chap 6. enzymes
Chap 7. carbohydrates and glycobiology

# Quizes
3/23 (Mon): Chapter 2 and 3
4/6 (Mon): Chapter 4 and 5

3 | Amino Acids, Peptides, Proteins

© 2013 W. H. Freeman and Company

CHAPTER 3
Amino Acids, Peptides,
Proteins
Learning goals:



Structure and naming of amino acids
Structure and properties of peptides
Ionization behavior of amino acids and peptides
Methods to characterize peptides and proteins

Proteins: Main Agents of Biological Function • Catalysis – enolase (in the glycolytic pathway) – DNA polymerase (in DNA replication) • Transport – hemoglobin (transports O2 in the blood) – lactose permease (transports lactose across the cell membrane) • Structure – collagen (connective tissue) – keratin (hair. cell motility) . nails. horns) • Motion – myosin (muscle tissue) – actin (muscle tissue. feathers.

Proteins serve a wide range of biological functions .

Amino Acids: Building Blocks of Protein • Proteins are linear heteropolymers of -amino acids • Amino acids have properties that are well-suited to carry out a variety of biological functions – – – – Capacity to polymerize Useful acid-base properties Varied physical properties Varied chemical functionality .

Amino acids share many features. differing only at the R substituent .

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the fourth substituent is also hydrogen .Most -amino acids are chiral • The -carbon always has four substituents and is tetrahedral • All (except proline) have: – an acidic carboxyl group – a basic amino group – an -hydrogen connected to the -carbon • The fourth substituent (R) is unique – In glycine.

All amino acids are chiral (except glycine) Proteins only contain L amino acids .

L system . in relation to D-and L-glyceraldehyde => D.Absolute configuration: the configuration of four different substituent groups around an asymmetric carbon atoms.

Amino Acids: Atom Naming • Organic nomenclature: start from one end • Biochemical designation: – start from -carbon and go down the R-group .

aliphatic (7) • Aromatic (3) • Polar.Amino Acids: Classification Common amino acids can be placed in five basic groups depending on their R substituents: • Nonpolar. uncharged (5) • Positively charged (3) • Negatively charged (2) .

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These amino acid side chains absorb UV light at 270–280 nm .

Cysteine can form disulfide bonds. .These amino acids side chains can form hydrogen bonds.

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Aromatic R Groups These amino acid side chains absorb UV light at 270-280 nm FIGURE 3-6 Absorption of ultraviolet light by aromatic amino acids .

Protein concentration measurements? Measurement of light absorption by a spectrophotometers used to detect and identify molecules and to measure their concentration in solution .

The fraction of the incident light absorbed by a solution at a given wavelength is related to the thickness of the absorbing layer (path length) and the concentration of the absorbing species. A Io is the intensity of the incident light I is the intensity of the transmitted light I/Io (the inverse of the ratio in the equation) is the transmittance ε is the molar extinction coefflcient (in units of liters per mole-centimeter) c is the concentration of the absorbing species (in moles per liter) l is the path length of the light-absorbing sample (in centimeters) . These two relationships are combined into the Lambert-Beer law Io log = εcl I => absorbance.

.BOX 3-1 FIGURE 1 The principal components of a spectrophotometer.

Absorbance A is directly proportional to the concentration
of the absorbing solute.

ε varies with the nature of the absorbing compound, the
solvent, and the wavelength, and also with pH if the lightabsorbing species is in equilibrium with an ionization state
that has different absorbance properties.

Cysteine can form disulfide bonds

FIGURE 3-7Reversible formation of a disulfide bond by the oxidation of two molecules of
cysteine. Disulfide bonds between Cys residues stabilize the structures of many proteins.

You should memorize

- One letter codes
- Three letter codes
- Chemical structure of 20 amino acids

Uncommon Amino Acids in Proteins
• Not incorporated by ribosomes
 except for Selenocysteine
• Arise by post-translational modifications of
proteins
• Reversible modifications, especially
phosphorylation, are important in regulation and
signaling

Modified Amino Acids Found in Proteins .

Reversible Modifications of Amino Acids .

the amino group is neutral –NH2 and the amino acid is in the anionic form.Ionization of Amino Acids • At acidic pH. Zwitterion: a dipolar ion with spatially separated positive and negative charges. • At alkaline pH. the carboxyl group is protonated and the amino acid is in the cationic form. . such ions are called Zwitterions. • At neutral pH. The net charge is zero. the carboxyl group is deprotonated but the amino group is protonated.

FIGURE 3-9 Nonionic and zwitterionic forms of amino acids. .

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FIGURE 3-10 Titration of an amino acid. Cation  Zwitterion  Anion .

Chemical Environment Affects pKa Values -carboxy group is much more acidic than in carboxylic acids -amino group is slightly less basic than in amines .

