Essential Cell Biology 3rd: Ch 4 Protein Structure and Function

INTRO

• Not only do proteins make up the majority of the dry weight of the cell, but they execute almost all of its myriad functions.

• All enzymes are proteins, but not all proteins are enzymes.

• The many functions that proteins carry out are possible do to the huge number of different shapes they are capable of adopting.

THE SHAPE AND STRUCTURE OF PROTEINS

• By understanding the structure at the atomic level allows us to describe how the exact shape of each protein determines its function in the cell.

• The shape of a protein is specified by its amino acid sequence.

• Proteins are composed of a combination of the 20 different amino acids linked together by covalent peptide bonds. Because of this proteins are referred to as either polypeptides or polypeptide chains. 

 

• Each protein's amino acids are present in a specific unique order, called the amino acid sequence. This sequence is identical from one molecule of the protein to the next.

• The side chains of the amino acid are what give it its unique properties  

 

• Each polypeptide contains a polypeptide backbone that supports the amino acid side chains. This backbone is made from the repeated core sequences of the amino acids.

• The shape of the protein chain is constrained by different sets of noncovalent bonds that involve not only the polypeptide chain but also the amino acid side chains.

• Hydrogen bonds, electrostatic attractions, and van der Waals attraction.

• Hydrophobic interactions also play a role in the shape of proteins. In the aqueous environment of the cell, hydrophobic nonpolar side chains of particular amino acids tent to be forced together to minimize the disruptive effect on the hydrogen bonded network of the surrounding water molecules.

• These amino acids are phenylalanine, leucine, valine, and tryptophan tend to cluster in the interior of the folds of the protein chains and tend to form hydrogen bonds with other amino acid side chains or the polypeptide backbone.

• Proteins fold into a conformation of lowest energy

• The conformation of a protein is determined by the order of the amino acid chain. The structure can be denatured if treated with solvents that disrupt the noncovalent interactions, but when the denaturing solvent is removed the protein will return, or renature, into its original conformation.

• This shows that the information needed to determine the three dimensional structure of a protein is determined by its amino acid sequence.

• Each protein normally folds into one conformation, which minimizes the free energy of the structure (G). The proper folding of proteins can usually happen on its own, but in cells its generally assisted by special proteins called molecular chaperones.

• This structure of the protein may change slightly though when it interacts with other molecules, and is crucial to it's function.

• If the protein folds incorrectly they can form what is called Aggregated proteins, which can damage the cell or even whole tissues. Prion diseases, Alzheimer's, and Huntington's diseases are examples of these. And in some cases aggregated proteins can cause normal proteins to change into aggregated proteins.

• The reasons the protein chaperones are so important is because they prevent the newly synthesized protein chains from associated with the wrong proteins in the crowded cytoplasm. But the overall structure of the protein is still determined by the amino acid sequence.

• Proteins come in a wide variety of complicated shaped

• Proteins are the most structurally diverse macromolecules in the cell.

• On average they are between 50 and 2000 amino acids, but they can range anywhere from 30 to 13000. 
• these are just some of the proteins that we will use in this course... 

 

• We can now easily determine the amino acid sequence of proteins by sequencing the gene that encodes it and using the universal code to determine the order of the amino acids in the polypeptide chain.

• Even though we know the sequence of proteins we have not yet figured out how to reliably predict the three dimensional conformation of proteins from only the sequence of the amino acids. The only way to currently determine this shape is by x-ray crystallography or nuclear magnetic resonance (NMR)

• There are four different ways to model protein's three dimensional structure

• Polypeptide backbone model

• Ribbon model 

• Wire model that includes the amino acid side chains

 

• Space filling model

• The Alpha Helix and the Beta sheet are common folding patterns

• Even though each protein folds in its own way, there are two common folding patters.

• Alpha helix

• Beta (pleated) sheet 

 

• These two patterns are common because they result from hydrogen bonds that form between the N-H and C=O groups in the backbone. Since the backbone is involved in this and not the side chains, these two forms can be generated by many different amino sequences.

• Helices form readily in biological structures

• A helix is made by placing similar subunits next to each other in the same repeated relationship to the one before, and it can be either right handed or left handed.

• And Alpha helix is made from a structurally rigid cylinder, where there is a hydrogen bond between every fourth amino acid linking the C=O of one peptide bond to the N-H of another.

• Sometimes two or three Alpha helices will wrap around one another to form a particularly stable coiled-coil structure. This structure forms when Alpha helices have more of their nonpolar (hydrophobic) side chains on one side so they can twist around each other to minimize their contact with the cytosol.

