Chapter 3 Proteins

Protein Structure

1. The Primary Structure- essentially, just the sequence of amino acids. (going from an mRNA to a ribosome and the tRNA brings the amino acids and puts them and links them together) So, it is the order of amino acids. 2. Secondary Structure- due to interactions of the peptide backbone. Beta pleated sheet- is a common motif of regular secondary structure in proteins (can be parallel or anti-parallel). Alpha-helix is another common motif in the secondary structure of protein, has a right-spiral conformational "helix". 3. Tertiary Structure- due to side chain interactions. Amino acid side chains may interact and bond in a number of ways. The interactions and bonds of side chains within a particular protein determine its tertiary structure 4. Quaternary structure- proteins that contain multiple polypeptide subunits. Spatial arrangement of each subunit in these proteins

Anfinsen experiment: Protein Folding

1. The experiment demonstrated ribonuclease A could be completely denatured (using b-mercaptoethanol to reduce the disulfide bonds) and then fully renatured, essentially a full return of the catalytic activity was obtained. 2. The experiment showed that all the information necessary to determine the three-dimensional fold was incorporated in the primary structure (amino acid sequence) caveats 1. cannot refold MOST proteins in vitro to active conformation 2. Even for those that can be refolded, it is as a MUCH slower processes than what must happen in the cell

Three characteristic traits of all proteins

1. They adopt at least two stable three-dimensional shapes. 2. They bind to at least one molecular target 3. They perform at least on cellular function

Lysosomes

Another way of degradation. Both membrane and soluble proteins can be carried by vesicles that then fuse with and are digested by lysosomes. Phagosomes containing pathogens also fuse to lysosomes and are destroyed by lysomal proteinases. Once primary lysosome fuse with phagosome they form a secondary lysosome.

Soluble proteinases

How do you destroy proteins outside the cell? Soluble proteinases are key to degrading extracellular proteins, regulated by inhibitors. 1. Cells secrete proteinase enzymes that digest extracellular proteins especially those in the extracellular matrix (ECM) 2. Cells also secrete proteinase inhibitor proteins, which bind to the proteinases and prevent them from functioning. This prevents the proteinases from digesting healthy proteins and neighboring cells. 3. The relative concentration of proteinases and inhibitors in the extracellular fluid determines how much digestion occurs and for how long. This balance can vary during wound healing, tissue remodeling and growth, cancer, and so on.

Regulating Protein Function and Conformation

Key Concepts: To be functional, proteins must be able to be activated and inactivated; these two functional states are reflected by different shapes (conformations) 4 Mechanisms for controlling protein activity (duration and mechanisms vary per protein) 1. Chemical modification of amino acids- in a protein can cause switch between states 2. Binding of small molecules to allosteric sites- can switch states 3. Binding of regulatory proteins- to a protein can cause switching between states 4. Protein degradation- controls how much protein in a cell, hence how much activity

5 classes of amino acids based on chemistry of side group

Need to know one example from each class. 1. Non-polar, aliphatic side groups- Methionine 2. Polar, uncharged side groups- Threonine 3. Aromatic side groups- Phenylalanine. 4. Positively charged side groups- Lysine 5. Negatively charge side groups- Glutamate

Quarternary Structure

The quaternary structure refers to how these protein subunits interact with each other and arrange themselves to form a larger aggregate protein complex. The final shape of the protein complex is once again stabilized by various interactions, including hydrogen-bonding, disulfide-bridges and salt bridges.

Mech. #1 Chemical Modification

There are a lot of way the amino acid side chains can get modified. Know these 7 most common types of covalent modifications that can switch the protein from one modification to another. Not every aa can get modified. 1. Disulfide bonds (CYS) 2. Ubiquitin (LYS) 3. Sugars (ASN, SER, THR) 4. Lipds (CYS) 5. Phosphate groups (TYR, SER,THR) 6. Methyl groups (LYS, ARG) 7. Acetyl groups (LYS) many chemical modifications are reversible. Proteins can switch back and forth between active and inactive sate many times.