Amino acids can act as buffers Amino acids with uncharged side chains.6 It can act as a buffer in two pH regimes. .34 The pKa of the -amino group is 9. have two pKa values: The pKa of the -carboxyl group is 2. such as glycine.

Buffer Regions .

Amino acids carry a net charge of zero at a specific pH (the pI) • Zwitterions predominate at pH values between the pKa values of the amino and carboxyl groups • For amino acids without ionizable side chains. the net charge is zero –AA does not migrate in electric field . pI) is pK1  pK 2 pI  2 • At this point. the Isoelectric Point (equivalence point.

Ionizable side chains can show up in titration curves • Ionizable side chains can be also titrated • Titration curves are now more complex • pKa values are discernable if two pKa values are more than two pH units apart .

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How to Calculate the pI When the Side Chain is Ionizable • Identify species that carries a net zero charge • Identify pKa value that defines the acid strength of this zwitterion: (pK2) • Identify pKa value that defines the base strength of this zwitterion: (pK1) • Take the average of these two pKa values What is the pI of histidine? .

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Formation of Peptides • Peptides are small condensation products of amino acids • They are “small” compared to proteins (Mw < 10 kDa) .

Peptide ends are not the same Numbering (and naming) starts from the amino terminus AA1 AA2 AA3 AA4 AA5 .

Naming peptides: start at the N-terminus • Using full amino acid names – Serylglycyltyrosylalanylleucine • Using the three-letter code abbreviation – Ser-Gly-Tyr-Ala-Leu • For longer peptides (like proteins) the oneletter code can be used – SGYAL .

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Sample problem Draw the chemical structure of the following peptide at pH 7. CHEMIST .

111 11. 8. Net Electric Charge of Peptides A peptide has the sequence Glu–His–Trp–Ser–Gly–Leu–Arg–Pro–Gly (a)What is the net charge of the molecule at pH 3.) (b) Estimate the pI for this peptide. and 11? (Use pKa values for side chains and terminal amino and carboxyl groups as given in Table 3–1. .p.

. e.g. toxins – amanitin (mushrooms) – conotoxin (cone snails) – chlorotoxin (scorpions) .Peptides: A Variety of Functions • Hormones and pheromones – insulin (think sugar) – oxytocin (think childbirth) – sex-peptide (think fruit fly mating) • Neuropeptides – substance P (pain mediator) • Antibiotics – polymyxin B (for Gram – bacteria) – bacitracin (for Gram + bacteria) • Protection.

Proteins are: • Polypeptides (covalently linked -amino acids) + possibly: • cofactors  functional non-amino acid component  metal ions or organic molecules • coenzymes  organic cofactors  NAD+ in lactate dehydrogenase • prosthetic groups covalently attached cofactors heme in myoglobin • other modifications .

Polypeptide size and number varies greatly in proteins .

Classes of Conjugated Proteins .

What to Study about Peptides and Proteins What is its sequence and composition? What is its three-dimensional structure? How does it find its native fold? How does it achieve its biochemical role? How is its function regulated? How does it interacts with other macromolecules? How is it related to other proteins? Where is it localized within the cell? What are its physico-chemical properties? .

A mixture of proteins can be separated • Separation relies on differences in physical and chemical properties – – – – – – Charge Size Affinity for a ligand Solubility Hydrophobicity Thermal stability • Chromatography is commonly used for preparative separation .

salt concentration – (now) column chromatography . temperature.Protein separation • Crude extract – released proteins into a solution after breaking open the cells • Separate proteins into different fractions – (old fashion) Protein solubility: pH.

Column Chromatography .

Separation by Charge .

Amino acids placed on a cation-exchange resin (see Fig. (a) Asp and Lys (b) Arg and Met (c) Glu and Val (d) Gly and Leu (e) Ser and Ala .0 buffer.p. determine which will be eluted first from an ion-exchange column by a pH 7. and (2) hydrophobic interactions between amino acid side chains and the strongly hydrophobic backbone of the polystyrene resin. Separation of Amino Acids by Ion-Exchange Chromatography Mixtures of amino acids can be analyzed by first separating the mixture into its components through ion-exchange chromatography. 3–17a) containing sulfonate (-SO3.111 5.) groups flow down the column at different rates because of two factors that influence their movement: (1) ionic attraction between the sulfonate residues on the column and positively charged functional groups on the amino acids. For each pair of amino acids listed.

Separation by Size .

Separation by Affinity .

higher quality chromatographic materials => Reducing the transit time on the column.Modern chromatographic methods employing HPLC High pressure pump. limit diffusional spreading .

Electrophoresis for Protein Analysis Separation in analytical scale is commonly done by electrophoresis – Electric field pulls proteins according to their charge – Gel matrix hinders mobility of proteins according to their size and shape .

