Amino Acids and Peptides Andy Howard Introductory Biochemistry, Fall 2008 IIT

Amino Acids and PeptidesAmino Acids and Peptides

Andy Howard

Introductory Biochemistry, Fall 2008IIT

08/28/08 Biochemistry: Amino Acids

p. 2 of 89

Here’s the rest of the Here’s the rest of the thermodynamics lecture…thermodynamics lecture…

We didn’t quite finish that, so here’s the part that we didn’t get to.

I’ll also offer some help in getting the homework done.

Then we’ll move on to today’s topic, which is amino acids and peptides.


p. 3 of 89

Free energy as a source of Free energy as a source of workwork

Change in free energy indicates that the reaction could be used to perform useful work

If Go < 0, we can do workIf Go > 0, we need to do work to

make the reaction occur


p. 4 of 89

What kind of work?What kind of work?

Movement (flagella, muscles) Chemical work:

– Transport molecules against concentration gradients

– Transport ions against potential gradients

To drive otherwise endergonic reactions– by direct coupling of reactions– by depletion of products


p. 5 of 89

Coupled reactionsCoupled reactions

Often a single enzyme catalyzes two reactions, shoving them together:reaction 1: A B Go

1 < 0 reaction 2: C D Go

2 > 0

• Coupled reaction:A + C B + D: Go

C = Go1 + Go

2

• If GoC < 0,

then reaction 1 is driving reaction 2!


p. 6 of 89

How else can we win?How else can we win?

Concentration of product may play a role As we’ll discuss in a moment, the actual free

energy depends on Go and on concentration of products and reactants

So if the first reaction withdraws product of reaction B away,that drives the equilibrium of reaction 2 to the right


p. 7 of 89

QuantitationQuantitation

Concentration affects GG is the actual determiner of

spontaneity:G < 0 means the reaction will proceed

from left to rightG > 0 means the reaction will proceed

from right to left


p. 8 of 89

How does How does GG relate to relate to GGoo’?’?

We’ll look at this in more detail around mid-semester;

But here’s the equation:G = Go’ + RT lnKeq

G = Go’ + RT ln[products]/[reactants]For a simple reaction A B:

G = Go’ + RT ln[B]/[A]For A + C B + D:

G = Go’ + RT ln([B][D])/([A][C])


p. 9 of 89

What does that mean?What does that mean?

It means that if the concentration of products is high and the concentration of reactants is low, equilibrium will be shifted leftward;

If the concentration of products is low and the concentration of reactants is high, equilibrium will be shifted rightward


p. 10 of 89

Adenosine TriphosphateAdenosine Triphosphate

ATP readily available in cells Derived from catabolic reactions Contains two high-energy phosphate

bonds that can be hydrolyzed to release energy: O O-

|| |(AMP)-O~P-O~P-O-

| || O- O


p. 11 of 89

Hydrolysis of ATPHydrolysis of ATP

Hydrolysis at the rightmost high-energy bond:ATP + H2O ADP + Pi

Go = -33kJ/mol• Hydrolysis of middle bond:

ATP + H2O AMP + PPi

Go = -33kJ/mol• BUT PPi 2 Pi, Go = -33 kJ/mol• So, appropriately coupled,

we get twice as much!


p. 12 of 89

ATP as energy currencyATP as energy currency

Any time we wish to drive a reaction that has

Go < +30 kJ/mol, we can couple it to ATP hydrolysis and come out ahead

If the reaction we want hasGo < +60 kJ/mol, we can couple it toATP AMP and come out ahead

So ATP is a convenient source of energy — an energy currency for the cell


p. 13 of 89

Coin analogyCoin analogy

Think of store of ATPas a roll of quarters

Vendors don’t give changeUse one quarter for some reactions, two

for othersInefficient for buying $0.35 items


p. 14 of 89

Other high-energy compoundsOther high-energy compounds

Creatine phosphate: ~ $0.40Phosphoenolpyruvate: ~ $0.35So for some reactions, they’re more

efficient than ATP


p. 15 of 89

Dependence on ConcentrationDependence on Concentration

Actual G of a reaction is related to the concentrations / activities of products and reactants: G = Go + RT ln [products]/[reactants]

• If all products and reactants are at 1M, then the second term drops away; that’s why we describe Go as the standard free energy


p. 16 of 89

Is that realistic?Is that realistic?

