Chapter 3

Antibodies

'Y' shaped. Proteins that circulate in the blood and are made by the immune system. -light and heavy chain arms linked by disulfide bond.

Coiled Ribbons

(solid cylinders for @ helices and flat ribbons or arrows for Beta strands, and flexible thin strands for Beta turns, coils, and loops) -a protein representation that shows how Beta strands and @ helices are organized in the protein.

The principal secondary structures of proteins

-alpha helix (major internal support elements in most proteins) -beta sheet (major internal support elements in most proteins) [found in the floor of a binding pocket of or a hydrophobic core; in proteins embedded in membranes the B sheets curve around and form a hydrophilic central pore through which ions and small molecules flow through - short U-shaped beta turn [found on surface of a protein that reverse the direction of the polypeptide backbone towards proteins interior] [helped large proteins fold into highly compact structures].

The Diverse Functions of Proteins

-binding to one another or other macro molecules. (induces shape change in proteins and thus alter/influences its activity. -enzymatic catalysis -folding into a channel or pore within membrane. (allowing molecules and ions to flow)

3 ways to regulate protein activity

1. cells increase/decrease the steady-state level of the protein by altering its rate of synthesis, its rate of degradation, or both. 2. cells can change in intrinsic activity, as distinct from the amount, of the protein. [can change the time a protein is an active/inactive state.] 3. there an be a change in location or concentration within the cell of the protein itself, the target f the protein's activity (i.e. enzyme substrate) or some other molecule required for the proteins activity (i.e. enzyme cofactor)

Two important roles for protein degradation

1. degradation removes proteins that are potentially toxic, improperly folded or assembled, or damaged- including the products of mutant genes and proteins damaged by chemically active cell metabolites or stress (heat shock) --(Even with chaperones, some new proteins are degraded before they even fold properly) (There is 1-2%/hour degradation in mammalian cells. 2. the controlled destruction of otherwise normal proteins provides a powerful mechanism for maintaing the appropriate levels of the proteins and their activities and for permitting rapid changes int hess levels to help the cells respond to changing conditions.

2 most frequently used mass spec methods

1. matrix assisted laser desorption/ionization (MALDI) 2. Electrospray (ES)

three most widely used characteristics for separating proteins:

1. size- defined either as length or mass 2. net electronic charge 3. binding affinity

Features of proteins that limit their ability to form other potential conformations [besides native]

1. size- if a chain is too large, it could sterically hinder/block another region from packing too close together against another region. 2. charges- if a chain has a positive charge it'll attract a complimentary negative charge from another side chain and simply repel other 'like' charges 3. Aliphatic side chains- like with heptad repeats help form helices associations and coiled coils.

Quaternary Structure

4th level of a protein; describes the number (stoichiometry) and relative positions of the subunits in multimeric proteins. -flu virus hemagglutinin is an ex of a trimer of 3 identical subunits (homotrimer)

Nascent Protein

A protein as it is being formed by a ribosome before it folds into its active shape

3 most common structural motifs

A) Coiled-coil motif; B) EFhand/helix-loop-helix motif; C) Zinc-finger motif

Site the allosteric effector ligand binds too

Allosteric binding site.

Comparing sequences and structures of proteins

Allows for scientists to deduce much about what these [unknown] proteins do.

reducing agents

Beta mercaptoethanol that breaks disulfide bonds can further destabilize disulfide-containing proteins.

Four Ways to visualize Protein structures

C(a) backbone trace, ball and stick model, ribbons, solvent-accessible surface

Complementarity-determining regions

CDRs which are the antigen binding sites. Unique sequences specific to an antibody.

Allosteric switches

Ca2+ (calmodulin) and GTP control protein activity through noncovalent binding.

Protein Domain

Distinct regions of protein structure; there are 3 classes; functional, structural, and topographical.

Ca2+/Calmodulin-mediated switching

EF Hand/Helix-loop-helix motif. -calmodulin is a widely dispersed cytosolic protein that contains 4 Ca2+ binding sites at each of its EF hands. regulate the other activities of proteins by acting as a switch to turn off/on. (Ca2+ concentration INTRAcellular typically kept low)

SDS-Polyacrylamide Gel Electrophoresis [PAGE]

Electrophoresis; separates proteins primarily on the basis of their masses. 1. treatment with detergent denatures polypeptide chains. 2. during electrophoresis, the complexes migrate through the gel. 3. smaller complexes move through PORES faster than larger ones. Thus proteins separate into bands based on their size as they migrate. 4. Dye proteins to ID

Substrates

Enzymatic ligands are...?

Molecular Chaperones types

Hsp70 (Heat Shock Protein 70 in mitochondrial matrix. found in all organisms. BiP in the ER. DnaK in bacteria.

Homology

In Protein sequences there is a similarity which is often reflected in similar structures.

Michaelis constant Km

Is a measure of the affinity of an enzyme for its substrate.

Hsp90 Function

Molecular Chaperon. Functions as a dimer in a cycle in which ATP binding, hydrolysis, and ADP release are coupled to major conformational changes and to binding, activation, and release of clients. -ATP activates binding domain to form "closed" formation and its hydrolysis of activates client proteins and their release from the MC.

