Protein Molecules Have Functions Binding

Regulation

Catalysis

Structural

Petsko & Ringe

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Protein structure determines function

Growth of PDB—RCSB Annual Report 2006 2

Protein structures are organized in hierarchy – – – –

Primary: amino acid sequence Secondary: recurring structure stabilized through main chain hydrogen bonds Tertiary: packing of secondary structures Quaternary: assembly of multiple polypeptide chains in a protein complex

– Imagine a parallel with human language: E.g. primary Î syllable secondary Î word tertiary Î sentence quarternary Î sentence with semicolon (?)

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Proteins are made of amino acids Side chain

Amino

C=carbon N=nitrogen O=oxygen H=hydrogen R=anything

Carboxylic acid Of the virtually infinite number of possible amino acids, 20 are used in all three kingdoms of life to make naturally occurring proteins Only small alpha amino acids are used (MW 75 – 204 Da) R is called the side chain Stereochemistry is important: L vs. D amino acid Carbon atoms in the side chain are designated using Greek letters: beta, gamma, 4 delta, epsilon, zeta, eta

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Amino acid side chains exhibit a range of chemical and biophysical properties

A = Ala C = Cys D = Asp E = Glu F = Phe G = Gly H = His I = Ile K = Lys L = Leu M = Met N = Asn P = Pro Q = Gln R = Arg S = Ser T = Thr V = Val W =6 Trp Y = Tyr

Hydrophobicity

Hydrophobic surface repels water Similarly, hydrophobic amino acids are non-polar and will repel water

Hydrophobicity of a molecule is determined by measuring the partitioning of a molecule between a polar solvent, e.g. water, and a nonpolar solvent, e.g. octanol

Opposite of “hydrophobic” is “hydrophilic” Amino acids that are hydrophilic are polar and/or charged

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Hydrophobic amino acids (residues)

Gly, G

Ala, A

Val, V

Leu, L

Ile, I Met, M

e.g. Pro, P Ile, Val are β-branched Proline is really an imino acid

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Aromatic amino acids

Trp, W (tWo rings)

Tyr, Y (tYrosine)

Phe, F

His, H

Conjugated planar π system Aromatic interactions Pi Pi interaction (aromatic stacking) Edge to face interaction

UV absorption: Trp and Tyr concentration measurement

Trp fluorescence spectroscopy—folding and unfolding 9

Charged amino acids

Lys, K

His, H

Asp, D

Glu, E

Arg, R (aRginine) pKa of arg guanido group ~ 12.0 pKa of lys side chain amine ~ 10.4 – 11.1 pKa of his imidazole ~ 6.0 – 7.0 pKa of asp side chain carboxyl ~ 3.9 – 4.0 pKa of glu side chai carboxyl ~ 4.3 – 4.5

HA(aq) + H2O(l) ⇌ H3O+(aq) + A−(aq) or HA(aq) ⇌ H+(aq) + A−(aq)

[A ] pH = pKa + log -

[HA]

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Hydroxyl containing amino acids

Amide containing amino acids

Asn, N (asparagiNe) Ser, S

Tyr, Y (tYrosine)

Thr, T

Gln, Q (Qlutamine) 11

Sulfur containing residues

Cys, C Met, M

12 © I. Samish

Grouping of amino acids based on their physical properties

Suggested substitution for (buried) amino acid

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(Exposed) amino acids

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Post Translational Modification There are further chemical modifications of amino acids after their incorporation into a protein » Phosphorylation, acetylation, methylation, hydroxylation, glycosylation ,alkylation, biotinylation » Ubiquitination, SUMOylation » Prenylation » Disulfide formation » Proteolytic cleavage

Reduction

Oxidation

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Amino acids are used to make other chemicals Serotonin : Trp derivative GABA γ-aminobutyric acid: derived from glutamate Histamine : His derivative Dopamine : Tyr derivative

GABA

Dopamine Histamine 16

Peptide bond Two or more amino acids linked together by a peptide bond constitutes a polypeptide—i.e. a polymer containing amino acids in peptide bonds

this step takes energy

Peptide bond is thermodynamically unstable but kinetically stable: a property that is common to many biomacromolecules including protein, DNA and RNA Hydrolysis of a peptide bond results in two fragments with a large increase in total entropy 17

Peptide chain has a direction: starts at the amino terminus (“Nterminus”) and ends at the carboxy terminus (“C-terminus”)

Backbone N-terminus

C-terminus

Ramachandran and Sasisekharan, Adv. Protein Chem. 23, 283 (1968) 18

Peptide bond (ω) is “flat” 40% double bond characteristics

A * sin2δ where A ~ 30 kcal/mol Î 0.9 kcal = 10 deg 3.5 kcal = 20 deg

Edison, Nat Struct Biol 8, 201 (2001)