No, but it doesn’t matter; as long as we can define the concentrations,we can correct for them

Often we can rig it so[products]/[reactants] = 1even if all the concentrations are small

Typically [ATP]/[ADP] > 1 so ATP coupling helps even more than 33 kJ/mol!


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How How doesdoes this matter? this matter?

Often coupled reactions involve withdrawal of a product from availability

If that happens, [product]/[reactant]shrinks, the second term becomes negative, and G < 0 even if Go > 0


p. 18 of 89

How to solve energy problems How to solve energy problems involving coupled equationsinvolving coupled equations

General principles:– If two equations are added, their

energetics add– An item that appears on the left and

right side of the combined equation can be cancelled

This is how you solve the homework problem!


p. 19 of 89

A bit more detailA bit more detail

Suppose we couple two equations:A + B C + D, Go’ = xC + F B + G, Go’ = y

The result is:A + B + C + F B + C + D + GorA + F D + G, Go’ = x + y

… since B and C appear on both sides


p. 20 of 89

What do we mean by What do we mean by hydrolysis?hydrolysis?

It simply means a reaction with waterTypically involves cleaving a bond:U + H2O V + W

is described as hydrolysis of Uto yield V and W


p. 21 of 89

Phew. We’re done with Phew. We’re done with thermodynamics… for now!thermodynamics… for now!

We’ll come back to this after from time to time in the semester

We’ll cover kinetics in some detail when we discuss enzyme dynamics and mechanisms


p. 22 of 89

Let’s begin, chemically!Let’s begin, chemically!

Amino acids are important on their own and as building blocks

We need to start somewhere:– Proteins are made up of amino acids– Free amino acids and peptides play

significant roles in cells, even though their resting concentrations are low

– We’ll build from small to large


p. 23 of 89

PlansPlans

iClicker stuff Acid-base

equilibrium Amino acid

structures Chirality Acid/base

chemistry

Side-chain reactivity

Peptides and proteins

Side-chain reactivity in context

Disulfides


p. 24 of 89

iClicker quiz!iClicker quiz!

1. The correct form of the free energy equation is generally given as:– (a) H = G - TS– (b) PV = nRT– (c) G = H - TS– (d) S = H - G– (e) none of the above

(20 seconds for this one)


p. 25 of 89

iClicker quiz, problem 2iClicker quiz, problem 2

2. Suppose a reaction is at equilibrium with H = -6 kJ mol-1 andS = -0.02 kJ mol-1K-1.Calculate the temperature.– (a) 250K– (b) 280K– (c) 300K– (d) 310K– (e) 340K

45 seconds for this one


p. 26 of 89

iClicker quiz, problem 3iClicker quiz, problem 3

3. Suppose the reaction AB is endergonic with Go = 37 kJ/mol. What would be a suitable exergonic reaction to couple this reaction to in order to drive it to the right?– (a) hydrolysis of ATP to AMP + PPi

– (b) hydrolysis of glucose-1-phosphate– (c) hydrolysis of pyrophosphate– (d) none of the above

30 seconds for this one


p. 27 of 89

That’s the end of this part That’s the end of this part of your iClicker quiz!of your iClicker quiz!

Note that the scores don’t make much difference to your final grade, but being present does matter somewhat

Two more questions later in the lecture


p. 28 of 89

Acid-Base EquilibriumAcid-Base Equilibrium

In aqueous solution, the concentration of hydronium and hydroxide ions is nonzero

Define:– pH -log10[H+]– pOH -log10[OH-]

Product [H+][OH-] = 10-14 M2 (+/-) So pH + pOH = 14 Neutral pH: [H+] = [OH-] = 10-7:

pH = pOH = 7.


p. 29 of 89

So what’s the equilibrium So what’s the equilibrium constant for this reaction?constant for this reaction?

Note that the equation isH2O H+ + OH-

Therefore keq = [H+][OH-] / [H2O]But we just said that

[H+] = [OH-] = 10-7MWe also know that [H2O] = 55.5M

(= (1000 g / L )/(18 g/mole))So keq = (10-7M)2/55.5M = 1.8 * 10-16M


p. 30 of 89

Henderson-Hasselbalch Henderson-Hasselbalch EquationEquation

If ionizable solutes are present, their ionization will depend on pH

Assume a weak acid HA H+ + A-

such that the ionization equilibrium constant is Ka = [A-][H+] / [HA]

Define pKa -log10Ka

Then pH = pKa + log10([A-]/[HA])


p. 31 of 89

The Derivation is Trivial!The Derivation is Trivial!

