Proteins

1. Proteins

2. Proteins • The most important type of macromolecule. • Roles: • Structure: collagen in skin, keratin in hair, crystallin in eye. Also slimy substances like mucus and the bacterial capsule. • Enzymes: all metabolic transformations, building up, rearranging, and breaking down of organic compounds, are done by enzymes, which are proteins. • Other macromolecules such as carbohydrates and lipids are created by enzymes from smaller molecules. • Transport: oxygen in the blood is carried by hemoglobin, everything that goes in or out of a cell (except water and a few gasses) is carried by proteins. • Also: nutrition (egg yolk), hormones, defense, movement • Enzymes are usually roughly globular, while structural proteins are usually fiber-shaped. Proteins that transport materials across membranes have a long segment of hydrophobic amino acids that sits in the hydrophobic interior of the membrane.

3. Levels of Structure • A polypeptide is one linear chain of amino acids. Each gene produces one polypeptide. A protein may contain one or more polypeptides. Proteins also sometimes contain small helper molecules (co-factors) such as heme. • The primary structure (1o) is just the sequence of amino acids in the polypeptide. • The secondary structure (2o) is local folding patterns, mostly alpha helix and beta sheet. • The tertiary structure (3o) is the overall folding pattern of the entire polypeptide. • The quaternary structure (4o) is the joining of individual polypeptides (subunits) into an active protein. Proteins that are just a single monomeric polypeptide have no quaternary structure.

4. Amino Acids • Amino acids are the subunits of proteins. • “Amino acid” is a very general term, but we mostly refer to the 20 amino acids coded in DNA. • Or 22: some bacteria also code for selenocysteine and pyrolysine. • The properties of the protein are determined by the R-groups on the amino acids. • A few types: • hydrophobic (found in membranes and protein interiors): Leucine, isoleucine, valine, methionine, phenylalanine, tryptophan • positively charged (basic): lysine, arginine, histidine • negatively charged (acidic): aspartate, glutamate • polar but uncharged: serine, threonine, asparagine, glutamine • chain bending (imino acid): proline • disulfide bridge forming: cysteine • Small: glycine, alanine, serine

6. Another View of Amino Acid Properties

7. One Letter Amino Acid Codes

8. Protein Folding • After the polypeptides are synthesized by the cell, they spontaneously fold up into a characteristic conformation which allows them to be active. The proper shape is essential for active proteins. For most proteins, the amino acids sequence itself is all that is needed to get proper folding. • However, chaperone proteins assist the folding of some proteins, and they help re-fold denatured proteins after events like heat shock. • Proteins fold up because they form hydrogen bonds between amino acids. The need for hydrophobic amino acids to be away from water also plays a big role. Similarly, the charged and polar amino acids need to be near each other. • The joining of polypeptide subunits into a single protein also happens spontaneously, for the same reasons. • Proteins fold into a configuration that minimizes their free energy. • Denaturation (e.g. by heating or alkaline conditions) means the unfolding of proteins into different, non-functional conformations. • It should be noted that some proteins have more than one stable configuration, prions for example. It is also possible that certain regions of proteins have no single stable configuration, more like a glass than a crystal.

9. Prions • A prion is an “infectious protein”. • Prions are the agents that cause mad cow disease (bovine spongiform encephalopathy), chronic wasting disease in deer and elk, scrapie in sheep, and Creutzfeld-Jakob syndrome in humans. • These diseases cause neural degeneration. In humans, the symptoms are approximately those of Alzheimer’s syndrome accelerated to go from onset to death in about 1 year. Fortunately, the disease is very hard to catch and very rare, and they usually have a long incubation time. No cure is known, and not enough is known about how it is spread to do a thorough job of preventing it. Avoid eating brains is a good start though. • The prion protein (PrP) is normally present in the body. Like all proteins, it is folded into a specific conformation, a state called PrPC. Prion diseases are caused by the same protein folded abnormally, a state called PrPSc. A PrPSc can bind to a normal PrPC protein and convert it to PrPSc. This conversion spreads throughout the body, causing the disease to occur. It is also a form of inheritance that does not involve nucleic acids.

