• There is a huge number of different three-dimensional shapes possible, determined by the amino acid sequence of the polypeptide.

• Function follows structure.

• There is an enormous versatility in protein structure and therefore function.

Proteins are made up of amino acids covalently bonded together by peptide bonds.

alpha carbon

carboxyl terminus

amino terminus

Each protein has a unique sequence of amino acids. This amino acid sequence specifies the shape of the protein due to the fact that each protein folds into the most energetically favorable conformation

Many of the covalent bonds allow free rotation of the atoms they join. so that the polypeptide backbone can in principle fold up in an enormous number of ways. However each chain, depending on the sequence of amino acids will be constrained by many different sets of weak noncovalent bonds formed both by atoms in the polypeptide backbone and the atoms in the amino acid side chains.

These weak bonds include hydrogen bonds, ionic bonds, van der Waals attractions. A fourth weak force important in protein folding is hydrophobic/hydrophilic interactions

The distribution of polar and nonpolar amino acids is important in how a protein folds. The nonpolar side chains tend to cluster in the interior of a molecule, avoiding contact with water, while the polar side chains arrange themselves near the outside.

Hydrophobic areas also tend to be found spanning the lipid bilayer of membranes like the plasma membrane.

Large numbers of hydrogen bonds form between adjacent regions of the polypeptide chain and help stabilize its three-dimensional shape.

enzyme lysozyme

Each protein normally folds into a single stable conformation – of lowest energy. This conformation will change slightly during interactions with other molecules (as in enzyme-substrate complexes). This change in shape is often crucial for the function of the protein. Ex. receptor proteins

This conformation (the 3-D shape) is specified by its amino acid (aa) sequence. The non-covalent bonds and hydrophobic/philic interactions which hold a protein in the most energetically favorable conformation depend entirely on the aa sequence.

See question 5-1

Molecular chaperones assists protein folding and prevent newly synthesized protein chains from associating with the wrong partners. Make protein folding more reliable.

However, all the information required for proper protein folding is contained in its amino acid sequence.

Proteins in a cell are found in a range of sizes. Protein sequencing has been replaced by DNA sequencing, which is much easier.

Three-dimensional structure is determined by x-ray crystallography and NMR specroscopy. Panel 5-6, pg. 165.

phosphocarrier protein HPr, a transport proteins that facilitates sugar transport into bacterial cells

polypeptide backbone modelN-

terminus

C-terminus

ribbon model

wire model includes amino acid side chains

space-filling model

Find two regular folding patterns: alpha helix and beta sheets. Both result from hydrogen-bonding between the N-H and C=O groups in polypeptide backbone, without involving side chains

Alpha helix is found in alpha-keratin, abundant in skin, hair, nails

Every 4th peptide bond

1/2 bonds with 4-5

Complete turn every 3..6 aa

The polypeptide chains are held together by hydrogen bonds between peptide bonds in different strands. The amino acid side chains in each strand alternately project above and below the plane of the sheet.

Antiparallel

Parallel

Coiled-coilEx. Alpha-keratin, forms intracellulr fibers that reinforce the outer layer of the

skinAnd myosin molecules in muscle cells

Hydrophobic helices also tend to be found spanning the lipid bilayer of membranes like the plasma membrane.

Channel proteins often have hydrophobic exteriors and hydrophilic interiors.

Domains are produced by any part of a polypeptide chain that can fold independently into a compact, stable structure - a modular unit. Proteins often have more than one

domain - each with a specific function.

Catabolite activator protein (CAP)

Binds DNA

Binds cyclic AMP (intracellular signaling molecule)

Turns genes on or off

• CAP is a bacterial signal transduction molecule

• The large domain binds cyclic AMP, an intracellular signaling molecule. When cyclic AMP binds it causes a conformational change in the protein that enables the small domain to bind to a specific DNA sequence and turn on adjacent genes.

Cytochrome b, a single-domain protein involved in electron transfer in E. coli

NAD-binding domain of the enzyme lactic dehydrogenase

The variable domain of the immunoglobulin (antibody) light chain - a beta barrel

Notice loops at each turn

• The polypeptide chain generally passes back and forth across the entire domain, making sharp turns only at the protein surface. The protruding loop regions often form the binding sites for other molecules.

• For each protein, a single conformation is extremely stable and has the exact chemical properties that enable the protein to perform a particular catalytic or structural function.

• Proteins are so precisely built that the change of even a few atoms in one amino acid can sometimes disrupt the structure and the function of a protein.

• Proteins can be grouped into families with very similar sequences and structures, probably due to genes duplicating and evolving.

Serine protease family: elastase, trypsin, chymotrypsin, and some proteases in blood clotting.