Ho hum:pKa = -log([A-][H+]/[HA])

= -log([A-]/[HA]) - log([H+])= -log([A-]/[HA]) + pH

Therefore pH = pKa + log([A-]/[HA])

Often writtenpH = pKa + log([base]/[acid])


p. 32 of 89

How do we use this?How do we use this?

Often we’re interested in calculating [base]/[acid] for a dilute solute

Clearly if we can calculate log([base]/[acid]) = pH - pKa

then you can determine[base]/[acid] = 10(pH - pKa)

A lot of amino acid properties are expressed in these terms

It’s relevant to other biological acids and bases too, like lactate and oleate


p. 33 of 89

Reading recommendationsReading recommendations

If the material on ionization of weak acids isn’t pure review for you, I strongly encourage you to read the relevant sections of chapter 2 in Garrett & Grisham

We won’t go over this material in detail in class because it should be review, but you do need to know it!


p. 34 of 89

So: let’s look at amino acidsSo: let’s look at amino acids

The building blocks of proteins are of the form H3N+-CHR-COO-;these are -amino acids.

But there are others,e.g. beta-alanine:H3N+-CH2-CH2-COO-


p. 35 of 89

These are zwitterionsThese are zwitterions

Over a broad range of pH:– the amino end is protonated and is therefore

positively charged– the carboxyl end is not protonated and is

therefore negatively charged

Therefore both ends are charged Free -amino acids are therefore highly

soluble, even if the side chain is apolar


p. 36 of 89

At low and high pH:At low and high pH:

At low pH, the carboxyl end is protonated

At high pH, the amino end is deprotonated

These are molecules with net charges


p. 37 of 89

Identities of the R groupsIdentities of the R groups

Nineteen of the twenty ribosomally encoded amino acids fit this form

The only variation is in the identity of the R group (the side chain extending off the alpha carbon)

Complexity ranging from glycine (R=H) to tryptophan (R=-CH2-indole)


p. 38 of 89

Let’s learn the amino acids.Let’s learn the amino acids.

We’ll walk through the list of 20, one or two at a time

We’ll begin with proline because it’s weird

Then we’ll go through them sequentiallyYou do need to memorize these, both

actively and passively


p. 39 of 89

Special case: prolineSpecial case: proline

Proline isn’t an amino acid: it’s an imino acid

Hindered rotation around bond between amine N and alpha carbon is important to its properties

Tends to abolish helicity because of that hindered rotation


p. 40 of 89

The simplest amino acidsThe simplest amino acids

Glycine

AlanineCN+HHHHCCOO-HHH

CN+HHHHCOO-H

methyl


p. 41 of 89

CN+HHHHCCOO-CCHHHHCHHHHH

CN+HHHHCCOO-CHCHHHHCHHHH

Branched-chain aliphatic aasBranched-chain aliphatic aas

Valine

Isoleucine

Leucine

CN+HHHHCCOO-CCHHHHHHH

isopropyl


p. 42 of 89

Hydroxylated, polar amino acidsHydroxylated, polar amino acids

Serine Threonine

CN+HHHHCCOO-OHHH

CN+HHHHCCOO-OCHHHHH

hydroxyl


p. 43 of 89

Amino acids with carboxylate Amino acids with carboxylate side chainsside chains

Aspartate Glutamate

CN+HHHHCCOO-CHHO-O

CN+HHHHCCOO-HHCHHCO-O

carboxylate

methylene


p. 44 of 89

Amino Acids with amide side Amino Acids with amide side chainschains

asparagine glutamineCN+HHHHCCOO-HHCHHCNOHH

CN+HHHHCCOO-HHCNOHH

Note: these are uncharged!