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SDS PAGE: Molecular Weight • SDS – sodium dodecyl sulfate – a detergent • SDS micelles bind to and unfold all the proteins – SDS gives all proteins an uniformly negative charge – The native shape of proteins does not matter – Rate of movement will only depend on size: small proteins will move faster .

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SDS-PAGE can be used to calculate the molecular weight of a protein .

. this time with molecular masses of 160. 90. When electrophoresis is carried out in the presence of SDS and dithiothreitol. and 60 kDa. The two 90 kDa subunits (possibly identical) are linked by one or more disulfide bonds. and 60 kDa. 90. Answer The protein has four subunits. Subunit Composition of a Protein A protein has a molecular mass of 400 kDa when measured by gel filtration.p. When subjected to gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). with molecular masses of 160. three bands are again formed. Determine the subunit composition of the protein. the protein gives three bands with molecular masses of 180. 90. and 60 kDa. 160. 111 10.

Isoelectric focusing can be used to determine the pI of a protein .

Isoelectric focusing and SDS-PAGE are combined in 2D electrophoresis .

Quantification of unseparated proteins Activity: total units of enzymes in a solution Specific activity: the number of enzyme units per milligram of total protein .

Specific activity (activity/total protein) can be used to assess protein purity .

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Quaternary structure : arrangement in space of 2 or more polypeptide subunits . -S-S-) : sequence of amino acid residues -Secondary structure : particular structural arrangements of amino acids giving rise to recurring structural patterns -Tertiary structure : 3D folding of a polypeptide chain .Levels of structure in proteins -Primary structure : a description of all covalent bonding (peptide bonding.

FIGURE 3-23 Levels of structure in proteins. .

. the function may also be changed.  But.The function of a protein depends on its amino acid sequence • Each type of protein has a unique amino acid sequence. • Amino acid sequence determines 3D structure and ultimately its function.  If primary structure alters. 20-30% of the proteins in humans are polymorphic.  Functionally similar proteins from different species often have similar amino acid sequence.

The amino acid sequences of millions of proteins have been determined • Determination of amino acid sequence from polypeptide chain is difficult • Amino acid sequence in protein is related to nucleotide sequence • DNA sequence => decoding genetic code => protein amino acid sequence .

cleavage.Protein Sequencing • It is essential to further biochemical analysis that we know the sequence of the protein we are studying • Actual sequence generally determined from DNA sequence • Edman Degradation (Classical method) – Successive rounds of N-terminal modification. and identification – Can be used to identify protein with known sequence • Mass Spectrometry (Modern method) – MALDI MS and ESI MS can precisely identify the mass of a peptide. and thus the amino acid sequence – Can be used to determine post-translational modifications .

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Edman’s Degradation .

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FIGURE 3-27 Cleaving proteins and sequencing and ordering the peptide fragments. .

Amino acids sequences can also be deduced by other methods Mass spectrometry Analytes  Ionization in vacuum: generating charged analytes  apply them into an electric/magnetic field paths through the field are a function of mass-to-charge ratio (m/z) Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS)  Light absorbing matrix: laser light  protein ionization and desorption into vacuum Electrospray ionization mass spectrometry (ESI MS)  Analytes pass through a charged needle kept at high electric potential  dispersing the solution into a find mist of charged microdroplets. .

. The droplets evaporate. and the ions (with added protons in this case) enter the mass spectrometer for m/z measurement.Electrospray mass spectrometry of a protein. A protein solution is dispersed into highly charged droplets by passage through a needle under the influence of a high-voltage electric field.

with each successive peak (from right to left) corresponding to a charged species increased by 1 in both mass and charge.Electrospray mass spectrometry of a protein The spectrum generated (b) is a family of peaks. Inset: a computer-generated transformation of this spectrum. .

Tandem MS (MS/MS)  To sequence short polypeptide  peptide separation by first MS  fragmentation by high-energy impact with “collision gas”  b-type ion: charge retained on N-terminal side  y-type ion: charge retained on C-terminal side  measure m/z ratio by second MS .

BOX 3-2 FIGURE 2a Obtaining protein sequence information with tandem MS. .

.Obtaining protein sequence information with tandem MS.

Protein Sequences as Clues to Evolutionary Relationships • Sequences of homologous proteins from a wide range of species can be aligned and analyzed for differences • Differences indicate evolutionary divergences • Analysis of multiple protein families can indicate evolutionary relationships between organisms. ultimately the history of life on Earth .

Chapter 3: Summary In this chapter. we learned about: • The many biological functions of peptides and proteins • The structures and names of amino acids found in proteins • The ionization properties of amino acids and peptides • The methods for separation and analysis of proteins .

• Quiz: 3/23 (Mon) Chap 2 and 3 .