Roadmap The topics: basic concepts of molecular biology more on Perl overview of the field biological databases and database searching sequence alignments phylogenetics structure prediction microarray data analysis

Protein Synthesis the national health museum Process whereby DNA encodes for the production of amino acids and proteins. This process can be divided into two parts: 1. Transcription Before the synthesis of a protein begins, the corresponding RNA molecule is produced by RNA transcription. One strand of the DNA double helix is used as a template by the RNA polymerase to synthesize a messenger RNA (mRNA). This mRNA migrates from the nucleus to the cytoplasm. During this step, mRNA goes through different types of maturation including one called splicing when the non-coding sequences are eliminated. The coding mRNA sequence can be described as a unit of three nucleotides called a codon. 2. Translation The ribosome binds to the mRNA at the start codon (AUG) that is recognized only by the initiator tRNA. The ribosome proceeds to the elongation phase of protein synthesis. During this stage, complexes, composed of an amino acid linked to tRNA, sequentially bind to the appropriate codon in mRNA by forming complementary base pairs with the tRNA anticodon. The ribosome moves from codon to codon along the mRNA. Amino acids are added one by one, translated into polypeptidic sequences dictated by DNA and represented by mRNA. At the end, a release factor binds to the stop codon, terminating translation and releasing the complete polypeptide from the ribosome. One specific amino acid can correspond to more than one codon. The genetic code is said to be degenerate. the national health museum

Proteins

Proteins Proteins perform a vast array of biological functions including: Transport: hemoglobin (delivers O2 to lungs) Mechanical support: collagen Storage: ferritin (stores iron) Regulation: repressor proteins (gene expression) Antibodies: immunoglobulin Catalysis: SOD (superoxide dismutase) … Misfold: mad cow disease, Alzheimer's disease, …

Amino acid composition Basic Amino Acid Structure: The side chain, R, varies for each of the 20 amino acids C R C H N O OH Amino group Carboxyl group Side chain

The Peptide Bond Dehydration synthesis Polypeptide with repeating backbone: N–C –C –N–C –C

Side chain properties What make amino acids having different properties ? Carbon does not make hydrogen bonds with water easily – hydrophobic O and N are generally more likely than C to h-bond to water – hydrophilic The amino acids forms three general groups: Hydrophobic Polar Charged (positive/basic & negative/acidic)

The Hydrophobic Amino Acids Proline severely limits allowable conformations!

The Charged Amino Acids Krane & Raymer

The Polar Amino Acids Krane & Raymer

More Polar Amino Acids and

Peptidyl polymers A few amino acids in a chain are called a polypeptide. A protein is usually composed of 50 to 400+ amino acids.

Primary & Secondary Structure Primary structure = the linear sequence of amino acids comprising a protein: AGVGTVPMTAYGNDIQYYGQVT… Secondary structure Regular patterns of hydrogen bonding in proteins result in two patterns that emerge in nearly every protein structure known: the -helix and the -sheet The location of direction of these periodic, repeating structures is known as the secondary structure of the protein

Levels of Protein Structure Secondary structure elements combine to form tertiary structure Quaternary structure occurs in multi-enzyme complexes Many proteins are active only as homodimers, homotetramers, etc.

Dihedral angles

 Helix Most abundant secondary structure 3.6 amino acids per turn Hydrogen bond formed between every fourth reside Avg length: 10 amino acids, or 3 turns Varies from 5 to 40 amino acids

 Helix Normally found on the surface of protein cores Interact with aqueous environment Inner facing side has hydrophobic amino acids Outer-facing side has hydrophilic amino acids Every third amino acid tends to be hydrophobic Pattern can be detected computationally Rich in alanine (A), gutamic acid (E), leucine (L), and methionine (M) Poor in proline (P), glycine (G), tyrosine (Y), and serine (S)

 Sheet

 Sheet Hydrogen bonds between 5-10 consecutive amino acids in one portion of the chain with another 5-10 farther down the chain Interacting regions may be adjacent with a short loop, or far apart with other structures in between Directions: Same: Parallel Sheet Opposite: Anti-parallel Sheet Mixed: Mixed Sheet Alpha carbons (and R side groups) alternate above & below the sheet Prediction difficult, due to wide range of  and  angles

Ramachandran Plot (alpha)

Ramachandran Plot (beta)