amide


p. 45 of 89

Sulfur-containing amino acidsSulfur-containing amino acids

Cysteine Methionine

CN+HHHHCCOO-HHSH

CN+HHHHCCOO-HHSCCHHHHH

sulfhydryl


p. 46 of 89

Positively charged side chainsPositively charged side chains

Lysine Arginine

CN+HHHHCCOO-HHCCCHHHHHHN+HHH CN+HHHHCCOO-HHCNCHHHHHCNN+HHHH

Guani-dinium


p. 47 of 89

Aromatic Amino AcidsAromatic Amino Acids

Phenylalanine TyrosineCN+HHHHCCOO-HHCCCCCCHHHHH

CN+HHHHCCOO-HHCCCCCCHHHHOH

phenyl


p. 48 of 89

Histidine: a special caseHistidine: a special case

Histidine

imidazole


p. 49 of 89

Tryptophan: the biggest of allTryptophan: the biggest of all

TryptophanCN+HHHHCCOO-HHCCCNCCHHCCHHHH

indole


p. 50 of 89

ChiralityChirality

Remember:any carbon with four non-identical substituents will be chiral

Every amino acid except glycine is chiral at its alpha carbon

Two amino acids (ile and thr) have a second chiral carbon: C


p. 51 of 89

All have the same handedness at the alpha carbon

The opposite handedness gives you a D-amino acid– There are D-amino acids in many organisms– Bacteria incorporate them into structures of their

cell walls– Makes those structures resistant to standard

proteolytic enzymes, which only attack amino acids with L specificity

Ribosomally encoded amino Ribosomally encoded amino acids are L-amino acidsacids are L-amino acids


p. 52 of 89

The CORN mnemonicThe CORN mnemonicfor L-amino acidsfor L-amino acids

Imagine you’re looking from the alpha hydrogen to the alpha carbon

The substituents are, clockwise:C=O, R, N:


p. 53 of 89

Abbreviations for the amino Abbreviations for the amino acidsacids

3-letter and one-letter codes exist– All the 3-letter codes are logical– Most of the 1-letter codes are too

6 unused letters, obviously– U used for selenocysteine– O used for pyrrollysine– B,J,Z are used for ambiguous cases:

B is asp/asn, J is ile/leu, Z is glu/gln– X for “totally unknown”

http://www.chem.qmul.ac.uk/iupac/AminoAcid/A2021.html

CN+HHHHCOO-CHHSeH


p. 54 of 89

Letters A-F: acid-base propertiesLetters A-F: acid-base properties

AminoAcid

Side-chain

3-lettabbr.

1-let

pKa,COO-

pKa, NH3

+

alanine CH3 ala A 2.4 9.9

* asx Bcysteine CH2SH cys C 1.9 10.7aspartate CH2COO- asp D 2.0 9.9glutamate (CH2)2COO- glu E 2.1 9.5phenyl-alanine

CH2-phe phe F 2.2 9.3


p. 55 of 89

Letters G-LLetters G-L

AminoAcid

Side-chain

3-lettabbr.

1-let

pKa,COO-

pKa, NH3

+

glycine H gly G 2.4 9.8histidine -CH2-

imidazolehis H 1.8 9.3

isoleucine CH(Me)Et ile I 2.3 9.8

Ile/leu * lex? J 2.3 9.7-9.8lysine (CH2)4NH3

+ lys K 2.2 9.1leucine CH2CHMe2 leu L 2.3 9.7


p. 56 of 89

Letters M-SLetters M-S

methionine (CH2)2-S-Me met M 2.1 9.3

asparagine CH2-CONH2 asn N 2.1 8.7

pyrrol-lysine

see above pyl O 2.2 9.1

proline (CH2)3CH (cyc) pro P 2.0 10.6glutamine (CH2)2CONH2 gln Q 2.2 9.1

arginine (CH2)3-guanidinium

arg R 1.8 9.0

serine CH2OH ser S 2.2 9.2


p. 57 of 89

Letters T-ZLetters T-Z

threonine CH(Me)OH thr T 2.1 9.1

seleno-cysteine

CH2SeH Sec U 1.9 10.7

valine CH(Me)2 val V 2.3 9.7tryptophan CH2-indole trp W 2.5 9.4unknown Xaa X

tyrosine CH2-Phe-OH tyr Y 2.2 9.2

glu/gln (CH2)2-COX glx Z


p. 58 of 89

Remembering the abbreviationsRemembering the abbreviations

A, C, G, H, I, L, M, P, S, T, V easy F: phenylalanine sounds like an F R: talk like a pirate D,E similar and they’re adjacent N: contains a nitrogen W: say tryptophan with a lisp Y: second letter is a Y You’re on your own for K,O,Q,J,B,Z,U,X


p. 59 of 89

Do you need to memorize these Do you need to memorize these structures?structures?