Tertiary Structure Continued

What are these R groups and how do they affect the shape of protein? 1. hydrophobic side chains are not going to want to be on the outside of a protein molecule that's inside of an aqueous solution(exposed to water), so this may "bend" the protein structure and affect its shape. (ex. Valine) 2 .a hydroxl group on a side chain, making it polar, also making it more hydrophilic, so it might sit on the outside of a protein in contact with an aqueous solution. Or it may form hydrogen bonds with other side chains. (ex. Serine) 3. There may be ionic side chains, side chains that have a charge. One R chain may have a positive, another R chain may have a negative charge, and therefore they are going to be attracted to each other and have an ionic bond. 4. under certain conditions, side chains may form covalent bonds with other side chains. 5. most proteins have on highly stable tertiary structure, which is often organized around a core region of hydrophobic residues.

Mech #3 Binding regulatory proteins

proteins can bind and regulate other proteins. ex. Binding of alpha-I (protein) to AC (adenylate cyclase) inhibits AC activity. Binding of alpha-s protein to AC activates AC activity. ex. Protein kinase A activity is regulated indirectly by small molecule (cAMP) and directly by a regulatory subunit that inactiviates it. <---example of Mech 2 and 3

Proteins are constructed from...

1. 20 different amino acids 2. These 20 are classified into 3 or 4 groups, according to the chemical nature of their side chains

Antibody Structure

1. Antibodies are immune system-related proteins called immunoglobulins. 2. Each antibody consists of 4 polypeptides-two heavy chains and two light chains joined to form a "Y" shaped molecule. 3. antibodies are multimers: aggregate of multiple molecules that is held together with non-covalent bonds. Although there are also covalent bonds too. 4. The amino acid sequence in the tips of the "Y" varies amongst different antibodies, the variable region. This variable region gives the antibody its specificity for binding antigen. 5. The constant region determines the mechanism used to destroy antigen.

3 Broad Classes of Protein based on FUNCTION

1. Cellular change- a. enzymes (catalyzes things. change proteins or other type molecules)(enzymes are proteins and/or RNAs that catalyze cellular reactions); b. regulatory proteins (affect gene expression including protein activity, binding to DNA, can affect RNA polymerase); c. motility proteins (effect movement of cells or movement within cells) 2. Response to environment- a. Transport proteins (move molecules in and out of cell or organelle); b. Hormonal proteins (serves as signals between cells); c. Receptor proteins (recognizes such as binding to hormones or other signals 3. Cellular maintenance - a. Defensive proteins (protects cells against pathogens); b. Storage proteins; c. Structural proteins (scaffold giving cells/organelles their shape/size)

Chaperonins

1. Chaperonins are protein complexes that bind and help fold newly synthesized. 2. Chaperonins can bind to a polypeptide even as it is being synthesized by a ribosome, thereby helping prevent irreversible misfolding. 3. After the entire polypeptide has been synthesized, chaperonins assist its folding into the most stable functional shape (the native conformation) 4. In some cases, chaperonins form a large complex called microcages, and polypeptides can enter these complexes to help them fold properly. 5. in vivo, proteins requre both chaperonins and the primary sequence to fold properly/efficiently

Alpha- carbon

1. Each amino acid contains a central carbon atom called the "___", attached to four different molecular structures. 2. Two of these are functional groups ( an amino acid and a carboxylic acid group, hence the term amino "acid") and one is a single hydrogen atom. The fourth structure, often called an amino acid side chain (commonly abrv. R or R group) differed in each different amino acid. The R group plays a big role in defining the shape of the proteins.

