Cell biology Protein Structure and Function Chapter 4

Tightly bound small molecuels add extra functions to proteins

proteins often emply small nonprotein molecules to perform functions that would be difficult or impossible using amino acids alone.

Enzymes greatly accelerate the speed of chemical reactions

The rate of an enzyme reaction increases as the substrate concentration increases, until a maximum value (Vmax) is reached. At this point, all substrate-binding sites on the enzyme molecules are fully occupied, and the rate of the reaction is limited by the rate of the catalytic process on the enzyme surface. For most enzymes, the concentration of substrate at which the reaction rate is half maximal (Km) is a direct measure of how tightly the substrate is bound, with a large value of Km (a large amount of substrate is needed) corresponding to weak bonding. Michaelis Constant (Km): A small Km indicates that a substrate binds very tightly to the enzyme due to a large number of noncovalent interactions. A large Km indicates weak bonding.

Large protein molecules often contain more than one polypeptide chain

The same type of weak noncovalent bonds that enable a polypeptide chain to fold into a specific confromation also allow protines to bind to each other to produce larger structures in the cell. Binding site: Any region on a proteins surface that interacts with another molecule through sets of noncovalent bonds -if a binding site recognizes the surface of a second protein, the tight binding of two folded polypeptide chains at this site will create a larger protein which quaternary structure has a precisely defined geometry Subunit: each polypeptide chain in such a protein as aboce, each subunit may contain ore than one domain Dimer: two identcial folded polypeptide chains forming a symmetrical complex of two protein subunits held together by interactions between two identical binding sites

B sheets form rigid structures at the core of many proteins

A B sheet is made when hydrogen bonds form between segments of a polypeptide chain that lie side by side. When the neighobring segments run in the same orientation, the structure forms a parallel B sheet. When they run in opposite directions the structure forms an antiparallel sheet

The catalytic activities of enzymes are often regulated by other molecules

A common type of control occurs when a specific molecules other than a substrate binds to an enzyme at a spcial regulartory site, altering the rate at which the enzyme converts its substrate to product. In feedback inhibition for exmaple: An enzyme acting early in a reaction pathway is inhibited by a molecule produced later in that pathway. Thus, when ever large quantities of the final product begin to accumulate, the product binds to an earlier enzyme and slows down it scatalytic action, limiting further entries of substrates into that reaction pathway feedback inhibition can occur almost instantaneously and is rapidly reversed when product decreases Negative regulation: Prevents an enzyme from acting Positive regulation: Enymes activity is stimulated by a regulatory molecule rather than being suppressed -positive regulation occurs when a product in one branch of the metabolic pathway stimulates the activity of an enzyme in another pathway

Helices form readily in biological structures

A helix is generated simply by placing many similar subunits next to one another, each in the same strictly repeated relationshio to the one before. Since it is very rare for subunits to join up in a striaght line, this arrangement will generaly result in a structure that resembes a spiral staircase an A helix is generated when a single polypeptide chain turns around itself to form a structurally rigid cylinder. -a hydrogen bond is made between every fourth amino acid linking C=O of one peptide bond to the N-H of another. -right handed -Short regions of a helix are especially abundant in proteins that are embedded in cell membranes, such as transport proteins and receptors. -portions of a transmembrane protein that cross the lipid bilayer usually form an a helix composed largely of amino avids with nonpolar side chains. The polypeptide bakbone which is hydrophilic is hydrogen bonded to itself inside the a helix, where it is shielded form the hydrophobic lipid environment of the membrane by the protruding nonpolar side chains -Sometimes two or three a helices will wrap around one another to form a particularly stable structure called a coiled coil. THis structure forms when a helces have most of their nonpolar (hydrophobic) side chains along one side, so they can twist around each other with their hydrophobic side chains facing inward-minimizing contact with the aquesous cytosol

ATP hydrolysis allows motor proteins to produce directed movements in cells

Conformational changes also enable certain specialized proteins in eukaryotic cells to drive directed movements of cells and their components Motor proteins: Generate the forces responsible for muscle contraction and most other eukaryotic cell movements to force a protein to proceed in a single direction, the conformational changes must be unidirectional. To achieve such directionality, one of the steps must be made irreversible. For most porteins that are able to move in a single direction for long distances, this irreversibility is achieved by coupling one of the conformational changes to the hydrolysis of an ATP molecule that is tightly bound to the protein whichis why motor proteins are also ATPases. A great deal of free energy is released when ATP is hydrolyzed making it very unlikely that the protein will undergo a reverse shape change as required for moving backward.

