Protein Structure and Folding

1. Protein Structure and Folding

2. Life … is a relationship between molecules. Linus Pauling, as quoted in T. Hager, Force of Nature: The Life of Linus Pauling (1997), p. 542

3. 4.1 Introduction

4. Proteins are found in all living systems, ranging from bacteria and archaea through the unicellular eukaryotes, to plants, fungi, and animals. • In all life forms, proteins are made up of the same building blocks―amino acids. • Each cell contains thousands of different genes and makes thousands of different proteins.

5. What is a gene? • In the late 1930s… “A molecule of living stuff made up of many atoms held together.”

6. What is a gene? • A specific stretch of nucleotides in DNA (or in some viruses, RNA) that contains information for making a particular RNA molecule that in most cases is used to make a particular protein.

7. Primary structure: amino acids and the genetic code

8. The 22 amino acids found in proteins • Proteins are chain-like polymers of amino acids specified by the genetic code. • Each amino acid has an amino group (NH3+) and a carboxyl group (COO) attached to a central carbon called the -carbon. • The only difference between two amino acids is in their different side chain or “R group.”

9. At pH 7 the amino and carboxyl groups of amino acids are charged. • Over a pH range from 1 to 14 these groups exhibit binding and dissociation of a proton. • The weak acid-base behavior of amino acids provides the basis for many techniques for amino acid identification and protein separations.

10. Protein primary structure • Amino acids joined together by peptide bonds form the primary structure of a protein. • The amino group of one molecule reacts with the carboxyl group of the other in a condensation reaction.

11. When joined in a series of peptide bonds, amino acids are called residues. • A short sequence of amino acids is called a peptide; the term polypeptide applies to longer chains of amino acids. • The arrangement of amino acids, with their distinct side chains, gives each protein its characteristic structure and function.

12. The peptide bond has a partial double bond character as a result of resonance. • Free rotation occurs only between the -carbon and the peptide unit. • Trans and cis-configurations are possible about the rigid peptide bond. • The peptide chain is flexible, but it is more rigid than it would be if there were free rotation about all of the bonds.

13. Translating the genetic code • How is the genetic code translated into a specific sequence of amino acids?

14. A DNA sequence is read in triplets using the antisense (non-coding) strand as a template that directs synthesis of RNA via complementary base pairing. • An open reading frame (ORF) in the mRNA indicates the presence of a start codon followed by codons for a series of amino acids and ending with a termination codon.

15. The genetic code • Each “codon box” is composed of four three-letter codes, 64 in all. • 61 codons are recognized by tRNAs for the incorporation of the 20 common amino acids. • 3 codons signal termination, or code for selenocysteine and pyrrolysine.

20. The genetic code is degenerate • tRNAs specific to a particular amino acid recognize multiple codon triplets that differ only in the third letter. e.g. leucine is coded for by 6 different codons, while methionine has only one codon

21. The “wobble hypothesis” • Pairing between codon and anticodon at the first two codon positions always follows the usual rule of complementary base pairing. • Exceptional “wobbles” (non-Watson-Crick base pairing) can occur at the third position.

22. The genetic code is not universal • In certain organisms and organelles the meaning of select codons has been changed. e.g. Tetrahymena reads UAA and UAG as glutamine (Gln)

23. The 21st and 22nd genetically encoded amino acids The UGA code for selenocysteine is found in: • >15 genes in prokaryotes that are involved in redox reactions. • >40 genes in eukaryotes that code for various antioxidants and the type I iodothyronine deiodinase. The UAG code for pyrrolysine has been found in: • a few archaebacteria and eubacteria.

24. Modified nucleotides and codon bias • “Wobbles” can occur at the third position. • When bases in the anticodon are modified, further pairing patterns are possible. • Examples: Inosine can pair with U, C, and A. 2-thiouracil restricts pairing to A alone.

25. Implications of codon bias for molecular biologists • The frequencies with which different codons are used vary significantly between different organisms and between proteins expressed at high or low levels within the same organism. • Expression of functional proteins in heterologous hosts is a cornerstone of molecular biology research. • Codon bias can have a major impact on the efficiency of expression of proteins if they contain codons that are rarely used in the desired host.

