Bioch/BIMS 503 Lecture 1 Structure and Properties of Amino Acids and the Peptide

Backbone

August 26, 2008

Robert NakamotoMol. Physiology & Biol PhysicsTel: 982-0279, rkn3c@virginia.eduSnyder 380 (Fontaine)

Major topics –• Names, abbreviations, general structure of amino acids

• Amino acid chemical classes (polar, hydrophobic, acidic, basic, aromatic, S-containing)

• Amino acid structural classes/affinity

• Amino acid evolutionary classes• pK - Henderson-Hasselbach equation• Structure of the peptide bond• Proteomics – MS protein sequencing

Further Reading –• Lehninger, Chapter 3 pp 75-86, 102-106

• MvHA, Chapter 5, pp 126-142• Brandon & Tooze, Ch. 1• Aebersold R, Mann M. (2003) Mass spectrometry-based proteomics. Nature. 422:198-207 PMID:12634793

Hierarchies of protein structure

secondarystructure

super-secondarystructure

ternaryfold

ternary structure

-helix -strand

4-helix bundles

Rossman fold

-meander Greek key

primarystructure

MVDFYYLPGSSPCRSVIMTAKAVGVELNKK

Does this structural hierarchy reflect the folding process?

Secondary structure first, or last?

Examples of protein folds and complexes:

Many bacterial toxin proteins undergo conformational changes that insert into host cell membrane: for example, Anthrax toxin protein/Protective protein

complex

From Santelli et al., 2004, Nature 430, 905-908

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Currently 10,340 protein fold families in Pfam[http://pfam.sanger.ac.uk/]

Pfam is a comprehensive collection of protein domains and families, represented as multiple sequence alignments and as profile hidden

Markov models. Generally does not include membrane proteins.

What defines the 3-dimensional fold of a protein?

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Structure and properties ofAmino-acids

Alanine Ala A Leucine Leu LArginine Arg R Lysine Lys KAsparagine Asn N Methionine Met MAspartic acid Asp DPhenylalanine Phe FCysteine Cys C Proline Pro PGlutamine Gln Q Serine Ser SGlutamic acid Glu E Threonine

Thr TGlycine Gly G Tryptophan Trp WHistidine His H Tyrosine Tyr YIsoleucine Ile I Valine Val V

Asp/Asn Asx B Glu/Gln Glx Z

Amino-acid Chirality

L-glyceraldehyde D-glyceraldehyde

Amino-acid Chiralitythe “CORN” rule

CO

R

N

H CC

R

N

O

O H

Figure 5.3: The amino acids found in proteins.

CYCLIC AMINO ACID

AROMATIC AMINO ACIDS

H

Leucine

Alanine

H

H

CO

R

N CO

R

N

Proline - a cyclic amino-acid

CO

R

N

H

CO

R

N

CO

R

N

H

CO

R

N

Pro

Ala

Classifications of amino-acids

• Abundance• Hydrophobicity• Mutability• Structural preference

• Charge properties

us.expasy.org/cgi-bin/protscale.pl

Molecular weight Number of codon(s) Bulkiness Polarity / Zimmerman Polarity / Grantham Refractivity Recognition factors Hphob. / Eisenberg et al. Hphob. OMH / Sweet et al. Hphob. / Hopp & Woods Hphob. / Kyte & Doolittle Hphob. / Manavalan et al. Hphob. / Abraham & Leo Hphob. / Black Hphob. / Bull & Breese Hphob. / Fauchere et al. Hphob. / Guy Hphob. / Janin Hphob. / Miyazawa et al. Hphob. / Rao & Argos Hphob. / Roseman Hphob. / Wolfenden et al. Hphob. / Welling & al Hphob. HPLC / Wilson & al Hphob. HPLC / Parker & al Hphob. HPLC pH3.4 / Cowan Hphob. HPLC pH7.5 / Cowan Hphob. / Rf mobility HPLC / HFBA retention HPLC / TFA retention HPLC / retention pH 2.1 HPLC / retention pH 7.4 % buried residues % accessible residues Hphob. / Chothia Hphob. / Rose & al Ratio hetero end/side Average area buried Average flexibility alpha-helix / Chou & Fasman beta-sheet / Chou & Fasman beta-turn / Chou & Fasman alpha-helix / Deleage & Roux beta-sheet / Deleage & Roux beta-turn / Deleage & Roux Coil / Deleage & Roux alpha-helix / Levitt beta-sheet / Levitt beta-turn / Levitt Total beta-strand Antiparallel beta-strand Parallel beta-strand A.A. composition A.A. comp. in Swiss-Prot Relative mutability