• Beta sheets form rigid structures at the core of many proteins.

• Beta sheets are made when hydrogen bonds form between segments of polypeptide chains lying side by side.

 

• Parallel beta sheets are formed when the structure consists of neighboring polypeptide chains that run in the same orientation, from the N-terminus to the C-terminus. When the sheet is folded back and forth over itself, it forms an antiparallel beta sheet.

• Both sheets form a rigid pleated structure that forms the core of many proteins.

• Proteins have several levels of organization

• The Alpha helix and Beta sheets are only the first level of protein structure, there are other levels of structure build upon structures forming the final protein's conformational structure.

• Primary structure - the amino acid sequence.

• Secondary structure - the Alpha helix and the Beta sheet

• Tertiary structure - the full three dimensional conformation of the entire polypeptide chain.

• Quaternary structure - the complex of more than one polypeptide chain interacting with each other.

• Protein domain - any segment of a polypeptide chain that can fold independently into a compact, stable structure.

• A domain usually consists of about 100-250 amino acids, and are often associated with different function. Ie. the polymerase involved in DNA replication.

• Few of the many possible polypeptide chains will be useful.

• While the possible number of combinations being 20^n, the actual stable, biologically useful combinations are much less than this number.

• Proteins have adapted over time, and some are even conserved across species.

• A change in even a few amino acids in a protein can disrupt the structure of the protein and eliminate its function.

• Proteins can be classified into families

• Many proteins can be grouped into families, showing us the evolution of proteins through time.

• Protein families - each family member has an amino acid sequence and a three-dimensional conformation that closely resembles that of the other family members.

• Large protein molecules often contain more than one polypeptide chain.

• The noncovalent bonds that help to hold the protein in its conformation, can also allow proteins to bind to each other to produce larger structures.

• Binding site- any region on a proteins surface that can interact with another molecule through sets of noncovalent bonds.

• Subunit- each individual protein that makes up a larger protein through noncovalent binding.

• DIMER- a molecule or molecular complex consisting of two identical molecules linked together

• Proteins can assemble into filaments, sheets, or spheres

• It is possible to dissociate structures into their constituent macromolecules. If we then mix these molecules back together, they will reassemble spontaneously into the original structure.

• This shows that the cell structure is self-organizing.

• Some types of proteins have elongated fibrous shapes

• Most proteins previously mentioned are globular proteins - more of a rounded compact shape. But others are simple, elongated three dimensional structures referred to as fibrous proteins.

• Examples would be Keratin, the extracellular matrix, Collagen, and Elastin. 

 

• Extracellular proteins are often stabilized by covalent cross-linkages

• Many protein molecules are either attached to the outside of a cell's plasma membrane or secreted as part of the extracellular matrix.

• Proteins are often strengthened by covalent cross-linkages to help prevent their deformation do to extracellular conditions.

• The most common cross-linkage bond is the Sulfur-Sulfur, disulfide bond.

 

• These bonds are catalyzed during formation by an enzyme in the ER. 

• S-S bonds do not change the shape of the protein, but act more like a staple, to help reinforce the favored conformation.

• These bonds are not formed in the cytosol, as the extra reinforcement is not needed in the mild environment of the cell.

HOW PROTEINS WORK

• A protein's function depends on its ability to bind specifically to other molecules. This can allow them to act as catalysts, structural supports, signal receptors, and tiny motors.

• All proteins bind to other molecules

• Regardless of the strength of the bond between molecules, the selectivity for the bond is always very specific.

• Each protein molecule can bind to just one, or a few, molecules out of the many thousands of different molecules it may encounter.

• Ligand- any substance that is bound to a protein, whether it is an ion, a small molecule, or a macromolecule.

• An encounter that will result in a bond will only occur if the surface contours of the ligand molecule fit very closely to the protein.

• Binding site- the region of the protein that associates with a ligand. And is usually formed by a cavity in the protein surface, formed by a particular arrangement of amino acids.

• But changes to interior amino acids that do not provide binding sites matter as well, these can distort the shape of the binding sites and proteins resulting in loss of function.

• The binding sites of antibodies are especially versatile.

• Antibodies - immunoglobulins, are proteins produced by the immune system in response to foreign molecules. 

• These will bind very tightly to the target either inactivating it or marking it for destruction.

• Antigen - the antibodies target

• Antibodies are Y shaped with two identical binding sites that complement a small portion of the surface of the antigen molecule. 

 

• The amino acid sequence in these loops can be changed by mutation without altering the basic structure of the antibody.

• Enzymes are powerful and highly specific Catalysts.

