Biochemistry I Lecture Notes PDF

BIOL or CHEM 3361 Biochemistry I Proteins: Primary structure, Sequencing, Mass spectrometry and Sequence Alignments Reading: Chapter 5 Nature of Protein Sequences The no. of possible sequences is astronomically large Probability that two proteins will by ch...

BIOL or CHEM 3361 Biochemistry I Proteins: Primary structure, Sequencing, Mass spectrometry and Sequence Alignments Reading: Chapter 5 Nature of Protein Sequences The no. of possible sequences is astronomically large Probability that two proteins will by chance have similar sequences is negligible Therefore, sequence similarity implies evolutionary relatedness Proteins with similar sequences are described as homologous - orthologous proteins are from different species (ancestral gene) - paralogous proteins are from the same species (gene duplication) Sequence Alignments Computer programs optimally align sequences in databases e.g. BLAST or Basic Local Alignment Search Tool Mutations represent changes at one or more sites in the amino acid sequence of a protein Score alignment based on matching or similar amino acids Also put in gaps which bring penalties Alignment can be local or global ATP Synthase Nucleotide-binding Site BLAST & Alignments Phylogeny of Cytochrome c The number of amino acid differences between two cytochrome c sequences is proportional to the phylogenetic difference between the species from which they are derived This observation can be used to build phylogenetic trees of proteins This is the basis for studies of molecular evolution Phylogeny of Cytochrome C This phylogenetic tree depicts the evolutionary relationships among eukaryotic organisms as determined by the similarity of their cytochrome c sequences. Numbers are the amino acid changes between hypothetical branch points and to the extant species at the tips. Protein Structure and Function Because structure depends on sequence, and because function depends on structure, it is tempting to imagine that all proteins of similar structure should share a common function, but this is not always true (a)Some proteins share similar structural features but carry out different functions. (b) Proteins with different structures can carry out similar functions. How to Proteins get Structure? Peptide Bond Links Amino Acids Partial double bond character - coplanar O, C and N - limited rotation NH acts as a donor and O as an acceptor for H-bonds Phi (Φ) and Psi (Ψ) Angles Only two degrees of freedom for peptide backbone (dihedral angles) 1. Angle about the Cα-N bond is denoted φ (phi) 2. Angle about the Cα-C bond is denoted ψ (psi) Positive values are clockwise rotations as view from Cα and starting at 0° Restriction of Phi (Φ) and Psi (Ψ) Angles Some values forbidden due to steric crowding φ = 0°, ψ = 180° φ = 180°, ψ = 0° φ = 0°, ψ = 0° Display φ and ψ on a Ramachandran Plot The colored regions are “allowed” Acetyltransferase RTT109 Proline has a fixed phi angle of -60° Particular angles give secondary structures Protein Folding – 2°, 3°, and 4° Weak interactions stabilize proteins van der Waals are ubiquitous hydrogen bonds form wherever possible ionic bonds occur on protein surface hydrophobic interactions occur in protein interior/core and drive folding Most stable structure has largest no. of interactions Hydrogen bonding involves backbone CO and NH, plus some sidechain groups Polarity of H-bonding groups must be neutralized if they are in the hydrophobic core. This occurs via secondary structures. Secondary and Supersecondary Structures Secondary structures: Helices (particularly alpha) Beta sheets Beta turns Random coil Supersecondary structures: Coiled coils Beta hairpin Beta-alpha-beta Helix-turn-helix, Greek keys, alpha-alpha hairpin, EF hand etc. The α-Helix  The α-Helix 3.6 residues per turn φ = −60°, ψ = −45 to -50° Diameter of 6 Å (without sidechains) Helix pitch or rise of 5.4 Å (1.5 Å per res.) Different helix lengths Average of 10 residues per helix Almost always right-handed due to close approach of side chains of amino acids (for L- amino acids, the left-handed α-helix has a close approach between the carbonyl oxygen and the β-carbon) Left-handed helices are shorter and contain glycine The α-Helix Net Dipole Moment H-bonds point in the same direction Gives a partial positive charge at the amino end Gives a partial negative charge at carboxyl end Exploited in recognition Amino acids can be classified as helix- formers or helix breakers * Breakers to remember * Based on studies with poly-aa Preference not * Can bend or break strong enough to be correct 100% of the time The Helical Wheel Reveals non-polar or polar character of helices Amphipathic helices often found on protein surface with polar residues in solvent, and non-polar residues hidden in the core Top: An amphiphilic helix in flavodoxin Middle: A nonpolar helix in citrate synthase: Bottom: A polar helix in calmodulin The “non-α” Helices The β-sheet H-bonds between C=O and N-H residues on different strands Strands from different regions of polypeptide Strands are: - parallel (top) with bent H- bonds - anti-parallel (bottom) with straight H-bonds Antiparallel is more favorable The β-Sheet is Pleated Cα atoms are a little above/below the plane of the sheet (zig-zag structure) Sidechains project up/downward The β-sheet Parallel φ = −120°, ψ = 105°(small range) > 5 strands Hydrophobic sidechains on both sides Anti-parallel φ = −135°, ψ = 140°(large range) < 5 strands Hydrophobic sidechains on one sides (alternate in sequence) Rise is 3.47 Å (0.695 Å per 2 res.) for antiparallel strands and 3.25 Å (0.65 Å per 2 res.) for parallel strands with 5 residues Each strand of a β-sheet is 5-10 residues long The β-sheet Can be mixed parallel and anti-parallel Often have a right-handed twist Topology Diagrams Useful to compare structures Topology Diagrams