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Carbohydrates

Supplemental Slides

Biochem 5613

Figures: Lehninger Principles of Biochemistry 6th Ed., Nelson & Cox

Essentials of Biochemistry, 3rd Ed. OSU Custom Edition

Voet & Voet, Biochemistry 3rd Ed.

Voet, Voet & Pratt, Fundamentals of Biochemistry 2nd Ed.

Berg, Tymoczko, & Stryer, Biochemistry 2001

Glycoproteins

N-linked glycoproteins: Large, heterogeneous oligosaccharides linked to asparagine side chains

O-linked glycoproteins: Smaller carbohydrates (usually 1-5 saccharides) linked to hydroxyl groups of Ser and/or

Thr side chains

What is wrong with

these structures?

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Glycoproteins Carbohydrate may be a large amount of the glycoprotein mass

Carbohydrate is hydrophilic; usually on

surface of a folded protein

Protein is dark grey; carbohydrate groups are

in light grey

(a) Hemagglutinin

(b) CD2

(c) RNase B

Imperiali & OConnor, 1999

Glycoproteins

Oligosaccharide substituents of proteins are highly dynamic

RNase B protein is linked

to a heptasaccharide

substituent

Model displaying all permitted structures of oligosaccharide in

yellow and

superimposed on

protein core to

demonstrate freedom

of motion

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N-Linked Protein Glycosylation

Oligosaccharyl transferase (OST) is a 9 subunit, membrane bound protein complex that covalently attaches a conserved

oligosaccharide to the protein substrate

The OST enzyme is required to activate the Asn amide to allow it to serve as a good nucleophile. Why?

OST: Domain Structure

Hua et al (2008) Structure

The nine domains of OST serve different roles:

Stt3p: Catalytic domain

Stt3p, Ost1p, Ost2p, Wbp1, and Swp1p essential for function

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N-Linked Protein Glycosylation

N-linked glycosylation occurs in the endoplasmic reticulum (ER) and is co-translational; may assist protein folding

N-Linked Protein Glycosylation

Transferred tetradecasaccharide is built up unit-by-unit by glycotransferases (enzymes that transfer sugar groups

from diphosphate donors)

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N-Linked Protein Glycosylation

Transferred carbohydrate is a conserved oligosaccharide:

Glc3Man9GlcNac2

GlcNAcGlcNAc

Man

Man

Man

Glc

GlcGlc

Man

Man

Man

Man

Man

Man

In light grey: 5-sugar conserved core of all N-linked glycoproteins

Maturation: Glycosidases and glycotransferases work together

to modify the final glycan

N-Linked Glycosylation

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O-Linked Glycosylation

O-linked glycosylation of proteins occurs in the Golgi apparatus

Sugar groups form a glycosidic bond with the side chain hydroxyl group of Ser or Thr

One major purpose is immunological identification:

For example: Mucins are heavily glycosylated

proteins which are a significant component of mucus

Many cell-surface proteins are O-glycosylated

O-Linked Glycosylation

Core is not as conserved as in N-linked glycosylation

Gal-GalNAc-Ser/Thris the most commonly

observed core

However, other cores are observed

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Protein Sequencing

Chapter 3.4

Biochem 5613

Figures: Lehninger Principles of Biochemistry 6th Ed., Nelson & Cox

Essentials of Biochemistry, 3rd Ed. OSU Custom Edition

Voet & Voet, Biochemistry 3rd Ed.

Voet, Voet & Pratt, Fundamentals of Biochemistry 2nd Ed.

Berg, Tymoczko, & Stryer, Biochemistry 2001

Amino Acid Analysis

Historically, protein content was determined by breaking the

protein into individual amino acids and quantitating the products:

Resuspend protein in 6 M HCl and boil overnight

Amino acid analysis provides information about content,

NOT the sequence

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AAA: Acid Hydrolysis

Partial degradation of Cys, Ser, Thr, and complete degradation

of Trp; do NOT get accurate numbers for these amino acids!

Complete conversion

H3N+ COOH

Asn -> Asp

Gln -> Glu

H3N+ COOH

HO

Ser, Thr

Dehydrate

Dehydroalanine

How do we determine a protein sequence?