• Many proteins just bind to other molecules and their job is done, but for enzymes, this is only the first step.

• Enzymes can be grouped into functional classes based on the chemical reactions they catalyze.

Enzyme Class	Biochemical Function
Hydrolase	General term for enzymes that catalyze a hydrolytic cleavage reaction.
Nuclease	Breaks down nucleic acids by hydrolyzing bonds between nucleotides
Protease	Breaks down proteins by hydrolyzing peptide bonds between amino acids.
Synthase	General name used for enzymes that synthesize molecules in anabolic reactions by condensing two molecules together.
Isomerase	Catalyzes the rearrangement of bonds within a single molecule.
Polymerase	Catalyzes polymerization reactions such as the synthesis of DNA and RNA.
Kinase	Catalyze the addition of phosphate groups to molecules. Protein kinases are an important group of kinases that attach phosphate groups to proteins.
Phosphatase	Catalyzes the hydrolytic removal of a phosphate group from a molecule.
Oxido-reductase	General name for enzymes that catalyze reactions in which one molecule is oxidized while the other is reduced. Enzymes of this type are often called oxidases, reductases, or dehydrogenases.
ATPase	Hydrolyzes ATP. Many proteins with a wide range of roles have an energy-harnessing ATPase activity as part of their function, including motor proteins such as myosin and membrane transport proteins such as the sodium-potassium pump.

• Lysozyme illustrates how and enzyme works.

• Lysozyme severs the polysaccharide chains that form the cell walls of bacteria through hydrolysis, causing the cell wall to rupture.

• They lysozyme holds six sugars with its active site distorting only one of the sugar-sugar bonds, then catalyzes the addition of a water molecule to the distorted bond, breaking it.

• This does not happen on its own partially because of the activation energy required for this to occur, but also because the molecule would have to be distorted into a particular shape, the transition state, for the water to interact with the bond.

• Step by step

1. The enzyme stresses its bound substrate by bending some critical chemical bonds in one sugar, so that the shape of the sugar more closely resembles the shape of high-energy transition states formed during the reaction. 
2. The negatively charged aspartic acid reacts wit the C1 carbon atom on the distorted sugar, breaking this sugar-sugar bond and leaving the aspartic acid covalently linked to the site of the bond cleavage.  
3. Aided by the negatively charged glutamic acid, a water molecule reacts with the C1 carbon atom, displacing the aspartic acid and completing the process of hydrolysis.

 

• Most drugs inhibit enzymes

• Pharmaceutical companies often target enzymes of interest to develop medications to help treat or prevent illness.

• Tightly bound small molecules add extra function to proteins.

• Proteins often use non-protein molecules to preform functions that would be difficult of impossible using amino acids alone.

• Ex. Retinal is attached via a lysine side chain to rhodopsin, the purple light sensitive pigment made by the rod cells. The retinal will change its shape when it absorbs a photon, which is then amplified by the rhodopsin, triggering a network of enzymatic reactions that lead to an electrical signal being sent to the brain.

• Enzymes, like other proteins, usually have a small molecule or metal atom associated with their active site that assists with their catalytic functions.

HOW PROTEINS ARE CONTROLLED

• Protein and enzyme activity are controlled at many levels.

• Gene expression

• Confining sets of enzymes in subcellular compartments

• The proteins shape can be altered, effectively altering its function.

• The catalytic activities of enzymes are often regulated by other molecules.

• Feedback inhibition- an enzyme acting early in a reaction pathway is inhibited by a late product of that pathway.

• This occurs when a molecule other than a substrate specifically binds to an enzyme at a regulatory site outside of the active site, which can alter the rate at which the enzyme works.

• This works almost instantaneously, and is rapidly reversed when product levels fall.

• Feedback inhibition is a negative regulation, it prevents an enzyme from acting.

• Positive regulation - the enzyme's activity is stimulated by a regulatory molecule rather than being shut down.

• This tends to occur when one metabolic branch stimulates the activity of an enzyme in another pathway.

• Allosteric enzymes have binding sites that influence one another.

• Enzymes have active sites and at one or more sites that recognize regulatory molecules, and the substrate and the regulatory sites must somehow "communicate" in a way that allows the catalytic events at the active site to be influenced by the binding of the regularity molecule at the separate site.

• This "communication" is accomplished through conformational changes that take place in the enzyme. The binding of the regulatory molecule causes a conformational change, shifting the active binding site.

• Allosteric - they (proteins) can adopt two or more slightly different conformations. By shifting from one to the other, activity can be regulated.

• This is true for enzymes as well as many proteins including receptors, structural proteins, and motor proteins.