Amino acid frequencies in proteins

+ Ala A 0.0780Arg R 0.0512Asn N 0.0448Asp D 0.0536

- Cys C 0.0192Gln Q 0.0426

+ Glu E 0.0629+ Gly G 0.0737- His H 0.0219Ile I 0.0514

+ Leu L 0.0901Lys K 0.0574

- Met M 0.0224- Phe F 0.0385Pro P 0.0520

+ Ser S 0.0711Thr T 0.0584

- Trp W 0.0132- Tyr Y 0.0321+ Val V 0.0644

Amino acid Hydropathicity/Hydrophobicity

Arg:-4.5Lys:-3.9Asp:-3.5Glu:-3.5Gln:-3.5Asn:-3.5His:-3.2Pro:-1.6Tyr:-1.3Trp:-0.9Ser:-0.8Thr:-0.7Gly:-0.4Ala: 1.8Met: 1.9Cys: 2.5Phe: 2.8Leu: 3.8Val: 4.2Ile: 4.5

Arg: 3.0Lys: 3.0Asp: 3.0Glu: 3.0Ser: 0.3Gln: 0.2Asn: 0.2Pro: 0.0Gly: 0.0Thr:-0.4His:-0.5Ala:-0.5Cys:-1.0Met:-1.3Val:-1.5Leu:-1.8Ile:-1.8Tyr:-2.3Phe:-2.5Trp:-3.4

Hopp T.P., Woods K.R. (1981) PNAS. 78:3824-3828.

Kyte J., Doolittle R.F. (1982). J. Mol. Biol. 157:105-132

D. M. Engelman, T. A. Steitz, A. Goldman, (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 321

Arg:12.3Asp: 9.2Lys: 8.8Glu: 8.2Asn: 4.8Gln: 4.1His: 3.0Tyr: 0.7Pro: 0.2Ser:-0.6Gly:-1.0Thr:-1.2Ala:-1.6Trp:-1.9Cys:-2.0Val:-2.6Leu:-2.8Ile:-3.1Met:-3.4Phe:-3.7

Hopp/Woods

Kyte/Doolittle

GES

Amino-acid classes from evolution/mutation

GSTM1_HUMAN MPMILGYWDIRGLAHAIRLLLEYTDSSYEEKKYTMGDAPDYDRSQWLNEKFKLGLDGSTM2_HUMAN MPMTLGYWNIRGLAHSIRLLLEYTDSSYEEKKYTMGDAPDYDRSQWLNEKFKLGLDGSTM4_HUMAN MPMILGYWDIRGLAHAIRLLLEYTDSSYEEKKYTMGGAPDYDRSQWLNEKFKLGLDGSTM5_HUMAN MPMTLGYWDIRGLAHAIRLLLEYTDSSYVEKKYTMGDAPDYDRSQWLNEKFKLGLDGTM1_MOUSE MPMILGYWNVRGLTHPIRMLLEYTDSSYDEKRYTMGDAPDFDRSQWLNEKFKLGLDGTM2_MOUSE MPMTLGYWDIRGLAHAIRLLLEYTDTSYEDKKYTMGDAPDYDRSQWLSEKFKLGLDGTM3_MOUSE MPMTLGYWNTRGLTHSIRLLLEYTDSSYEEKRYVMGDAPNFDRSQWLSEKFNLGLDGTM4_MOUSE MSMVLGYWDIRGLAHAIRMLLEFTDTSYEEKRYICGEAPDYDRSQWLDVKFKLDLDGTM3_RABIT MPMTLGYWDVRGLALPIRMLLEYTDTSYEEKKYTMGDAPNYDQSKWLSEKFTLGLD

Given a set of (closely) related protein sequences...

