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Protein Molecules Have Functions

Binding

Catalysis

Regulation

Structural

Petsko & Ringe

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Growth of PDB—RCSB Annual Report 2006

Protein structure determines function

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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 syllable

secondary wordtertiary sentencequarternary sentence with semicolon (?)

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Proteins are made of amino acids

Amino

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, delta, epsilon, zeta, eta

C=carbonN=nitrogenO=oxygenH=hydrogenR=anything

Side chain

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Amino acid side chains exhibit a range of chemical and biophysical properties A = AlaC = CysD = AspE = GluF = PheG = GlyH = HisI = IleK = LysL = LeuM = MetN = AsnP = ProQ = GlnR = ArgS = SerT = ThrV = ValW = TrpY = Tyr

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Hydrophobicity

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

Hydrophobic surface repels water

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

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

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Hydrophobic amino acids (residues)Hydrophobic amino acids (residues)

Ala, A Val, V Leu, L Ile, I

IleIle, Val are , Val are ββ--branchedbranched

ProlineProline is really an is really an iminoimino acidacid

e.g.

Gly, G

Pro, P

Met, M

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Aromatic amino acidsAromatic amino acids

Conjugated planar Conjugated planar ππ systemsystemAromatic interactions

Pi Pi interaction (aromatic stacking)Pi Pi interaction (aromatic stacking)Edge to face interactionEdge to face interaction

UV absorption: UV absorption: TrpTrp and and TyrTyrconcentration measurement

Trp fluorescence spectroscopy—folding and unfolding

Trp, W (tWWo rings)

Tyr, Y (tYYrosine)

Phe, F His, H

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Charged amino acidsCharged amino acids

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

Lys, KKArg, RR (aRRginine)

His, H

[ ][ ]HAAlogpKapH

-

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

Asp, D Glu, E

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Hydroxyl containing amino acidsHydroxyl containing amino acids Amide containing amino acidsAmide containing amino acids

Ser, S

Thr, T

Asn, N (asparagiNNe)

Gln, Q(QQlutamine)

Tyr, Y (tYYrosine)

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Sulfur containing residuesSulfur containing residues

Cys, C

Met, M

© I. Samish

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Suggested substitution for (buried) amino acid

(Exposed) amino acids

Grouping of amino acids based on theirphysical properties

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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

OxidationReduction

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Serotonin : Trp derivativeGABAGABA γ-aminobutyric acid: derived from

glutamateHistamine : His derivativeDopamine : Tyr derivative

Histamine

Dopamine

GABA

Amino acids are used to make other chemicals

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

Peptide bond

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

this step takes energy

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

BackboneN-terminus

C-terminus

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

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Peptide bond (ω) is “flat”

40% double bondcharacteristics

A * sin2δwhere A ~ 30 kcal/mol 0.9 kcal = 10 deg3.5 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 peptidechain, we know what the entire protein looks like

Dihedral angle

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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

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

Non-Gly or Pro

Gly

Pro

Pyruvate kinase

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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)

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

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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 ~20% prolines and 0.1% of other amino acids in cis conformation

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Side chain dihedral angles are namedchi1, chi2, chi3, …

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

Eclipsed = high energy

Conformational analysis

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

t = 180°

pos dihedral

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

g+ = -60° g- = 60°

Val, Ile, Thr

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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 Codons Observed Frequencyin Vertebrates

Alanine GCU, GCA, GCC, GCG 7.4 % Arginine CGU, CGA, CGC,CGG, AGA, AGG 4.2 % Asparagine AAU, AAC 4.4 % Aspartic Acid GAU, GAC 5.9 % Cysteine UGU, UGC 3.3 % Glutamic Acid GAA, GAG 5.8 % Glutamine CAA, CAG 3.7 % Glycine GGU, GGA, GGC, GGG 7.4 % Histidine CAU, CAC 2.9 % Isoleucine AUU, AUA, AUC 3.8 % Leucine CUU, CUA, CUC, CUG, UUA, UUG 7.6 % Lysine AAA, AAG 7.2 % Methionine AUG 1.8 % Phenylalanine UUU, UUC 4.0 % Proline CCU, CCA, CCC, CCG 5.0 % Serine UCU, UCA, UCC, UCG, AGU, AGC 8.1 % Threonine ACU, ACA, ACC, ACG 6.2 % Tryptophan UGG 1.3 % Tyrosine UAU, UAC 3.3 % Valine GUU, GUA, GUC, GUG 6.8 % Stop Codons UAA, UAG, UGA ---

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Amino acid frequnecyThe 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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Units: Energy, heat joule (J)joule (J) kg.m2.s-2

Or calorie (cal) = 4.184 J

Prefixes for units:mega (M)mega (M) 106 kilo (K)kilo (K) 103 millimilli (m)(m) 10-3

micro (micro (μμ)) 10-6 nanonano (n)(n) 10-9 picopico (p)(p) 10-12

femtofemto (f)(f) 10-15

Constants: Avogadro num. Avogadro num. (N)(N) 6.022x1023 molec.mol-1

Gas constant Gas constant (R)(R) 8.3145 J.K-1.mol-1

BoltzmannBoltzmann constant constant (k(kbb)) 1.3807x10-23 J.K-1 (=R/N) Planck’s constant Planck’s constant (h)(h) 6.6261x10-34 J.s

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

ConstantsConstants