Chapter 4

Amino Acids

Instructor: Rashid Syed

Textbook: Biochemistry (4th Edition ) by Donald Voet Judith G. Voet

Amino Acids, Peptides and Proteins

Structure of 20 common amino acids

General structure of peptides and proteins

Ionization behavior of amino acids and peptides

Methods to characterize peptides and proteins

Amino Acids

Amino acids are building blocks for proteins

They have a central -carbon and -amino and -carboxyl groups

20 different common amino acids

Same core structure, but different side group (R)

The -C is chiral (except glycine); natural proteins contain only L-isoforms.

Amino acids are ampholytes, pKa of -COOH is ~2 and of -NH2 is ~ 9

At physiological pH most aa occur as zwitterions.

Ampholytes

A molecule containing ionizing groups with both acidic and basic pKa values is called an ampholyte.

The ionic form of each group in the compound is dependent on the pH of the solution.

If the pH of solution is greater than the pKa, the group is in the conjugate base form (deprotonated).

If the pH of solution is less than the pKa, the group is in the conjugate acid form (protonated).

General Structure of Amino Acids Two numbering systems (1,2 and ,,.) COOH group is written at top, chiral carbon is in the center, R-group is at the bottom Absolute configuration: L-aa : NH2 is on the left of the chiral C D-aa : NH2 is on the right of the chiral C

Classification of Amino Acids

Aliphatic / non-polar R group: glycine, alanine, valine, leucine, isoleucine, methionine, proline

Aromatic R group: phenylalanine, tyrosine, tryptophan

Polar R group (net charge 0 at pH 7.0) uncharged: serine, threonine, cysteine, asparagine, glutamine

Positively Charged R group (+ charge at pH 7.0): lysine, arginine, histidine

Negatively Charged R group ( - charge at pH 7.0): aspartate, glutamate

Amino Acid R-group Classification

Aliphatic: gly (G), ala (A) , val (V), leu (L), ile (I) Aromatic: Trp (W), Phe (F), Tyr (Y), Sulphur : Met (M), Cys (C) Hydroxyl: Ser (S), Thr (T), Tyr (Y) Cyclic: pro (P) Carboxyl: asp (D), glu (E) Amide: asn (N), gln (Q) Amine: lys (K), arg (R)

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.

H +

at biological pH

Ionization of Amino Acids

pKa values depend on chemical environment

Titration Curve for Glycine

At pH > pI, net charge = -

At pH < pI, net charge = +

Calculation of pI for Glycine

Use the Henderson-Hasselbalch equation to calculate the pI.

At isoelectric point, pH = pI

pI = pKCOOH + log [H3N+CH2COO-] [H3N+CH2COOH]

pI = pKNH3+ + log [H2NCH2COO-] [H3N+CH2COO-]

Adding up: 2pI = pKCOOH + pKNH3+ + log [H2NCH2COO-] [H3N+CH2COOH]

When pH=pI, [H2NCH2COO-]=[H3N+CH2COOH] 2pI = pKCOOH + pKNH3+ or pI = {pKCOOH + pKNH3+}/2

Titration Curve for Histidine

triphasic

pI = (pKR + pK2)/2

Chapter 6

Techniques of Protein Purification

Instructor: Rashid Syed

Textbook: Biochemistry (4th Edition ) by Donald Voet Judith G. Voet

Protein Biochemistry Methods

Methods can be analytical or preparative

Preparative: purification of protein of interest

Analytical: identification and characterization

Methods based on solubility, size, charge, binding affinity, activity

Protein Purification

Proteins can be purified from cell or tissue samples

Samples are homogenized and fractionated by differential centrifugation to isolate the fraction containing protein of interest

Protein purification is a multi-step procedure

Need to establish a specific method of identification. Can be enzymatic, binding or activity assay

With each step, the purification level and specific activity increases and the yield decreases

Specific Activity (activity/total protein) can be Used to Assess Protein Purity

Solubility

Salting Out: For most proteins, solubility decreases as salt concentration increases.

Greater the polarity, greater the solubility.

Proteins can be fractionated by sequential salt precipitation

Isoelectric Precipitation: Proteins are least soluble when pH = pI. (because at pI, net charge is 0)

Size

Dialysis: Diffusion through a semi-permeable cellulose membrane. Different pore sizes allow removal of molecules smaller than specific MW

Gel-Filtration Chromatography: Also called size-exclusion chromatography. Column packed with porous beads made of a cross-linked polymer. Can get different pore sizes. Smaller molecules get trapped within the porous beads and their flow down the column is retarded. Larger molecules are excluded from the beads and move down between loosely packed bead. Smaller the molecule longer the elution time.