• Phosphorylation can control protein activity by triggering a conformational change.

• Eucaryotic cells can also regulate protein activity by adding a phosphate to one of the amino acid chains.

• Protein phosphorylation - This will cause a major conformational change because the phosphate group carries two negative charges attracting a cluster of positively charged amino acid side chains.

• More than 1/3 of the proteins in mammals appear to use this at some point, and can either inhibit or stimulate protein activity. Inhibition or stimulation is dependent on the protein involved AND the site where it is being phosphorylated. 

 

• Protein phosphorylation involves the enzyme- catalyzed transfer of the terminal phosphate group of ATP to the hydroxyl group on a serine, threonine, or tyrosine and is catalyzed by a protein kinase. The removal, or dephosphorylation, is catalyzed by a protein phosphatase.

• The removal of the phosphate groups occurs in response to signals given when the state of the cell changes.

• GTP -binging proteins are also regulated by the cyclic gain and loss of a phosphate group

• This form of regulation uses either guanosine triphosphate (GTP) or guanosine diphosphate (GTP). The proteins are in their active states when GTP is bound to the protein. Then protein itself can then hydrolyzes the GTP to GDP (releasing a phosphate) and change to an inactive conformation. 

 

• Nucleotide hydrolysis allows motor proteins to produce large movements in cells

• Motor proteins - proteins whose major function is to move other molecules. 

• For the motor proteins to move backwards, the hydrolysis of ATP would have to be reversed as well. And since this is not thermodynamically favorable, it does not occur, and the motor protein is moved rapidly forward. 

 

• Proteins often form large complexes that function as protein machines.

• Larger proteins with many domains tend to have more elaborate functions compared to single domain proteins.

• Protein machines - a highly coordinated, linked set of 10 or more proteins

• This is driven by hydrolysis or bound ATP or GTP

• Covalent modification controls the location and assembly of protein machines

• Most protein machines form a specific sites in the cell and are activated only when and where they are needed, and is generally accomplished by the covalent addition of a modifying group to one or more specific amino acid side chains on the participating proteins.

• There are more than 200 types of covalent modifications that can occur in the cell, each helping to regulate protein function.

• Phosphorylation can not only inhibit or increase protein activity, but it can also promote the assembly of  proteins into larger complexes.

• Regulatory protein code - a set of covalent modifications that a protein contains at any moment.

• The attachment or removal of these modifying groups controls the behavior of a protein, changing its activity or stability, its binding partners, or its location inside the cell.

HOW PROTEINS ARE STUDIED

• Cells can be grown in a culture dish

• In vitro - reactions carried out in a test tube in the absence of cells.

• In vivo - any reaction taking place inside a living cell, even cells that are growing in culture.

• Cultured cells are accessible to study in ways that are often not possible in intact tissues.

• The cultured cells can also provide a ready source of raw materials.

• Purification techniques allow homogeneous protein preparations to be obtained from cell homogenates.

• Steps to purification of proteins through chromatography. 

 

1. Break open the cells to release their contents. The contents is called a cell homogenate.
2. Initial fractionation to separate out the class of molecules of interest.
3. Chromatography. This can be used to separate the individual components into different portions, which can then be put through an assay to determine which fractions contain the protein of interest.
4. Additional chromatography can then be used to reach the pure form of the protein.

• The most common forms of chromatography separate polypeptides on the basis of their size, charge, or their ability to bind to a particular chemical group.

• Antibodies can sometimes be used to help extract proteins of interest. 

• This can also be used to isolate proteins that interact physically with the protein as well. To do this the purified protein is attached to the matrix of the chromatography column, and the proteins that bind to this protein will collect in the column and can then be eluted by changing the composition of the washing solution.

• Electrophoresis can also be used to separate proteins. 

 

1. A mixture of proteins are loaded into a polymer gel 
2. This is then subjected to an electric field. The proteins will them migrate through the gel at different speeds depending on their size and net charge.   
3. If the proteins are too similar, or the number too great, a two dimensional gel electrophoresis can be used.  
4. The bands or spots can be visualized by staining

• Large amounts of almost any protein can be produced by genetic engineering techniques.

• Advances in genetic engineering now allows us to produce a large amount of almost any desired protein

• We can use these proteins in research, medicine, and other types of therapy.

• We can also genetically engineer techniques to design proteins that preform novel tasks.

• Automated studies of protein structure and function are increasing the pace of discovery.

• Proteomics - the large scale study of cellular proteins in which the activities or structures or hundreds, even thousands, of proteins are analyzed by highly sensitive, automated techniques.