… how often is one amino-acid replaced by another?

Relative mutability of amino acids (Ala=100)

Ala: 100.0 Arg: 65.0 Asn: 134.0 Asp: 106.0 Cys: 20.0 Gln: 93.0 Glu: 102.0 Gly: 49.0 His: 66.0 Ile: 96.0 Leu: 40.0 Lys: 56.0 Met: 94.0 Phe: 41.0 Pro: 56.0 Ser: 120.0 Thr: 97.0 Trp: 18.0 Tyr: 41.0 Val: 74.0

Dayhoff M.O., Schwartz R.M., Orcutt B.C.(1978) In "Atlas of Protein Sequence and Structure", Vol.5, Suppl.3

Mutation frequencies after 1% change X

100,000

A 98754R 30 98974N 23 19 98720D 42 8 269 98954C 11 22 7 2 99432Q 23 125 35 20 4 98955E 65 18 36 470 3 198 99055G 130 99 59 95 43 19 87 99350H 6 75 89 25 16 136 6 5 98864I 20 12 25 6 9 5 6 3 9 98729L 28 35 11 6 21 66 9 6 51 209 99330K 21 376 153 15 4 170 105 16 27 12 8 99100M 13 10 7 4 7 10 4 3 8 113 92 15 98818F 6 2 4 2 31 2 2 2 16 35 99 2 17 99360P 98 37 8 8 7 83 9 13 58 5 52 11 8 9 99270S 257 69 342 41 152 37 21 137 50 27 40 32 20 63 194 98556T 275 37 135 23 25 30 19 20 27 142 15 60 131 7 69 276 98665W 1 18 1 1 16 3 1 8 1 1 7 1 3 8 1 5 2 99686Y 3 6 22 15 67 8 2 3 182 10 8 3 6 171 3 20 7 23 99392V 194 12 11 20 41 13 29 31 8 627 118 9 212 41 15 25 74 17 11 98761 A R N D C Q E G H I L K M F P S T W Y V

Jones D.T., Taylor W.R. and Thornton J.M. (1992) CABIOS 8:275-282

The PAM250 matrixPAM: Point Accepted Mutation

Cys 12Ser 0 2Thr -2 1 3Pro -1 1 0 6Ala -2 1 1 1 2Gly -3 1 0 -1 1 5Asn -4 1 0 -1 0 0 2Asp -5 0 0 -1 0 1 2 4Glu -5 0 0 -1 0 0 1 3 4Gln -5 -1 -1 0 0 -1 1 2 2 4His -3 -1 -1 0 -1 -2 2 1 1 3 6Arg -4 0 -1 0 -2 -3 0 -1 -1 1 2 6Lys -5 0 0 -1 -1 -2 1 0 0 1 0 3 5Met -5 -2 -1 -2 -1 -3 -2 -3 -2 -1 -2 0 0 6Ile -2 -1 0 -2 -1 -3 -2 -2 -2 -2 -2 -2 -2 2 5Leu -6 -3 -2 -3 -2 -4 -3 -4 -3 -2 -2 -3 -3 4 2 6Val -2 -1 0 -1 0 -1 -2 -2 -2 -2 -2 -2 -2 2 4 2 4Phe -4 -3 -3 -5 -4 -5 -4 -6 -5 -5 -2 -4 -5 0 1 2 -1 9Tyr 0 -3 -3 -5 -3 -5 -2 -4 -4 -4 0 -4 -4 -2 -1 -1 -2 7 10Trp -8 -2 -5 -6 -6 -7 -4 -7 -7 -5 -3 2 -3 -4 -5 -2 -6 0 0 17 C S T P A G N D E Q H R K M I L V F Y W

Solvent Exposed Area (SEA)

http://www.cmbi.kun.nl/swift/future/aainfo/access.htm

Probability that a particular residue will be positioned in real proteins so that its solvent exposed area meets the particular criterion in the columns title.

The data for this table was calculated from data taken from 55 proteins in the Brookhaven data base, coming from 9 molecular families: globins, immunoglobins, cytochromes c, serine proteases, subtilisins, calcium binding proteins, acid proteases, toxins and virus capsid proteins. Red entries are found on the surface of a proteins on > 70% of occurrences and blue entries are found inside of a protein of < 20% of occurrences.