Green portion: aa sequence is the same

Notice the structural similarity and active site in red.

Each cleaves different proteins or the bondsbetween different peptides

Serine

Larger protein molecules may contain more than one polypeptide chain or subunit. The region that interacts with another molecule through noncovalent bonds is the binding site.

Hemoglbin contains two alpha globin subunits and two beta globin subunits.

Heme is the site where oxygen is carried

There are many large multi-subunit proteins in cells.

Globular proteins

Proteins with one binding site can form a dimer

Proteins with two different binding sites will often form a long helical filament

or a closed ring

An actin filament, a helical array of actin proteins which can extent for micrometers in a cell - thousands of actin molecules

Many large structures such as viruses and ribosomes are built from a mixture of different proteins plus RNA or DNA molecules. These structures can be isolated, dissociated, and often spontaneously reassemble into the original structure. Much of the structure of a cell is self-organizing.

Can be right or left handed (screw=right (clockwise) same when turned upsidedown

Aminoacids - alpha helixActin molecules - actin filaments

Coiled-coilEx. Alpha-keratin, a dimer, forms intracellular fibers (cytoskeleton) that reinforce the outer layer of the skin

Capped by globular domains which are binding sites, allowing assembly into ropelike, stable, intermediate filaments in skin, hair,horns.

Globular proteins fold up into compact shapes, like irregular ball. Most enzymes, even large and complex enzymes are globular. Fibrous proteins are elongated.

Outside of the cell, fibrous proteins form the gel-like extracellular matrix. Secreted by cells, they assemble into sheets or long fibrils.

Collagen, the most abundant in animal tissues, consists of three long polypeptide chains, each with the nonpolar aa glycine at every third position. Wind around each other in long regular triple helix and bind to one another side-by-side and end-to-end

Hold tissues together.

Elastin is formed from relatively loose polypeptide chains which are covalently cross-liked into a stretchy meshwork

Skin, arteries lungs

Extracellular proteins are often stabilized by covalent cross-linkages. Esp disulfide bonds. These form as proteins are being exported from cells, catalyzed in the er by a special enzyme. Disulfide bonds do not typically form in the cell cytosol. They also do not change the protein’s conformation, but stabilize -reinforce - it.

The binding of a protein to another protein (its ligand) is highly selective. Many weak noncovalent bonds – hydrogen bonds, ionic bonds, van der Waals attractions - plus favorable hydrophobic interactions are needed. Therefore the ligand must fit precisely into the protein’s binding site.

The strength of the ligand/protein binding depends on the strength and number of covalent bonds and determines how long these molecules will stay together. Random movements due to thermal energy are always taking place.

The folding of the polypeptide chain typically creates a crevice or cavity on the protein surface. The amino acids involved in the binding site are often widely separated regions of the polypeptide chain brought together when the protein folds. These amino acids make many noncovalent bonds with the ligand

cyclic AMP

Other areas on the protein may contain binding sites for other ligand, some of which may regulate the proteins activity or place the protein in a particular location..

• Amino acids not in binding sites are usually important for the general shape of the protein, essential scaffold that gives the surface its contours and chemical properties.

• These areas are often the secondary structures and domains of the protein, that give it its 3- dimensional shape – beta-sheets and alpha-helices.

• Therefore, mistakes in the amino acids in these domains can change the 3-dimensional shape and destroy its ability to function

• Strong binding is required when molecules must remain tightly bound for long periods of time – for example, ribosomes or proteosomes

Antibodies (immunoglobuilins) are proteins made by our immune system that can bind virtually any molecule, including those on microorganisms. Each binds a target molecule (antigen) very tightly and with remarkable specificity.

The amino acid sequence in the loops is hypervariable, the DNA is actually changed in each one. The remainder of the domains are structural and contain binding sites for receptors on phagocytes.

beta-barrels

The equilibrium constant is a measure of the binding strength. The larger the equilibrium constant the tighter the binding between protein and ligand.

As ligands diffuse and bind to the antibody (or any protein binding site) and more and more antigen-antibody complexes take form, the reverse reaction will begin to take place. When association and dissociation take place at the same rate (free energy change = 0) the equilibrium constant will be

• Enzymes not only bind to specific ligands, called substrates, they also covert them into chemically modified products.

• The enzymes remain unchanged.• They speed up reactions, often by a factor

of a million or more.• Can be grouped into function classes.

Table 5-2.• Each enzyme is specific for a single type of

reaction.

Lysozyme is an antibiotic found in egg white, saliva, tears, and other secretions. It catalyzes the cutting of polysaccharide chains (hydrolysis) in the cell walls of bacteria, which results in their lysis.