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If we assume the peptide bond is “flat”, there are two remaining backbone dihedral angles, phi (φ) and psi (ψ) If we know the values of phi and psi for the entire peptide chain, we know what the entire protein looks like

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Dihedral angle

Distribution of main chain dihedral angles (phi and psi) in a protein is represented by the Ramachandran plot The ranges of phi, psi angles found in natural proteins are restricted to narrow regions of the phase space The distribution depends on amino acid type

Gly

Pro

Pyruvate kinase

Non-Gly or Pro 21

http://xray.bmc.uu.se/gerard/supmat/nonglypro_ramp.gif

Steric repulsion at short distances disallow parts of the Ramachandran phase space

If we assume all atoms are hard spheres of van der Waals radii, dashed regions of the dihedral space are not allowed due to steric clash Mandel et al. J Biol Chem 252, 4619 (1977) 22

Conformations of a double bond

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Amino acids are usually found in the trans conformation but prolines often (~ 6%) occur in the cis conformation—compare this with 0.04% of non-proline residue

Weiss, et al. Nat Struct Biol 5, 676 (1998)

However, based on free energy calculation in vacuum we may expect 24 ~20% prolines and 0.1% of other amino acids in cis conformation

Conformational analysis Side chain dihedral angles are named chi1, chi2, chi3, …

Staggered conformation has lower energy than eclipsed conformation (minimized 1-4 interaction)

Eclipsed = high energy

Staggered = low energy

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Newman projection shows three possible staggered conformations (trans, gauch+, gauche-) for chi1 in residues other than Ala, Gly, Pro Val, Ile, Thr

pos dihedral t = 180°

g+ = -60°

g- = 60°

•Chakraborty and Pal, Prog Biophy Mol Biol 76, 1 (2001)

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Main chain dihedrals (phi, psi) are correlated with chi1

In addition to 1-4 interaction, an optimum conformation must also minimize 1-5 interaction If the two internal dihedral angles of a sequence of five atoms have a combination of (g+, g-) or (g-, g+), the terminal atoms form a high energy syn-pentane conformation

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Amino acid composition Question: Does natural selection prefer certain amino acids over others? If a particular amino acid is in some way adaptive, then it should occur more frequently than expected by chance. Calculate the expected frequencies of amino acids and compare to observed frequencies

Amino Acids Alanine Arginine Asparagine Aspartic Acid Cysteine Glutamic Acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Stop Codons

Codons GCU, GCA, GCC, GCG CGU, CGA, CGC,CGG, AGA, AGG AAU, AAC GAU, GAC UGU, UGC GAA, GAG CAA, CAG GGU, GGA, GGC, GGG CAU, CAC AUU, AUA, AUC CUU, CUA, CUC, CUG, UUA, UUG AAA, AAG AUG UUU, UUC CCU, CCA, CCC, CCG UCU, UCA, UCC, UCG, AGU, AGC ACU, ACA, ACC, ACG UGG UAU, UAC GUU, GUA, GUC, GUG UAA, UAG, UGA

Observed Frequency in Vertebrates 7.4 % 4.2 % 4.4 % 5.9 % 3.3 % 5.8 % 3.7 % 7.4 % 2.9 % 3.8 % 7.6 % 7.2 % 1.8 % 4.0 % 5.0 % 8.1 % 6.2 % 1.3 % 3.3 % 6.8 % ---

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Amino acid frequnecy The frequencies of DNA bases in nature 30.3% adenine (A) 21.7% cytosine (C) 26.1% guanine (G) 22.0% thymidine (T) Compute the expected frequency of a particular codon (i.e. three DNA bases corresponding to one amino acid) by multiplying the frequencies of each DNA base comprising the codon. The expected frequency of an amino acid is calculated by adding the frequencies of each codon corresponding to that amino acid. Conclusions: Average amino acid composition passively reflects random permutations of the genetic code.

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Constants Units: Energy, heat joule (J) kg.m2.s-2 Or calorie (cal) = 4.184 J Prefixes for units: mega (M) 106 micro (μ) 10-6 femto (f) 10-15 Constants: Avogadro num. Gas constant Boltzmann constant Planck’s constant

kilo (K) 103 nano (n) 10-9

milli (m) 10-3 pico (p) 10-12

(N) 6.022x1023 molec.mol-1 (R) 8.3145 J.K-1.mol-1 (kb) 1.3807x10-23 J.K-1 (=R/N) (h) 6.6261x10-34 J.s

Conversions: Angstrom (Å) =10-10 m Calorie (cal) = 4.184 J Kelvin (K) = degrees Celsius (oC) + 273.15.

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