1) How many chains?

Total amino acid content N-terminal amino acid identification

2) Break protein down into peptide fragments

Disulfide bonds Sequence-specific digestion

Chemical Enzymatic (proteases)

3) Analyze peptide sequences

Edman degradation (Historically) Mass spectrometry (Modern Method)

4) Combine all of this into a primary sequence

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Insulin

First sequenced protein (Sanger)

Two chains complicates N-terminal identification

Three disulfide bonds

Protein Sequencing: Disulfides

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Protein Sequencing

Proteins are typically cleaved in multiple ways to allow the generation of overlapping peptide fragments

Fragmentation of Proteins

Determining the sequence of very large proteins is complicated It is common to break a protein into smaller peptide segments

to allow for easier sequencing

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Cyanogen BromideCleavage at methionine residues

Methyl thiocyanate

leaving group

Hydrolysis

1)

2)

3)

4)

Protease Cleavage: Trypsin

Trypsin cleaves after lysine and arginine (red)

AA C-terminal to cleavage site can be anything exceptproline (blue)

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Protease Cleavage: Chymotrypsin

Chymotrypsin cleaves peptide bonds C-terminal to large, hydrophobic amino acid side chains

High rate of cleavage: C-terminal to Phe, Tyr, Trp

VERY slight non-specific cleavage at other hydrophobic residues

Protease Terminology

Peptidase: breaks peptide bond

Exopeptidase: breaks peptide bond on a terminal amino acid

(exterior peptide bond)

Endopeptidase: breaks peptide bond somewhere in the

middle of (inside) the sequence

Carboxypeptidase:

Exopeptidase that hydrolyzes peptide bond at the C-terminus

(carboxyl terminus)

Aminopeptidase:

Exopeptidase that hydrolyzes peptide bond of the N-terminal

residue (amino terminus)

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Protease Specificities

Endo Lys-C

Endo Glu-C

Endo Asp-N

Name based on

specificity and

cleaved bond

Endo Lys-C

Endo Glu-C

Endo Asp-N

Trypsin

Chymotrypsin

Cyanogen Bromide

Carboxypeptidase A

Aminopeptidase

Where would each of the following species cleave the peptide below?

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Peptide SequencingEdman Degradation

= Edmans reagent

Phenylisothiocyanate

Peptide Sequencing Edman Degradation

Peptides can undergo multiple cycles of Edman degradation to

identify entire sequence

Edman degradation can be automated: protein sequencers

can carry out approximately one

round per hour

Up to 50 amino acids can be sequenced fairly reliably before

small errors build up

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Modern Sequencing Methods:

Mass Spectrometry

Ion source Mass analyzer Detector

All mass spectrometers have a common organization

All separate based on mass to charge ratio

Electrospray Ionization (ESI)

Generates population of protein ions with many charge states

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ESI-MS: Mass reconstruction/deconvolution

Mass to charge

ratio observed

Positive charge is

generated by

protonation, so

add H+ for each

charge, each H+

adds 1 Da

m + z

zObserved =

MALDI: Matrix Assisted Laser Desorption Ionization

HCCA

Several different

matrices are

commonly used:

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MALDI: Fewer Charge States

Example: Mix of 4 proteins; observed [M+H]+, [M+2H]2+

H4

11237

H2B

13496

H2A

13951

H3(pT118)

15354

m/Z

ESI vs MALDI

Electrospray MS: Syringe full of protein/peptide solution

Ionize by concentrating charges in vacuum Highly charged spectrum Often connected to liquid chromatography systems

MALDI MS: Crystallize protein/peptide in matrix

Ionize using laser Tend to see the parent mass Simpler analysis of mixtures of ions

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Carboxypeptidase: Sequencing Exopeptidase

Great for sequencing based on remaining parent ion!

MALDI/Carboxypeptidase Sequencing

Figure from John J. Lennon, ABRF News, 1997

Time

series

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Tandem Mass Spectrometry for Sequencing

Any ionization technique

MALDI is common

MS/MS Sequencing