The structure of lysozyme was worked by x-ray crystallography. It has a binding site (active site) that precisely fits the substrate. This is the site of the chemical reaction. The active site holds 6 linked sugars at the same time in an transition state – the atoms in the substrate are held in a slightly altered geometry. The enzyme quickly hydrolyzes the bond and releases the substrate

Conditions are created in the microenvironment of the lysozyme active site that greatly reduce the activation energy needed.

glutamic acid

aspartic acid

high conc of H+

D. Some enzymes briefly form a covalent bond between the substrate and a side chain of the enzyme. This bond is broken in the end, leaving the enzyme unchanged. Figure 4-5.

As the concentration of the substrate increases, the rate of the reaction increases in a linear fashion. As the enzymes active sites become saturated with substrate the rate increases only slightly until the maximum value is reached – Vmax.

The rate of product formation now depends only on the speed of the enzyme. This turnover number is often around 1000 substrate molecules per second! The concentration of substrate needed to allow efficient an enzyme rate is κM at which the enzyme works at half its max speed.

Tightly bound small molecules add functions to proteins.

rhodopsin = the purple light-sensitive pigment made by the rod cells in the retina which detects light by a small molecule retinal, embedded in the protein. Retinal changes its shape when it absorbs a photon of light and transmits this

shape change to the protein, which triggers a cascade of enzymatic reactions that lead to an electrical signal carried to the brain.

Hemoglobin contains a heme group which is

tightly bound to the protein.

• Enzymes often have non-protein small molecules as essential components.– retinal and heme

• Sometimes these small molecules are covalently attached – cell membrane proteins covalently attached to lipid

molecules - lipoproteins

– glycoproteins, cell membrane or secreted.

• Enzymes often have a small molecule or metal atom tightly associated with their active site that assists in the catalytic function.– carboxypeptidase – zinc

– biotin transfers a carboxylate group (a vitamin)

The metabolic system in the cell is so complex that elaborate controls are required to regulate when and how rapidly each reaction occurs. Regulation occurs at many levels. Transcriptional, Translational, Confining an enzyme to a particular space, enclosed in or a part of a membrane (mitochondria). Most rapid and general process is at the level of the enzyme itself.

1. Feedback inhibition

product inhibits one of the first enzymes in the pathway

Positive regulation occurs when a product in one branch stimulates activity of an enzyme in another pathway.

Ex. accumulation of ADP activates several enzymes involved in oxidation of sugar molecules.

multiple points of control

Allosteric regulation – the regulatory molecule binds to another binding site on the enzyme, rather than at the active site. The binding of the regulatory molecule changes the shape of the enzyme and the active site changes shape. In negative regulation, it is longer able to bind to its substrate.

induces a conformation change

This mechanism (conformation change) is important in the function of other proteins also. Receptors, structural proteins, motor proteins.

Conformational change in positive feedback.

2. A second regulatory method is to add a phosphate group covalently to one of the amino acid side chains of the enzyme (serine, threonine, or tyrosine). This causes a major conformational change which changes the activity of the enzyme. Reversible protein phosphorylation controls the activity of more than a third of the proteins in a typical mammalian cell. It occurs as a response to signals received from hormones, and neurotransmitters. Protein kinases phosphorylate enzymes and protein phosphatases dephosphorylate them.

3. GTP-binding proteins form molecular switches in response to a signal received by the cell.

are usually active when GTPoften bind to other proteins to control enzyme

activitiescrucial role in intracellular signaling pathways

Bacterial elongation factor EF-Tu

a GTP-binding protein

Allosteric transition = shape change A small change is magnified by conformational changes within the protein to produce a much larger movement.

• Enzymes are regulated by negative feedback inhibition (products inhibit an early enzyme) Fig. 32 and 33– Often by allosteric interactions in which binding of a

molecule at one site changes its shape at a different binding site

• Conformation (allosteric) change can be driven by protein phosphorylation (Fig. 36)

• Also by binding GTP (GTP-binding proteins) Fig 37 and 38

• Enzymes can also be activated by proteases which cleave off a segment – changing the shape of the enzyme.

Motor proteins

- muscle contraction, intracellular movement of organelles, chromosomes, enzymes along DNA, etc.

Without energy output, these movements will be random – back and forth

Not useful

Requiring energy output makes these series of conformational changes essentially irreversible.

Unidirectional

An orderly transition among three conformations driven by the hydrolysis of a bound ATP molecule.

Would require ADP ATP

Proteins often form large complexes that function as protein machines

EX. DNA replication, protein synthesis, vesicle budding, transmembrane signaling

Hydrolysis of ATP or GTP drives an ordered series of conformation changes. These changes effect each polypeptide chain.