Dialysis

Size-Exclusion Chromatography

Charge Ion Exchange

Chromatography: Columns are made of charged cellulose particles.

Carboxymethyl (CM) cellulose: - charge, cation exchange column

Diethylaminoethyl (DEAE) cellulose: + charge, anion exhange column

Proteins are eluted using a pH gradient

Binding Affinity Affinity

Chromatography: The column matrix is modified by covalent linkage to a compound with high specific binding affinity to protein of interest

Eg: Lectins, antibodies, ligands, substrates

3 steps: Specific binding of protein, washing unbound proteins, elution of bound protein by specific displacement, high salt or low pH

HPLC

High Pressure Liquid Chromatography

Enhanced version of all column chromatography techniques

Column material are very fine and closely packed for better resolution

High pressure has to be applied to maintain flow

Clean, rapid separation of very small samples

Electrophoresis

Primary analytical technique Electrophoresis is the movement of charged proteins in

an electric field Movement is from the electrode (cathode) to +

electrode (anode). Migration is related to charge: mass ratio. Generally, smaller proteins migrate further

Separation on slabs of polyacrylamide cross-linked with methylenebisacrylamide: inert, porous and readily formed

Visualization by staining (coomassie blue, silver)

Gel Electrophoresis

Determination of M. Wt. of Protein

Types of Electrophoresis Denaturing gels: SDS disrupts all non-covalent

interactions. Associates with all proteins and imparts high charge making native charge insignificant. Reducing agent opens disulfide linkages

Native gels: No SDS, native charge contributes to migration, usually protein activity maintained

Isoelectric focusing: Polyampholytes are used to form a pH gradient in the gel. Proteins focus where pH = pI, because at this point they have 0 net charge

2D electrophoresis: IEF + SDS-PAGE. Proteins separated by pI horizontally, then by size vertically for complete resolution of complex samples

Isoelectric Focusing can be Used to Determine the pI of a Protein

Isoelectric Focusing and SDS-PAGE are Combined in 2D Electrophoresis

Peptide Synthesis

The N-terminal aas NH2 group is blocked using 9-fluorenyl-methoxycarbonyl(Fmoc) group

The C-terminal aa is covalently attached to a solid support via its carboxlate group

The Fmoc group is removed (aa is deprotected) The n-1 aa is protected with Fmoc. The COOH group of n-1 aa is activated by

dicyclohexylcarbodiimide (DCC) The two are reacted to form a peptide bond The 2nd cycle starts with deprotection of n-1 NH2 After multiple cycles, the synthetic peptide is released from the

resin by HF hydrolysis

Chapter 7

Covalent Structures of Proteins

Instructor: Rashid Syed

Textbook: Biochemistry (4th Edition ) by Donald Voet Judith G. Voet

Proteins

Linear polymers of aa via amide linkages form peptides (1-10), polypeptides (11-100) and proteins (>100)

Eg: Aspartame (2), glutathione (3), vasopressin (9), insulin (51)

Proteins have an amino-end and carboxyl-end

In the lab, proteins can be hydrolyzed (to aa) by strong acid treatment

Physiologic hydrolysis by peptidases and proteases

Protein Sequence

Amino acid sequence determines primary structure

Unique for each protein; innumerable possibilities

Gene sequence determines aa sequence

Each aa is called a residue; numbering (& synthesis) always from NH2 end toward COOH end

Amino acids covalently attached to each other by an amide linkage called as a peptide bond.

The Peptide Bond

Numbering (and naming) starts from the amino terminus AA1 AA2 AA3 AA4 AA5

A Pentapeptide

Ser-Gly-Tyr-Ala-Leu SGYAL

serylglycyltyrosylalanylleucine

Ionization of Peptides & Net charge

Each ionizable group ionizes according to its pKa and solution pH.

Net charge is sum total of all charges on molecule.