The only clear trend in this table is that some residues, such as R and K, locate themselves so that they have access to the solvent. The so-called hydrophobic residues, such as L and F, show no clear trend: they are found near the solvent as often as they are found buried.

> 30 A2 < 10 A230 >SEA

> 10 A2

S 0.70 0.20 0.10

T 0.71 0.16 0.13

A 0.48 0.35 0.17

G 0.51 0.36 0.13

P 0.78 0.13 0.09

C 0.32 0.54 0.14

D 0.81 0.09 0.10

E 0.93 0.04 0.03

Q 0.81 0.10 0.09

N 0.82 0.10 0.08

L 0.41 0.49 0.10

I 0.39 0.47 0.14

V 0.40 0.50 0.10

M 0.44 0.20 0.36

F 0.42 0.42 0.16

Y 0.67 0.20 0.13

W 0.49 0.44 0.07

K 0.93 0.02 0.05

R 0.84 0.05 0.11

H 0.66 0.19 0.15

Ionization of Amino Acids in water

For all amino acids, there are two modes of ionization depending on the pH of the aqueous medium: (1) uncharged at low pH, –1 at high pH (acid), or (2) +1 at low pH, uncharged at high pH (base).

From the Henderson-Hasselbalchequation:

90% or 99% of the functional group is deprotonated (or protonated) when the pH is 1 or 2 pH units above (below) the pK.

€

pH = pKa + log[base−]

[acid]

log[base−]

[acid]= pKa − pH

[base−]

[acid]=10pKa − pH

pK1=2.3

pK2=9.6

The ionic properties of amino acids reflect the ionization of the COO–, NH3

+, and R-groups

cationzwitterion

(net charge 0)anion

When subjected to changes in pH, amino acids change from the protonated form with net positive charge in strongly acidic solution to the unprotonated form with net negative charge in strongly basic solution. During this transition, the amino acid will pass through a state with no net charge. The pH at which this occurs is the isoelectric point or pI. pI

can be calculated from pKa

values. For zwitteronic and acidic amino acids,

pI = 1/2(pK1+pK2). For basic

amino acids, pI =

1/2(pK2+pK3).

Ionic characteristics of amino-acids

€

C22+

€

C1+

€

A−

€

Zw

€

[C1+]

€

[Zw]

pH=7.4

pK2=6.

0

pH=7.4

pK2=7.

0

pH=6.8

pK2=6.

0

3.8% 28.2% 13.6%

94.7% 70.4% 86.0%€

pI =1

2(pK2 + pK3) =

1

2(6.0 + 9.2) = 7.6

Overall, the aa in solution is positively charged at pH < pI

pK1=1.8

pK2=6.0

pK2=9.2

pKa values of common amino acids

Amino Acid -COOH pKa -NH3+ pKa R group

pKa

Alanine 2.4 9.7

Arginine 2.2 9.0 12.5

Asparagine 2.0 8.8

Aspartic Acid

2.1 9.8 3.9

Cysteine 1.7 10.8 8.3

Glutamic Acid

2.2 9.7 4.3

Glutamine 2.2 9.1

Glycine 2.3 9.6

Histidine 1.8 9.2 6.0

Isoleucine 2.4 9.7

Leucine 2.4 9.6

Lysine 2.2 9.0 10.5

Methionine 2.3 9.2

Phenylalanine

1.8 9.1

Proline 2.1 10.6

Serine 2.2 9.2 ~13

Threonine 2.4 10.4 ~13

Tryptophan 2.4 9.4

Tyrosine 2.2 9.1 10.1

Valine 2.3 9.6

The planar nature of the peptide bond

MvHA Fig. 5.12

MvHA Fig. 5.8

Limited rotation around the peptide bond – cis- and trans-

proline

The 19 amino-acids other than proline strongly prefer (>99.7%) to have the C–carbons in the trans- configuration. Proline shows a weaker preference, with about 5% of Xaa-Pro in the cis- configuration.