Ramachandran Plot

Helices and Sheets

Loop Regions between  helices and  sheets Various lengths and three-dimensional configurations Located on surface of the structure Hairpin loops: complete turn in the polypeptide chain, (anti-parallel  sheets) More variable sequence structure Tend to have charged and polar amino acids

Coil Region of secondary structure that is not a helix, sheet, or loop

Determining Protein Structure There are O(100,000) distinct proteins in human proteome. Two methods for revealing positions of atoms in 3-D: X-Ray Crystallography X-ray diffraction pattern + mathematical construction Good protein crystal needed, good resolution of diffraction needed Nuclear Magnetic Resonance Small proteins only (< 250 residues) Inter-proton distances + geometric constraints

Bovine Ribonuclease Christian Anfinsen, 1957.

Disulfide Bonds Two cysteines in close proximity will form a covalent bond Disulfide bond, disulfide bridge, or dicysteine bond. Significantly stabilizes tertiary structure.

Forces Stabilizing Proteins All of the important tasks in living cells are carried out by proteins in which the polypeptide chain is tightly folded into a globular conformation that is essential for the biological function of the protein. The amino acid sequence of the protein determines the folded conformation of the protein. Using recombinant DNA technology, proteins can now be constructed with an desired amino acid sequence. The potential applications of this technology in health and other areas are almost unlimited. Consequently, it is essential that we learn to predict how a protein will fold given just the amino acid sequence, and that we learn how changes in the amino acid sequence will effect the function and stability of a protein. To this end, we have begun an in depth study of the energetics and mechanism of folding of several members of a family of microbial ribonucleases. The microbial RNases provide an excellent model system for protein folding studies. They are among the smallest enzymes known with around 100 amino acid residues and they fold into compact globular structures in which a the hydrophobic core is sandwiched between an a-helix and a ß-sheet. High resolution structures are available for most of these enzymes, and we have recently determined a 1.0 Å crystal structure for RNase Sa. We are using site-directed mutagenesis to make single changes in the amino acid sequence of these proteins. We then measure the conformational stability and thermodynamics of folding of these mutants. Our goal is to reach a better understanding of the forces that contribute to the conformational stability of globular proteins: hydrophobic interactions, hydrogen bonding, electrostatic interactions, and conformational entropy. We are using this and other information to try and increase the stability of enzymes for biotechnological applications.

Principles that govern the folding of protein chains - Christian Anfinsen, Science 1973

Ribonuclease

Disulfide Bonds 6 12 5 10 4 8 3 2 # of combinations # of S-S bonds # of cysteines 3 15 105 945 (n-1)*(n-3)*(n-5)*…*3 10395

Levinthal’s paradox How do proteins find the right conformation out of the simply endless number of potential three-dimensional forms that it could randomly fold into? Consider a 100 residue protein. If each residue can take only 3 positions, there are ? possible conformations. If it takes 10-13s to convert from 1 structure to another, exhaustive search would take ? years! 3100 = 5  1047 Golf course (blind person search holes, no guidance) 1.6  1027

Current Opinion in Structural Biology, 2004, 14, 70-75

What determines fold? Anfinsen’s experiments in 1957 demonstrated that proteins can fold spontaneously into their native conformations under physiological conditions. This implies that primary structure does indeed determine folding or 3-D structure. Exceptions exist Chaperone proteins assist folding Abnormally folded Prion proteins can catalyze misfolding of normal prion proteins that then aggregate

Other factors Physical properties of protein that influence stability & therefore, determine its fold: Rigidity of backbone Amino acid interaction with water Hydropathy index for side chains Interactions among amino acids Electrostatic interactions Hydrogen, disulphide bonds Volume constraints

Understand protein folding Structure: Given a sequence, what tertiary structure does it adopt? Global optimization, Monte Carlo, Molecular dynamics, Coarse-grained dynamics, etc. Thermodynamics: under mutation does the free energy of the native state change relative to native sequence? MC, MD, Free energy methods, etc. Kinetics: how fast does the protein fold? Does a different sequence fold faster and why? Lattice Monte Carlo, Molecular dynamics, Coarse-grained dynamics

CASP changed the landscape Critical Assessment of Structure Prediction competition. Even numbered years since 1994 Solved, but unpublished structures are posted in May, predictions due in September Various categories Relation to existing structures, ab initio, homology, fold, etc. Partial vs. Fully automated approaches Produces lots of information about what aspects of the problems are hard, and ends arguments about test sets. Results showing steady improvement, and the value of integrative approaches.