Amino Acids form Linear Polymers

1. Proteins are composed of polypeptides, which are linear polymers made up of 20 different amino acids 2. Amino acids in polypeptides are linked together by peptide bonds. 3. Amino acids have a characteristic structural polarity that is reflected in the polarity of polypeptides. 4. In many cases, several polypeptides must join together to form a functional protein. 5. All proteins exhibit three characteristic traits

Amino acids linked through peptide bonds

1. Side group confers unique properties on each of the 20 amino acids 2. Amino acids are linked by single bonds called peptide bonds, formed by the carboxylic acid group of one amino acid and the amino group of a second amino acid 3. Proteins, like nucleic acids, have a polarity. That is, overall protein, like each amino acid, has an N-terminus and a C-terminus. Conventionally, the N-terminus is represented to the left. N---->C

Non-polar and polar groups

1. The group containing the largest number of amino acids (9) is called the non-polar or hydrophobic group. 2. These amino acid side chains are composed almost entirely of carbon and hydrogen atoms 3. 6 amino acids belong to the polar group, and each of the polar side chains contains a polar functional group (hydroxyl, sulfhydrl, or amid) 4. Ionic group of 5 amino acids is sometimes separated into 2 subgroups: two containing a negatively charged carboxylic acid functional group( acidic group), and three containing a positively charged nitrogen-based functional group(amine or imine-basic subgroup). All 5 of these side chains contain one charged atom (O- or N-)

Mech. #4: Protein degradation

1. all those others were reversible. Protein degradation is irreversible. and often is acting on damaged or misfolded proteins. 2. protein degradation both regulates protein levels and recycles the amino acid subunits for use in new proteins 3. Three mechanisms that degrade proteins 1. Proteasomes- nucleus and cytosol 2. Lysosomes- organelle degrades usually proteins from vesicles 3. Soluble proteases- works outside the cell

Types of Peptides based on size/structure

1. dipeptide- two amino acids held together by a single peptide bond. Additionally are tripeptide (three amino), tetrapeptide (4 amino) notice that the number refers to the number of amino acids in the structure not the number of peptide bonds 2. Oligopeptides- designates a few amino acids help together by peptide bonds, think "group". Olio, di, tri, and tetra, help in cell-to-cell signals 3. polypeptide- typically 10 amino acids are more) e.g. newly synthesized polypeptides are not yet folded into protein 4. Protein- a polypeptide 40 or more amino acids and folded structure.

Proteasome

Cylinder shape. Has proteases on the surface that degrade the protein. This regulation happens largely on the caps of the Proteasome. Generally constructed of an alpha ring, then two beta rings, then another alpha ring. The interior is called the 20s core

5 Types of Chemical bonds between amino acids (Lecture)

Sometimes between R group and sometimes between the actual backbone. 1. Disulfide bond- the only covalent bond, so by far the most stable. Cystine amino acid forms a covalent bond with another Cystine. is the most stable and strong bond. 2. Hydrogen bonds- between the backbone of amino acids (how you get alpha-helix and beta-sheet) 3. ionic bonds- often in interior of protein. and they neutralize the charge. They occur in interior and they neuralize charged residues keeping interior hydrophobic. 4. Hydrophobic interactions- also in interior. it pulls structure together. Interactions between non-polar residues on one amino acid and non-polar residues on another amino acid. 5. van der Waals forces- another hydrophobic interaction. acts very short distances. amino acids are very close together. non-covalent bonds are important when protein changes its conformation.

Note about structural changes

These protein structural changes are rather subtle in appearance. Could be just one little loop of the protein that is changing

Mech #2: Small molecules bind allosteric sites

allosteric sites is a site on a different part of the protein. For instance ATP may bind to the site, allowing the protein to bind to the target. Allosteric binding site is actually different from the primary binding site. 2. Proteins bind and release these molecules extremely quickly from allosteric sites. So activation or inactive states are relatively short lived 3. Often used to vary the activity of enzymes. 4. Quite costly- cells expend a great deal of energy to maintain a steady supply of them. 5. An example could be how ATP can form noncovalent bonsd with amino acids in allosteric sites to increase the binding activity. Eventually the allosteric factor is relased and the protein changes back to the unbound shape.