Lysozomes illustrate how an enzyme works

Lysozyme: An enzyme that acts as a natural antibiotic in egg white, salive, tears, and other secretions by severing the polysaccharide chain walls of bacteria. Hydrolysis: Enzyme adds a molecule of water to a single bond between two adjacent sugar groups in the polysaccharide chain, thereby causing the bond to break. Reaction is favorable as the free energy of the severed chain is lower than the free energy of the intact chain. For colliding water molecule to break the bond linking two sugars, the polysaccharide molecule has to be distorted into the transition shape in which the atoms around the bond have an altered geometry and electron distribution. To distort the polysaccharide in this way requires a large input of energy which is where the enzyme comes in Lysozymes have a binding site on its surface termed the active site which is where catalysis takes place. Because its substrate is a polymer, lysozymes active site is a long groove that cradles six of the linked sugars in the polysaccharide chain at the same time. Once this enzyme -substrate forms the enzyme cuts the polyssaccharide by catalyzing the addition of a water molecule to one of its sugar-sugar bonds and the severed chains are quickly released freeing the enzyme for further cycles of cleavage. -lysosymes recognize its substrate through the formation of multiple noncovalent bonds Enzymes can encourage reactions in many ways: 1.) enzymbe binds to two substrate molecules and orients them precisely to encourage a reaction to occur between them 2.) binding of substrate to enzyme rearranges electrons in the substrate, creating partial negative and positive charges that favor a reaction 3.) enzyme strains the bound substrate molecule forcing it toward a transition state that favors a reaction

Many Interacting proteins are brought together by scaffolds

Many protein complexes are brought together by scaffold proteins,, large molecules that contain binding sitesrecognized by multiple proteins. By binding specific set of interacting proteins a scafold can greatly enhance the rate of a particular chemical reaction or cell process while also confining this chemistry to a particular area of the cell.

Covalent modifications also control the locations and interaction of proteins

Phosphorylation can do more than control a proteins activity it can create docking sites where other proteins can bind thus promoting the assembly of proteins into larger complexes Many proteins are modified by the addition of an acetyl group to a lysine side chain. modification of a protein at multiple sites can control its behavior The set of covalent modifications that a protein contains at any moment constitutes an important form of regulation. The attachment or removal of these modifying groups can change a proteins activity or stability, its binding partners or its location inside the cell

Proteins have several levels of organization

Primary structure: Amino acid sequence Secondary structure: A helices and B sheets that form within certain segments of the polypeptide chains Tertiary structure: The full three dimensional confroamtion formed by an entire polypeptide chain including the a helices, b sheets, and all other loops and folds that form between the N- and C- temini Quaternary structure: If the protein molecule exists as a complex of more than one polypeptide chain, then these interacting polypeptides form the quaternary structure Protein domain: Any segment of a polypeptide chain that can fold independenty into a compact stable structure. A protein domain usually consists of 40 and 350 amino acids folded into a helices and b sheets and other elements of strucute and it is th emodular unit from which many larger proteins are constructed

How proteins work

*All proteins bind to other molecules -the binding of a protein to other biological molecules always shows great specificity: each protein molecule can bind to just one or a few molecules out of the many thousands of different ones it encounters Ligand: Any substance that is bound by a protein, whether it is an ion, small organic molecule or a macromolecule the ability of a protein to bind selectively and with high affinity to a ligand is due to the formation of a set of weak noncovalent interactions, hydrogen bonds, electrostatic attractions, and van der waals attractions, plus favorable hydrophobic forces. -each individual noncovalent interaction is weak so that effective binding requires many such bonds to be formed simultaneously. This is possible only if the surface contours of the ligand molecule fit very closely to the protein when molecules have poorly matching surfaces few noncovalent interactions occur and the two molecuels dissociate as rapidly as they come together. This is what rpevents incorrect and unwanted associates from forming between mismatched molecules. Binding site: Cavity in the protein surface formed by a particular arrangement of amino acid side chains associated with a ligand

How proteins are controlled

-At the most fundamental level, the cell controls the amount of protein it contains. It can do so be controlling the expression of the gene that encodes that protein and it can regulate the rate that the protien is degraded -the cell also controls protein activities by confining the participating proteins to particular subcellular compartments. Some of these compartments are enclosed by membranes and others are created by the proteins that are drawn there -the activity of an individual protein can be rapidly adjusted at the level of the protein itself all of these mechanisms rely on the ability of proteins to interact with other molecules including other proteins. These interactions can cause proteins to adopt different conformations and thereby alter function