Yes, for the 20 major ones(not B, J, O, U, X, Z)

The only other complex structures I’ll ask you to memorize are:– DNA, RNA bases

– Ribose

– Cholesterol

– A few others I won’t enumerate right now.


p. 60 of 89

How hard is it to How hard is it to memorize the structures?memorize the structures?

Very easy: G, A, S, C, VRelatively easy: F, Y, D, E, N, QHarder: I, K, L, M, P, THardest: H, R, WAgain, I’m not asking you to memorize

the one-letter codes, but they do make life a lot easier.


p. 61 of 89

What amino acids are in ELVIS?What amino acids are in ELVIS?

(a) asp - lys - val - ile - ser(b) asn - lys - val - ile - ser(c) glu - leu - val - ile - ser(d) glu - lys - val - ile - ser(e) Thank you very much.

(25 seconds)


p. 62 of 89

Main-chain acid-base chemistryMain-chain acid-base chemistry

Deprotonating the amine group: H3N+-CHR-COO- + OH- H2N-CHR-COO- + H2O

Protonating the carboxylate:H3N+-CHR-COO- + H+ H3N+-CHR-COOH

Equilibrium far to the left at neutral pH First equation has Ka=1 around pH 9 Second equation has Ka=1 around pH 2


p. 63 of 89

Why does pWhy does pKKaa depend on the depend on the

side chain?side chain? Opportunities for hydrogen bonding or

other ionic interactions stabilize some charges more than others

More variability in the amino terminus, i.e. the pKa of the carboxylate group doesn’t depend as much on R as the pKa of the amine group


p. 64 of 89

How do we relate pHow do we relate pKKaa to to

percentage ionization?percentage ionization?Derivable from Henderson-Hasselbalch

equationIf pH = pKa, half-ionized

One unit below:– 90% at more positive charge state,– 10% at less + charge state

One unit above: 10% / 90%


p. 65 of 89

Don’t fall into the trap!Don’t fall into the trap!

Ionization of leucine:pH 1.3 2.3 3.3 8.7 9.7 10.7

%+ve 90 50 10 0 0 0

% neutral 10 50 90 90 50 10

%-ve 0 0 0 10 50 90

Main species

NH3+-

CHR-COOH

NH3+C

HR-COO-

NH3+

CHR-COO-

NH2-

CHR-COO-


p. 66 of 89

Side-chain reactivitySide-chain reactivity

Not all the chemical reactivity of amino acids involves the main-chain amino and carboxyl groups

Side chains can participate in reactions:– Acid-base reactions– Other reactions

In proteins and peptides,the side-chain reactivity is more important because the main chain is locked up!


p. 67 of 89

Acid-base reactivity Acid-base reactivity on side chainson side chains

Asp, glu: side-chain COO-:– Asp sidechain pKa = 3.9

– Glu sidechain pKa = 4.1

Lys, arg: side-chain nitrogen:– Lys sidechain –NH3

+ pKa = 10.5

– Arg sidechain =NH2+ pKa = 12.5


p. 68 of 89

Acid-base reactivity in histidineAcid-base reactivity in histidine

It’s easy to protonate and deprotonate the imidazole group


p. 69 of 89

Cysteine: a special caseCysteine: a special case

The sulfur is surprisingly ionizableWithin proteins it often remains

unionized even at higher pHCN+HHHHCOO-CHHSHCN+HHHHCOO-CHHS-H+H+pKa = 8.4


p. 70 of 89

Ionizing hydroxylsIonizing hydroxyls

X–O–H X–O- + H+ Tyrosine is easy, ser and thr hard:

– Tyr pKa = 10.5

– Ser, Thr pKa = ~13

Difference due to resonance stabilization of phenolate ion:


p. 71 of 89

Resonance-stabilized ionResonance-stabilized ion


p. 72 of 89

Other side-chain reactionsOther side-chain reactions

Little activity in hydrophobic amino acids other than van der Waals

Sulfurs (especially in cysteines) can be oxidized to sulfates, sulfites, …

Nitrogens in his can covalently bond to various ligands

Hydroxyls can form ethers, estersSalt bridges (e.g. lys - asp)


p. 73 of 89

PhosphorylationPhosphorylation

ATP donates terminal phosphate to side-chain hydroxyl of ser, thr, tyr

ATP + Ser-OH ADP + Ser-O-(P)Often involved in activating or

inactivating enzymesUnder careful control of enzymes called

kinases and phosphatases


p. 74 of 89

Peptides and proteinsPeptides and proteins

Peptides are oligomers of amino acidsProteins are polymersDividing line is a little vague:

~ 50-80 aa.All are created, both formally and in

practice, by stepwise polymerizationWater eliminated at each step


p. 75 of 89

Growth of oligo- or polypeptideGrowth of oligo- or polypeptide

CN+HHHHCOO-R1CN+HHHCOO-+H2OCN+HHHHCOR1CNCOO-HR2HR2H


p. 76 of 89

The peptide bondThe peptide bond

The amide bond between two successive amino acids is known as a peptide bond

The C-N bond between the first amino acid’s carbonyl carbon and the second amino acid’s amine nitrogen has some double bond character


p. 77 of 89

Double-bond character of Double-bond character of peptidepeptide

CN+HHHHCOR1NCHR2HCOCN+HHHHCO-R1N+CHR2HCO


p. 78 of 89

The result: planarity!The result: planarity!

This partial double bond character means the nitrogen is sp2 hybridized

Six atoms must lie in a single plane:– First amino acid’s alpha carbon– Carbonyl carbon– Carbonyl oxygen– Second amino acid’s amide nitrogen– Amide hydrogen– Second amino acid’s alpha carbon


p. 79 of 89

Rotations and flexibilityRotations and flexibility

Planarity implies = 180, where is the rotation angle about N-C bond

Free rotations are possible about N-C and C-C bonds– Define = rotation about N-C– Define = rotation about C-C

We can characterize main-chain conformations according to ,


p. 80 of 89

Ramachandran anglesRamachandran angles

G.N. Ramachandran


p. 81 of 89

Preferred Values of Preferred Values of and and

Steric hindrance makes some values unlikely

Specific values are characteristic of particular types of secondary structure

Most structures with forbidden values of and turn out to be errors


p. 82 of 89

How far from 180º can How far from 180º can vary? vary?

Remember what we said about the partial double bond character of the C-N main-chain bond

That imposes planarityIn practice it rarely varies by more than

a few degrees from 180º.


p. 83 of 89

Ramachandran plotRamachandran plot

Cf. figures in G&G If you submit a

structure to the PDB with Ramachandran angles far from the yellow regions, be prepared to justify them!


p. 84 of 89

How are oligo- and polypeptides How are oligo- and polypeptides synthesized?synthesized?

Formation of the peptide linkages occurs in the ribosome under careful enzymatic control

Polymerization is endergonic and requires energy in the form of GTP (like ATP, only with guanosine):

GTP + n-length-peptide + amino acid GDP + Pi + (n+1)-length peptide


p. 85 of 89

What happens at the ends?What happens at the ends?

Usually there’s a free amino end and a free carboxyl end:

H3N+-CHR-CO-(peptide)n-NH-COO-

Cyclic peptides do occurCyclization doesn’t happen at the

ribosome: it involves a separate, enzymatic step.


p. 86 of 89

Reactivity in peptides & proteinsReactivity in peptides & proteins

Main-chain acid-base reactivity unavailable except on the ends

Side-chain reactivity available but with slightly modified pKas.

Terminal main-chain pKavalues modified too

Environment of protein side chain is often hydrophobic, unlike free amino acid side chain


p. 87 of 89

What’s the net charge in ELVISWhat’s the net charge in ELVISat pH 7?at pH 7?

(a) 0(b) +1(c) -1(d) +2(e) -2


p. 88 of 89

DisulfidesDisulfides

In oxidizing environments, two neighboring cysteine residues can react with an oxidizing agent to form a covalent bond between the side chains

CHHSHCHHSH+(1/2)O2SSHCHHCHH2O


p. 89 of 89

What could this do?What could this do?

Can bring portions of a protein that are distant in amino acid sequence into close proximity with one another

This can influence protein stability

Documents

Amino Acids and Peptides Andy Howard Introductory Biochemistry, Fall 2008 IIT