4 Levels of Protein Structure

Protein structure is stabilized by non-covalent interactions and disulfide linkages

Disulfide Linkages

The Proteome Proteome is the protein equivalent of the genome

The proteome consist of all of the proteins expressed by a cell under specific conditions

The proteome of a cell depends on the cell type, its developmental stage, environment/stimuli, nutritional and metabolic status etc

The genome of a cell is fixed, the proteome is dynamic

The proteome is much larger than the genome. Each gene can translate into multiple isoforms of proteins

Protein Mass and Concentration

Protein mass is measured in Daltons (Da) or kDa

One Dalton is 1/12 the mass of a 12C atom

On average, the MW of each aa is 110 Da

Most proteins range from 30 to 80 kDa

Trp and Tyr have a high ability to absorb light with maximum absorption at 280 nm. Since most proteins contain these aa, protein concentration can be estimated spectrophotometrically.

Characterization of Protein Primary Structure

Amino Acid Composition

Complete hydrolysis for 24 hr at 110 oC in 6 M HCl Separation of amino acids by ion exchange

chromatography on sulfonated polystyrene resin Elution using a pH gradient Detection of amino acids by reaction with ninhydrin

or fluorescamine (spectrophotometry) Identification of amino acids by position of peak on

chromatogram Determination of amino acid ratios by size (height) of

each peak

Amino Acid Analysis

Identification of amino-terminal residue

Identification of N-terminal amino acid

The N-terminal aa can be identified by Sangers method. This method involves modification of the N-terminal residue by flurodinitrobenzene followed by complete hydrolysis of the peptide.

More recently, fluorescent compounds such as dansyl chloride or dabsyl chloride are used because of their higher sensitivity.

The N-terminal aa is the only modified aa and it is identified by chromatography.

The peptide is completely hydrolyzed and cannot be reused

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

The next cycle releases residue 2. It is possible to identify ~50 aa from each sample by this method

MS Procedures for Sequence IDs

MS Procedures for Sequence IDs

Proteins with disulfide linkages

Disulfides are reduced using DTT

-SH groups are blocked by treatment with iodoacetate to form carboxymethylated derivatives

Specific Cleavage of Polypeptides

Proteins larger than 50 aa are first hydrolyzed into shorter peptides

Chemical or enzymatic methods hydrolyze proteins at specific sites

Peptides are separated by chromatography

Peptides generated by 2 or more cleavage methods are each sequenced separately.

Sequences of individual peptides are overlapped together to deduce the entire protein sequence

Protein Sequencing Example Method 1 (Trypsin): ser-glu-phe-his-lys ala-ile-cys-asp-tyr-thr-ala gly-leu-pro-arg Method 2 (staphylococcal protease): gly-leu-pro-arg-ser-glu phe-his-lys-ala-ile-cys-asp tyr-thr-ala Overall protein sequence: Gly-leu-pro-arg-ser-glu-phe-his-lys-ala-ile-cys-asp-tyr-thr-ala

Protein Synthesis by Bruce Merrifield Method

Slide Number 1Amino Acids, Peptides and ProteinsAmino AcidsAmpholytesGeneral Structure of Amino AcidsClassification of Amino AcidsAmino Acid R-group ClassificationSlide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Ionization of Amino AcidsSlide Number 14Slide Number 15Slide Number 16Slide Number 17pKa values depend on chemical environmentTitration Curve for GlycineCalculation of pI for GlycineTitration Curve for HistidineSlide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Protein Biochemistry MethodsProtein PurificationSlide Number 41Slide Number 42Slide Number 43SolubilitySizeSlide Number 46Size-Exclusion ChromatographyChargeBinding AffinitySlide Number 50HPLCElectrophoresisGel ElectrophoresisDetermination of M. Wt. of ProteinTypes of ElectrophoresisIsoelectric Focusing can be Used to Determine the pI of a ProteinIsoelectric Focusing and SDS-PAGE are Combined in 2D ElectrophoresisPeptide SynthesisSlide Number 59ProteinsProtein SequenceThe Peptide BondSlide Number 63Ionization of Peptides & Net chargeSlide Number 65Disulfide LinkagesSlide Number 67The ProteomeProtein Mass and ConcentrationSlide Number 70Slide Number 71Amino Acid CompositionAmino Acid AnalysisIdentification of amino-terminal residueIdentification of N-terminal amino acidSlide Number 76Slide Number 77Slide Number 78Slide Number 79Proteins with disulfide linkagesSpecific Cleavage of PolypeptidesSlide Number 82Slide Number 83Protein Sequencing ExampleProtein Synthesis by Bruce Merrifield MethodSlide Number 86