Pro

Strategies for Protein Sequencing (Proteomics)

• Classic “Edman” sequencing– PTC-conjugation to N-terminal amino-acid

– Cleave N-terminal peptide bond

– Identify PTH amino-acid

– Repeat 20 - 30 cycles

• Sequencing with Mass-Spectrometry– isolate protein (or use mixture of proteins)

– cleave with trypsin (proteins don’t “fly”)

– separate on HPLC– separate peptides in MS(1)

– fragment peptides in collision cell

– separate peptide fragments in MS(2)

Protein primary structure can be determined by chemical

methods and from gene sequences

Edman degradation

Time-of-flight mass spectrometry measures the mass of proteins and

peptides

http://www.healthsystem.virginia.edu/internet/biomolec/

Most protein analysis done by Electrospray Ionisation (ESI) or Matrix Assisted Laser Desorption Ionisation (MALDI)

Positive ESI-MS m/z spectrum of lysozyme.

Figure 1 Generic mass spectrometry (MS)-based proteomics experiment. The typical proteomics experiment consists of five stages. In stage 1, the proteins to be analysed are isolated from cell lysate or tissues by biochemical fractionation or affinity selection. This often includes a final step of one-dimensional gel electrophoresis, and defines the 'sub-proteome' to be analysed. MS of whole proteins is less sensitive than peptide MS and the mass of the intact protein by itself is insufficient for identification. Therefore, proteins are degraded enzymatically to peptides in stage 2, usually by trypsin, leading to peptides with C-terminally protonated amino acids, providing an advantage in subsequent peptide sequencing. In stage 3, the peptides are separated by one or more steps of high-pressure liquid chromatography in very fine capillaries and eluted into an electrospray ion source where they are nebulized in small, highly charged droplets. After evaporation, multiply protonated peptides enter the mass spectrometer and, in stage 4, a mass spectrum of the peptides eluting at this time point is taken (MS1 spectrum, or 'normal mass spectrum'). The computer generates a prioritized list of these peptides for fragmentation and a series of tandem mass spectrometric or 'MS/MS' experiments ensues (stage 5). These consist of isolation of a given peptide ion, fragmentation by energetic collision with gas, and recording of the tandem or MS/MS spectrum. The MS and MS/MS spectra are typically acquired for about one second each and stored for matching against protein sequence databases. The outcome of the experiment is the identity of the peptides and therefore the proteins making up the purified protein population.

Aebersold R, Mann M. (2003) Nature. 422:198

FIG. 3.   Tandem mass (MS/MS) spectra resulting

from analysis of a single spot on a 2D gel. The first

quadrupole selected a single mass-to-charge ratio ( m/z) of 687.2 (A) or 592.6 (B), while the collision cell was filled

with argon gas, and a voltage which caused the peptide to undergo fragmentation by CID was applied. The third quadrupole scanned the

mass range from 50 to 1,400  m/z. The computer program Sequest (8) was utilized to

match MS/MS spectra to amino acid sequence by

database searching. Both spectra matched peptides

from the same protein, S57593 (yeast hypothetical

protein YMR226C). Five other peptides from the same

analysis were matched to the

same protein.

Gygi SP, et al. (1999) Mol Cell Biol. 19:1720

Search human protein (International Protein Index) database

20242509 residues in 65082 sequences FASTS (4.00 July 2001 (ajm)) function [MD20 matrix (18:-29)] ktup: 1 join: 58, gap-pen: -12/-2, width: 16 Scan time: 13.183

The best scores are: initn init1 bits E(65082)IPI00015759.1|SP:Q07244|NP:NP_112552 Het ( 463) 523 218 523 133 7.2e-36 3 46IPI00063875.1|NP:NP_112553;NP_002131|TR: ( 464) 523 218 523 133 6.2e-36 3 46IPI00059339.2|XP:XP_062032|ENSENSP000002 ( 482) 330 135 330 67 4.1e-16 3 46IPI00076129.1|XP:XP_087643 similar to he ( 161) 188 188 188 50 7.7e-11 1 19