Phosphorylation can control protein activity by causing a conformational change

Another method eukaryotic cells use to regulate protein activity involves attaching a phosphate group covalently to one or more of the proteins amino acid side chains. -since each phosphate group contains two negative charges the enzyme catalyzed addition of a phosphate group can cuase a conformational change by for example attracting a cluster of positively charged amino acid side chains form somewhere else in the same protein. This structural shift can in turn affect the binding of ligands elsewhere in the proteins surface altering the proteins actvity -removal of a phosphate group by a second enzyme will return it to tis original composition -addition and removal of a phosphate group from specific proteins often occur in response to signals that specify some change in a cell's state *protein phosphorylation involves the enzyme catalyzed transfer of the terminal phosphate group of ATP to the hydroxyl group on a serine, threonine, or tyrosine side chain of the protein and this reaction is catalyzed by a protein kinase removal of the phosphate group (dephosphorylation) is catalyzed by a protein phosphatase

Humans produce billions of different antibodies, each with a different binding site

Antibodies: Immunoglobulin proteins produced bu the immune system in response to foreign molecules, especially those on the surface of invading microorganisms. -each antibody binds to a particular target molecule extremely tightly, either inactivating the target directly or marking it for destruction Antigen: Antibodies target molecule Antibodies are y shaped molecuels wit htwo identical antigen binding sites each of which is compleemtnart to a small portion of the surgace of the antigen molecule.

Proteins often form large complexes that function as machines

As proteins grow from being small with one domain to large with many domains their functions become elaborate. Protein machines: Hydrolysis of bound nucleoside triphosphates (ATP or GTP) drives an ordered seriesof confomrational changes in some of the individual protein subunits, enabling the ensemble of proteins to move coordinately. the ensemble of proteins move coordinately. In these machine like complexes the appropriate enzymes can be positioned to carry out successive reactions in a series as during the synthesis of some ribosomes

Enzymes are powerful and highly specific catalysts

Enzymes: These molecules responsible for nearly all of the chemical transformations bind to one or more ligands called substrates and convert them into chemically modified products doing this over and over again without themselves being chnages. Enzymes act as catalysts that permit cells to make or break covalent bonds at will

Proteins can be purified from cells or tissues

First step is to break open cells to release their contents -resulting slurry is called a cell homogenate or extract this physical disruption is followed by an initial fractionation procedure to separate out the class of molecules of interest with this collection of proteins the job is then to isolate the desired protein *Standard approach involves purifying the protein through a series of chromatography steps which use different materials to separate the individual components of a complex mixture into portions or fractions based on the properties of the protein such as size, shape, or electrical charge. Chromatogrpahy is continued until pure Affinity chromatohraphy: The most efficient forms of protein chromatograpgy separating polypeptides on the basis of their ability to bind to a particular molecule. -can also be used to isolate proteins that interact physically with a protein being studied Electrophoresis: In this technique a mixture of proteins is loaded onto a polymer gel and subjected to an electric field. The polypeptides will then migrate through the gel at different speeds depending on size and net charge.

Some types of proteins have elongated fibrous shapes

Globular proteins: The polypeptide chain folds up into a compact shape like a ball with an irregular surface. Fibrous proteins: Proteins which have roles requiring it to expand a larger distance. These proteins generally have a relatively simple elongated three dimensional structure

Regulatory GTP binding proteins are switched on and off by the gain and loss of a phosphate group

Here the phosphate is not transferred from ATP to a protein. Instead, the phosphate is part of a guanine nucleotide guanosine triphosphate (GTP) that bind tightly various types of GTP binding proteins. These proteins act as molecular switches, they are in their active conformation when GTP is bound but they can hydrolyze this GTP to GDP which releases a phospgate and flips the protein to an inactive conformation.

Weak interactions between macromolecules can produe large biochemical subcompartments in cells

Intracellular condensate: Assemblies of macromelecules taking the form of spherical, liquid droplets that can be seen to break up and fuse.

Extracellular proteins are often stabilized by covalent cross linkages

Many protein molecules are atttaches to the surface of a cell's plasma membrane or secretes as part of the extracellular matrix, which exposes them to the potentially harsh conditions outside the cell. To help maintain their structures, the polypeptide chains in such proteins are often stabilized by covalent cross-linkages. These linkages can either tie together two amino acids in the same polypeptide chain or join together many polypeptide chains in a large protein complex Most common covalent cross links in proteins are sulfur-sulfur bonds. These Disulfide bonds (S-S bonds) are formed before a protein is secreted, by an enzyme in the endoplasmic reticulum that links together two -SH groups from cysteine side chains that are adjacent in the folded protein. -disulfide bonds dont change a proteins conformation but instead act as a sort of atomic staple to reinforce the proteins most favored conformation

Determining a proteins structure begins with determining its amino acid sequence

Mass spectrometry: This technique determines the exact mass of every peptide fragment in a purified protein which then allows the protein to be identified from a database that contains a lost of every protein though to be encoded by the genome of the relevant organism

Misfolded proteins can form amyloid structures which cause disease

Misfolded proteins are called prions. The misfolded prion form of a protein can convert properly folded version of the protein in an infected brain inot the abnormal conformation. THis allows the misfolded prions to form aggregates which can spread rapidly from cell to cell, eventually causing the death of the affected animal or human.

