Chapter 03_2015

BIOCHEMISTRY I

Midterm exam schedule (Chap 2-7)

4/24 (Fri) 8pm

No withdrawal allowed!

Key term report submission:

3/4 Chap 2. aqueous system.

3/9. Chap 3. amino acids, peptides, and proteins

3/16. Chap 4. tertiary and quaternary structure of proteins

3/23. Chap 5. protein functions

4/1. Chap 6. enzymes

4/8. 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

Structure and naming of amino acids

Structure and properties of peptides

Ionization behavior of amino acids and peptides

Methods to characterize peptides and proteins

Learning goals:

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, nails, feathers, horns)

Motion myosin (muscle tissue)

actin (muscle tissue, cell motility)

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

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, the fourth substituent is also hydrogen

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

Absolute configuration: the configuration of four different

substituent groups around an asymmetric carbon atoms, in

relation to D-and L-glyceraldehyde

=> D, L system

Amino Acids: Atom Naming

Organic nomenclature: start from one end

Biochemical designation:

start from -carbon and go down the R-group

Amino Acids: Classification

Common amino acids can be placed in five basic groups depending on their R substituents:

Nonpolar, aliphatic (7)

Aromatic (3)

Polar, uncharged (5)

Positively charged (3)

Negatively charged (2)

These amino acid side chains absorb UV light at 270280 nm

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

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. These two relationships are combined into

the Lambert-Beer law

IoI

log = cl

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

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)

=> absorbance, A

BOX 3-1 FIGURE 1 The principal components of a

spectrophotometer.

Absorbance A is directly proportional to the concentration

of the absorbing solute.

varies with the nature of the absorbing compound, the

solvent, and the wavelength, and also with pH if the light-

absorbing 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

Ionization of Amino Acids

At acidic pH, the carboxyl group is protonated and the amino acid is in the cationic form.

At neutral pH, the carboxyl group is deprotonated but the amino group is protonated. The net charge is zero; such ions are called Zwitterions.

At alkaline pH, the amino group is neutral NH2 and the amino acid is in the anionic form.

Zwitterion: a dipolar ion with spatially separated positive and

negative charges.

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

Cation Zwitterion Anion

FIGURE 3-10 Titration of an amino acid.

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, such as glycine, have two pKa values:

The pKa of the -carboxyl group is 2.34

The pKa of the -amino group is 9.6

It can act as a buffer in two pH regimes.

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 Isoelectric Point(equivalence point, pI) is

At this point, the net charge is zero

AA does not migrate in electric field

2

21 pKpKpI

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

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?

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 one-letter code can be used

SGYAL

Sample problem

Draw the chemical structure of the following peptide at pH 7.

CHEMIST

11. Net Electric Charge of Peptides A peptide has the sequence

GluHisTrpSerGlyLeuArgProGly

(a)What is the net charge of the molecule at pH 3, 8, and 11? (Use

pKa values for side chains and terminal amino and carboxyl groups as

given in Table 31.)

(b) Estimate the pI for this peptide.

p.111

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, e.g., toxins amanitin (mushrooms)

conotoxin (cone snails)

chlorotoxin (scorpions)

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 groupscovalently 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

Protein separation

Crude extract

released proteins into a solution after breaking open the cells

Separate proteins into different fractions

(old fashion) Protein solubility: pH, temperature, salt concentration

(now) column chromatography

Column Chromatography

Separation by Charge

5. 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. Amino acids placed on a cation-exchange

resin (see Fig. 317a) containing sulfonate (-SO3- ) 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, and (2) hydrophobic interactions between

amino acid side chains and the strongly hydrophobic backbone of the polystyrene

resin. For each pair of amino acids listed, determine which will be eluted first from

an ion-exchange column by a pH 7.0 buffer.

(a) Asp and Lys

(b) Arg and Met

(c) Glu and Val

(d) Gly and Leu

(e) Ser and Ala

p.111

Separation by Size

Separation by Affinity

Modern chromatographic methods employing HPLC

High pressure pump, higher quality chromatographic materials

=> Reducing the transit time on the column, 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

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

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

10. Subunit Composition of a Protein A protein has a molecular

mass of 400 kDa when measured by gel filtration. When subjected

to gel electrophoresis in the presence of sodium dodecyl sulfate

(SDS), the protein gives three bands with molecular masses of 180,

160, and 60 kDa. When electrophoresis is carried out in the

presence of SDS and dithiothreitol, three bands are again formed,

this time with molecular masses of 160, 90, and 60 kDa. Determine

the subunit composition of the protein.

p. 111

Answer The protein has four subunits, with molecular masses of

160, 90, 90, and 60 kDa. The

two 90 kDa subunits (possibly identical) are linked by one or

more disulfide bonds.

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

Levels of structure in proteins

-Primary structure

: a description of all covalent bonding (peptide bonding, -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

- Quaternary structure

: arrangement in space of 2 or more polypeptide subunits

FIGURE 3-23 Levels of structure in proteins.

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, the function may also be

changed.

Functionally similar proteins from different species often

have similar amino acid sequence.

But, 20-30% of the proteins in humans are polymorphic.

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

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, cleavage, 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

Edmans Degradation

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.

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. 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

The spectrum generated (b) is a family of peaks, with each successive

peak (from right to left) corresponding to a charged species increased by

1 in both mass and charge. 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