>>IPI00015759.1|SP:Q07244|NP:NP_112552 Heterogeneous nuc (463 aa) initn: 523 init1: 218 opt: 523 bits: 132.7 E(): 7.2e-36Smith-Waterman score: 523; 100.000% identity in 46 aa overlap (1-46:149-396) 10gi|108 LLIHQSLAGGIIGVK--------------- :::::::::::::::IPI000 ATSQLPLESDAVECLNYQHYKGSDFDCELRLLIHQSLAGGIIGVKGAKIKELRENTQTTI 120 130 140 150 160 170 20gi|108 -----------------------------IILDLISESPIK------------------- ::::::::::::IPI000 KLFQECCPHSTDRVVLIGGKPDRVVECIKIILDLISESPIKGRAQPYDPNFYDETYDYGG 180 190 200 210 220 230 30 40gi|108 -------------------GSYGDLGGPIITTQVTIPK :::::::::::::::::::IPI000 MAYEPQGGSGYDYSYAGGRGSYGDLGGPIITTQVTIPKDLAGSIIGKGGQRIKQIRHESG 360 370 380 390 400 410

Figure 3 Schematic representation of methods for stable-isotope protein labelling for quantitative proteomics. a, Proteins are labelled metabolically by culturing cells in media that are isotopically enriched (for example, containing 15N salts, or 13C-labelled amino acids) or isotopically depleted. b, Proteins are labelled at specific sites with isotopically encoded reagents. The reagents can also contain affinity tags, allowing for the selective isolation of the labelled peptides after protein digestion. The use of chemistries of different specificity enables selective tagging of classes of proteins containing specific functional groups. c, Proteins are isotopically tagged by means of enzyme-catalysed incorporation of 18O from 18O water during proteolysis. Each peptide generated by the enzymatic reaction carried out in heavy water is labelled at the carboxy terminal. In each case, labelled proteins or peptides are combined, separated and analysed by mass spectrometry and/or tandem mass spectrometry for the purpose of identifying the proteins contained in the sample and determining their relative abundance. The patterns of isotopic mass differences generated by each method are indicated schematically. The mass difference of peptide pairs generated by metabolic labelling is dependent on the amino acid composition of the peptide and is therefore variable. The mass difference generated by enzymatic 18O incorporation is either 4 Da or 2 Da, making quantitation difficult. The mass difference generated by chemical tagging is one or multiple times the mass difference encoded in the reagent used.

Aebersold R, Mann M. (2003) Nature. 422:198-207

Correlation between Protein and mRNA Abundance in Yeast –

Conclusions

• Correlation between mRNA and protein levels insufficient to predict protein expression levels (but good for very abundant proteins)

• 20-fold change in protein with little change in mRNA

• no change in protein with 30-fold change in mRNA

• codon bias does not predict protein or mRNA levels (but abundant proteins have biased codons)

Review questions –1. List the 20 amino acids, with their 1-letter

and 3-letter abbreviations.2. What are some of the most common amino-acids?

Least common?3. Which amino acids contain hydroxyl groups that

can be phosphorylated? (Why is this important?)4. Which amino-acids contain aromatic rings? 5. Which amino-acids are more likely to be on the

outside of proteins? On the inside? Why?6. Which amino-acid is likely to change its charge

state with pH changes within the physiological range (pH 6.5 – 8.0)? Why?

7. Outline the steps required for MS/MS protein identification

8. Which MS/MS protein sequencing techniques require a comprehensive protein sequence database?

Questions from previous exams –

1. Pick an acidic or basic amino-acid. (a) name the amino-acid; (b) draw the charge-structure of the amino-acid for each of the charge-states that it can assume (the actual covalent structure need not be correct, focus on the ionizable groups); (c) suggest an approximate pK for each of the ionizable groups. (d) Indicate the most abundant charge-state at pH 7.0.

2. The carboxyl group of amino acid alanine has a pKa value of 2.4 . In order to have 99% of the alanine in its COO form, what must the numerical relation be between the pH of the solution and the pKa of the carboxyl group of alanine.

3. Pick 5 amino acids including some that are more common and some that are less common. Construct a "PAM" amino-acid similarity matrix using those 5 amino acids, using +5 or +3 for identities, +1 for "conserved" amino acids (amino acids with similar properties), and -2 or -5 for non-conservative amino acids.