Proteins can be classified into families

Once a protein has evolced a stable conformation with useful properties, its tructure can be modified over time to enable it to perform new functions. Protein families: Each family member has an amino acid sequence and a three dimensional conformation that closely resembles those of the other family members

Shape of a protein is specified by its amino acid sequence

Protein molecules is made from a long chain of these amino acids, held together by covalent peptide bonds. -therefore proteins are referred to as polypeptides or polypeptide chains Amino acid sequence: With each type of protein, the amino acids are present in a unique order each polypeptide chain consists of a back bone adorned with a variety of chemical side chains. The polypeptide back bone is formed froma repeating sequence of core atoms (N-C-C) polypeptide chains have ends carrying an amino group and carboxy group N-Terminus: End carrying amino group C-terminus: End carrying the carboxyl group projecting from the polypeptide backbone are the amino acid side chains- the part of the amino acid that is not involved in forming peptide bonds long polypeptide chains are very flexible, as many of the covalent bonds that link the carbon atoms in the polypeptide backbone allow free rotation of the atoms they join. -thus proteins can in principle fold in enourmous number of ways shape of each of these folded chains is constrained by the combined effect of many noncovalent bonds including, hydrogen bonds, electrostatic attractions, and van der waals attractions bringing stability to the shape Fourth weak interaction (hydrophobic force): Plays a central role in determining the shape of a protein - in an aqueous environement, hydrophobic molecules, including the nonpolar side chains of particular amino acids, tend to be forced together to minimize their disruptive effect on the hydrogen bonded network of the surrounding water molecules -> this leads to more hydrophobic side chains being oriented twords the inside of the protein to avoid contact of aqueous environment

Proteins fold into a conformation of lowerst energy

The final folded structure, or conformation, adopted by any polypeptide chain is determined by energetic considerations: A protein generally folds into the shape in which its free energy (G) is minimized. -folding process is then energetically favorable as it releases heat and increases the disorder of the universe -a protein can be unfolded or denatured by treatment with solvents that disrupt the noncovalent interactions holding the foleded cgain together. Renaturation: After solvent is removed the protein refolds back into its original shape Chaperone proteins: Assist protein folding in living cells -some of these chaperone proteins bind to partly folded chains and help them to fold among the most energetically favorable pathway others form isolation chambers in which single polypeptides can fold without the risk of forming aggregates in the crowded conditions in cytoplasm

Proteins

The main building blocks from which cells are assembled, and they constitute most of the cells dry mass. in addition to providing the cell with shape and structure, proteins also execute nearly all its myriad functions. -enzymes promote intracellular chemical reactions by providing intricate molecular surfaces contoured with particular bumps and crevices that can craddle or exclude specific molecules -transporters and channels embedded in the plasma membrane control the passage of nutrients and other small molecules into and out of the cell. -other proteins carry messages from one cell to another or act as signal integrators that relay information from the plasma membrane to the nucleus

The a helix and b sheet are common folding patterns

The two folding patters are particularly common because they result from hydrogen bonds that form between N-H and C=O groups in the polypeptide backbone

Allosteric enzymes have two or more binding sites that influence one another

Two or more binding sites must be present on an enzyme: An active site that recognizes the substrates and one or more sites that recognizes regulatory molecules. These sites must somehow communicate to allow the catalytic events at the active site to be influenced by the binding of the regulatory molecule at a separate location. -the interaction between sites that are located in different regions on a protein molecule is now known to depend on a conformational change in the protein. The binding of a ligan to one of the sites causes a shift in the protein's structure from one folded shape to a slightly different folded shape and this alters the shape of a second binding site that can be far away. -many enzymes have two conformations that differ in activity, each of which can be stabilized by the binding of a different ligand. During feeback inhibition for expample an inhibiotr may bind to the enzyme placing its conformation in one which reduces bidnign to the active site Allosteric: An enzyme can adopt two or more slightly different conformations, and their activit can be regulated by a shift from one to another.

How proteins are studied

used to be difficult to get pure proteins as they were mixed with many different proteins and had to be separated with multiple steps of chromatography