9/9/13!1!Chapter 4 Protein Structure and Function Be Able To • Compare the four different levels of protein structure and which types of chemical bonds are utilized for each. • Describe the role of phosphorylation in protein regulation. • Compare the functional state of G-proteins bound to GDP vs. GTP. • Describe where feedback-inhibition would be used given a particular pathway. • Explain why disulfide bonds are utilized to stabilize protein structure. • Explain how a protein’s structure defines its function. Why are the arrangement of amino acids in an active site important? • Describe (in chemical terms) when alpha helices will partition into a membrane. What types of amino acids must be present? • Define protein domain. Why is this a useful unit for thinking about protein structure and function? • Compare three mechanisms to lower the activation energy of a reaction.9/9/13!2!Levels of Protein Structure • Primary Amino acid sequence • Secondary Stable folding patterns within a polypeptide • Tertiary Full three-dimensional conformation • Quaternary Complete structure of a complex formed between more than one polypeptide chain Primary Structure Amino acid sequence • Amino acids are joined together through condensation reactions to form peptide bonds • The order of amino acids in a linear polypeptide “string” is specific, and this sequence ultimately determines the three-dimensional structure of the protein • Proteins have structural polarity9/9/13!3!Protein folding As a polypeptide is being synthesized, it is very flexible. In principle, it could fold in many different ways. Most proteins fold into a stable three-dimensional molecule shortly after (and even during) synthesis Unstable folding leads to degradation or aggregation. What constrains protein folding?9/9/13!4!Protein folding Constrained by the formation of many weak noncovalent bonds • Polypeptide backbone • Amino acid side chains Ionic bonds Hydrogen bonds Hydrophobic interactions van der Waals attractions A single noncovalent bond may be weak, but many of them together form a strong arrangement9/9/13!5!Hydrophobic interactions constrain protein folding9/9/13!6!Protein folding in a cell is often assisted by molecular chaperones, or chaperonins A defect in protein folding can lead to aggregation, which underlies several disorders • Alzheimer’s • Huntington’s • Prion diseases Scrapie “Mad cow” Creutzfeldt-Jacob Prion disease9/9/13!7!Secondary Structure A few characteristic patterns occur frequently within folded proteins. These recurring shapes are particularly stable. α helix β sheet These structures are stabilized by hydrogen bonds forming between the amino and carbonyl groups in the polypeptide backbone Amino acid side chains are not involved in these hydrogen bonds so many different amino acid sequences can form these structures α Helix - The carbonyl group of one peptide bond is hydrogen bonded to the amino group of a peptide bond four amino acids away. This gives rise to a helix making a complete turn every 3.6 amino acids.9/9/13!8!A segment of α helix composed of many nonpolar amino acids can span a membrane bilayer. The hydrophilic polypeptide backbone (stabilized by H-bonding) is shielded from the hydrophobic lipid hydrocarbons Amphipathic α helices can form a coiled-coil9/9/13!9!β Sheet - The polypeptide strands are extended, giving the backbone a pleated shape. These strands associate through hydrogen bonding between peptide bonds in different strands. Antiparallel β sheet Parallel β sheet9/9/13!10!Tertiary Structure Three dimensional conformation The conformation formed by an entire polypeptide, including: α helices β sheets Other loops and folds Note: non-α helix and non-β sheet structures are often referred to as “random coils”. These structures are often not coiled and are almost certainly not random. Amino acids from distant regions of the primary structure may be closely associated in the final tertiary structure9/9/13!11!One polypeptide can have regions of both α helix and β sheet Modular structure Quaternary Structure Complex of more than one polypeptide • Each individual polypeptide is a subunit Example: two subunits=dimer • Subunits associate at specific sites Binding sites • Binding of subunits is enabled through weak noncovalent bonds (same as folding)9/9/13!12!Quaternary Structure - Identical subunits homotetramer homodimer Proteins can form large, complex structures through multimerization9/9/13!13!Quaternary Structure different subunits Hemoglobin “Occasionally it has been educational to write the structural formula for ribonuclease in full, in terms of its 1876 atoms of C, H, N, 0, and S. Portrayal of the complete molecule with all of the atoms of the amino groups, carboxyl groups, hydroxyl groups, guanido groups, imidazole rings, phenolic groups, indole rings, aromatic, aliphatic, and thioether sidechains, sulfhydryl groups, and disulfide bonds, helps in the visualization of the almost infinite number of ways in which such groups could be arranged. This characteristic of proteins makes it possible for nature to design catalysts for such a variety of specific reactions. There is no law that says that a nucleic acid or a polysaccharide could not be an enzyme. But it is understandable that the enzymes so far isolated have turned out to be proteins; a protein is equipped to participate, sometimes through cooperation with coenzymes, in the whole lexicon of organic reactions that require catalysis in the living cell.” – Stanford Moore and William Stein Science 180:458 (1973) Chemical Structures of Pancreatic Ribonuclease and Deoxyribonuclease9/9/13!14!A protein’s function is linked to its structure Proteins bind to other molecules (ligands) • Affinity (how tightly) • Specificity • Binding site What%are%the%primary%forces%that%determine%the%33D%protein%structure?%What%are%the%primary%forces%that%determine%the%folding%of%a%protein?%What%are%the%primary%forces%that%determine%ligand%affinity/specificity?%Shape determines affinity What happens if the noncovalent bonds between a protein and ligand are broken?9/9/13!15!A single aa change in hemoglobin has a profound effect on hemoglobin structure and red blood cell function Enzymes Binding is the first step in their function • Catalysts • Activation