26. What might happen if you tried to express a Tetrahymena gene that encodes a glutamine-rich protein in E. coli?

27. D- and L-amino acids in nature • D- and L-amino acids are enantiomers (sterioisomers that are mirror images of each other). • Living organisms are composed predominantly of L-amino acids. • Ribosomes only use L-amino acids to make proteins.

28. Exceptions: • D-amino acids are found in some peptides in microorganisms, but are synthesized by pathways that do not involve the ribosome. • D-amino acids are present in some peptides in other organisms, but are made from the genetically encoded L-amino acids by a post-translational process.

29. Examples: • D-amino acids are present in the venom of some bivalves, snails, spiders, amphibians, and the duck-bill platypus. • The presence of D-amino acids is linked to more potent venom.

30. The three-dimensional structure of proteins

31. Four Levels of Structure

32. There is tremendous variation in the size and complexity of proteins. • Dalton (Da) units are typically used to describe the molecular weight of proteins.

33. Typical polypeptide chains have molecular weights of 20 to 70 kDa (20,000 to 70,000 Da). • The average molecular weight of an amino acid is 110. • A typical polypeptide chain thus contains 181 to 636 amino acids.

34. Secondary structure • Interactions of amino acids with their neighbors gives a protein its secondary structure. • Primarily stabilized by hydrogen bonds. • Also depends on disulfide bridges, van der Waals interactions, hydrophobic contacts, and electrostatic interactions.

35. The three basic elements of protein secondary structure • -helix • -pleated sheet • Unstructured turns

36. -helix • Most common structural motif in proteins. • Tight helical structure stabilized by hydrogen bonding among near-neighbor amino acids. • Proline, the “helix-breaking residue”, cannot participate as a donor in hydrogen bonding.

37. -pleated sheet • Extended amino acids chains packed side by side to create a pleated, accordian-like appearance. • Stabilized by hydrogen bonding.

38. Parallel  structure • Two segments of a polypeptide chain (or two individual polypeptides) are aligned in the N-terminal to C-terminal direction or vice versa. Antiparallel  structure • One segment is N-terminal to C-terminal and the other is C-terminal to N-terminal.

39. Unstructured turns • “Turns” connect the -helices and -pleated sheets in proteins. • Relatively short loops that do not exhibit a defined secondary structure.

40. Tertiary structure • The folded three-dimensional shape of a polypeptide. • Most interactions are stabilized by noncovalent bonds: Hydrophobic interactions Hydrogen bonds

41. The principle covalent bonds within and between polypeptides are disulfide (S-S) bondsor “bridges” between cysteines.

42. Three main categories of tertiary structure • Globular proteins • Fibrous proteins • Membrane proteins

43. Globular proteins • The overall shape of most proteins is roughly spherical. e.g. the enzyme lysozyme folds up into a globular tertiary structure forming the active site.

44. Fibrous proteins • Long filamentous or “rod-like” structures. • Structural components of cells and tissues. • A number of major designs: - triple helical arrangement - “coiled coils” - antiparallel -pleated sheets

45. Membrane proteins • Differ from soluble proteins in the relative distribution of hydrophobic amino acid residues. • The seven transmembrane helix structure is a common motif in membrane proteins.

46. Prediction of protein structure • By comparing the sequences of proteins of unknown structure with those that have been determined, it is often possible to make structural predictions based on identified similarity.

47. Quaternary structure • A functional protein can be composed of one or more polypeptide subunits. • Can be identical or nonidentical subunits. • Stabilizing bonds are the same as those for tertiary structure.

48. Quaternary structure allows greater versatility of function. • Catalytic or binding sites are often formed at the interface between subunits. e.g. the two  and two  subunits in hemoglobin form a binding site for a heme group

49. Protein function and regulation of activity

50. Proteins larger than about 20 kDa are often formed from two or more domains with specific functions. • A single domain is usually formed from a continuous amino acid sequence. • e.g. DNA-binding domain • Domains can contain common structural-functional motifs.