10. Peptide Chain • The amino acids are linked together by peptide bonds, which are the same as amide bonds. • The ribosome synthesizes these bonds through a condensation/dehydration reaction • The peptide backbone is made up of the C and N involved in the peptide bond, plus the Cαthat links them. • The beginning of every protein is the N-terminus and the end is the C-terminus. • This means that there is a free amino group at the N terminus and a free acid group at the C terminus. • Although all proteins are synthesized with a methionine (or N-formyl methionine in bacteria) at the N terminus, this amino acid is often removed by an enzyme shortly after protein synthesis.

11. Peptide Bond Conformation • The peptide bond, the C=O bonded to N-H, is a rigid planar structure, with the O and N atoms in trans. • Due to delocalized electrons over the whole 4 atom group, similar to the aromatic ring • An exception: proline creates a bond in the cis configuration, because the N is bonded to the alpha carbon through the side chain. • Thus there are 2 bonds in the backbone that can rotate, the bonds leading to the Cα. • The bond to the N is called phi (φ) and the bond to the C is called psi (ψ). • Phi and psi have preferred angles, caused by hydrogen bonding and steric hindrance.

12. Ramachandran Diagrams • When the phi and psi angles from a large number of proteins are plotted, it is found that most amino acids fall into a small part of the possible bond angles. • These diagrams give rise to the notion of two main secondary structures, the alpha helix and the beta sheet. • There are also left-handed alpha helices and epsilon structures, but they are rarer. • These structures are held together by hydrogen bonds between the atoms of the peptide bond.

13. Secondary Structures in Proteins • Alpha helices are generally rather short, just one or a few turns of the helix. • Beta sheets come in a variety of forms, some containing just a single strand and others having several strands, sometimes parallel and sometimes antiparallel. • Beta sheets are rarely planar—they usually roll up in various ways • Connecting the strands of a beta sheet are tight turns called beta turns, which usually contain amino acids with very small side chains, such as glycine.

14. Protein Structure • Proteins can be described as having various regions of alpha helix and beta sheet, with loops in between that aren’t part of the regular secondary structures.

15. Between Secondary and Tertiary Structures • Secondary structures are local elements, primarily alpha helices and beta sheets. Tertiary structure is the folding pattern of the entire protein. • Alpha helices and beta sheets are often combined into specific elements called folds or supersecondary structures, which can be found in many different proteins. • The two shown here are the Greek key motif, composed of 4 beta sheet strands and the TIM barrel, composed of 8 alpha helices and 8 beta sheet strands. • Proteins are often thought of as being composed on one or more domains. Each domain is a relatively independent unit, separated from others on the linear polypeptide. Introns generally fall between domains in the DNA.

16. Bioinformatics of Proteins • The goal of bioinformatics is to determine the organism’s phenotype from the DNA sequence. From an evolutionary standpoint, phenotype is what natural selection acts on, and thus phenotype is the most conserved element between closely related species. • The phenotype is primarily determined by the organism’s proteins. In turn, protein function is determined by the three-dimensional structure of the protein. Structure is highly conserved in evolution. • It is very easy to go from a DNA sequence to a protein primary structure. The only real complications are determining the exact start site and location of introns. Protein sequence is more conserved than DNA sequence, due to the degeneracy of the genetic code and the fact that many amino acid substitutions have little effect on the protein. • However, going from primary sequence to three dimensional structure has proven to be quite difficult. Large amounts of resources are devoted to this problem, and we will look at some of this later. • But the point is, we try to infer function primarily by comparing protein sequences between genes of known function and new genes. Looking at protein structure would work much better, but it is currently too difficult.

17. Structure Conservation • A good example of 3-dimensional structure being conserved in the absence of sequence conservation is the fructose bis-phosphate aldolase, the glycolytic enzyme that splits fructose 1, 6-bis-phosphate into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (reversibly). • Eukaryotes, Bacteria, and Archaea all have this enzyme, but the lack of sequence homology caused them to be divided into class 1, class 1A, and class 2 enzymes. • X-ray crystallography shows that all three have a common structure, the TIM barrel, composed on alternating alpha helices and beta sheets.