Hsp70 [MC] function

Molecular Protein that helps pro tines fold into their proper 3D structure; -Transiently bind to a nascent polypeptide as it emerges from the ribosome or to unfolded proteins. -In its cycle, an unfolded protein substrate binds in rapid equilibrium to the open conformation of the substrate binding domain (SBD) of the monomeric chaperone, to which ATP is already bound to the nucleotide-binding domain. (NBD) 1.The substrate binding pocket is in the SBD/ Co Chaperone accessories DnaJ/Hsp40 stimulate the hydrolysis of ATP to ATD and conformational change in the Hsp70, resulting in the closed form in which the substrate is locked into the SBD and here is where the proper folding takes place. 2.ATP is exchanged for ADP via co-chaperone proteins GrpE/BAG1 and converts the Hsp70 back to its open form. 3. The properly folded substrate is released.

Hsp90 [MC]

Molecular chaperone that is present in all organisms except archaea. In eukaryotes, the most abundant proteins in the cytosol. These help cells cope with denatured proteins generated by stress (i.e. heat shock) and they ensure that some of their substrates- usually called "clients"- can be converted from an inactive to an active state or otherwise held in a functional conformation. -clients include transcription factor (receptors for hormones like testosterone/estrogen) -other clients are kinases (phosphorylation)

tertiary structure

Not rigidly fixed but undergoes continual, minute fluctuations, and some segments within the structure of a protein are so mobile they're considered disordered; that is lacking well defined 3D structure. -This variation in structure has important consequences for the function regulation of proteins. -stability can be increased with addition of disulfide bonds between side chains.

Membrane Bound Proteins

Permit the flow of ions and molecules across cellular membrane

Regulatory Proteins

Proteins that act as signals, sensors, and switches to control the activities of cells by altering the functions of other proteins and genes. -include: signaling proteins

Globular Proteins

Proteins that are generally water-soluble, compactly folded structures, often but not exclusively spheroidal, that comprise a mixture of secondary structures.

Fibrous Proteins

Proteins that are large, elongated, often stiff molecules. Often aggregate into multi protein fibers that do not readily dissolve in water, and play a structural role or participate in cellular movements.

Motor Proteins

Proteins that are responsible for moving other proteins, organelles, cells- even whole organisms.

Scaffold Proteins

Proteins that bring other proteins together into ordered arrays to perform specific functions more efficiently that if those proteins were not assembled together.

Enzymes

Proteins that catalyze chemical reactions.

Multimeric Proteins

Proteins that consist of two or more polypeptide chains, which in this context are referred to as subunits. -can be homomeric or heteromeric. -monomeric subunits can't function normally unless they are assembled into the full protein. [i.e. fatty acid synthases; polyketide synthases -ex: RNA polymerase

Structural Proteins

Proteins that determine the shapes of cells and their extracellular environments and serve as guide wires or rails to direct the intracellular movement of molecules and organelles. -formed by the assembly of multiple protein subunits into very large, long structures.

Homologs

Proteins that have a common ancestor. -common proteins belong to a "family" and can trace their lineages from comparison of their sequences. -+family=superfamily

Sulfur-35

Radioactive isotope most commonly used in proteins since it is commonly found in residues like cystine.

Specificity

Refers to the ability of a protein to bind one molecule or a very small group of molecules in preference to all other molecules.

Catalytic site

Site on enzyme that carries out the chemical reaction once the substrate has bound to it. -consists of amino acid side chains and backbone carbonyl and amino groups.

Two Properties of Protein characterization

Specificity and Affinity -Both specificity and affinity of a protein for a ligand depend on the structure of the ligand-binding site.

[@ helix based] Coiled-Coil Motif [or heptad repeat]

Structural motif characterized by 2 @ helices wound around each other. helix packing is stabilized by interactions between hydrophobic [through aliphatic (leucine/valine) intervention] side chains present at regular intervals along each strand and found along the seam of the intertwined helices. -@ helices exhibit characteristic heptad repeat sequence at the 1 and 4 positions. 1&4 are aliphatic -results in DNA-regulating proteins transcription factors and co factors; assemble into dimers and trimers too. -overall helical structure is amphipathic. -leucine zippers

Zinc-Finger motif

Structural motif present in many DNA binding proteins that help regulate transcription. contains 3 secondary structures: and @ helix and 2 Beta stands with antiparallel orientation that forms a fingerlike bundle held together by a zinc ion. -cysteine residues are at positions 3 and 6 -histidine residues are at positions 20 and 24.

Proteome

Term coined to refer to the entire protein complement of an organism. -However, variations in mRNA production, such as alternate splicing of protein modifications, generates many distinct human proteins.

Chaperones

The explanation for the cells remarkable efficiency in promoting proper proton folding is that cells make a set of proteins [these] that facilitate proper folding of nascent proteins. -Many are evolutionarily conserved.

turnover number

The maximum number of substrate molecules converted to product at a single enzyme active site per second.

Native State

The one (or closely related) confirmation the protein adopts [out of the many options it could choose] be sue for the vast majority of the proteins this state is the most stably folded form of the molecule and the one that permits it to function normally. -in thermodynamics, its the state with the lowest free energy (G)

denaturation

The process by which a protein's structure (including 2* and 3*) is disrupted. -can be induced by thermal energy from heat, extremes of pH that alter the charges on amino acid side chains, and exposure to agents such as urea or guanidine.[all of which disturb structure-stabilizing noncovalent interactions. increases the entropy of a system.

C(a) backbone trace

The simplest model of a protein that demonstrates how the polypeptide is tightly packed into a small volume. You trace the course of the backbone atoms with a solid line. -shows the overall fold of the polypeptide chain without consideration of the amino acid side chains.

How Enzymes increase the reaction rate

They decrease the energy of the transition state and therefore the activation energy required to reach it.

Random Coil

This applies to highly flexible parts of a polypeptide chain that have no fixed three-dimensional structure.

E3 [Ubiquitin]

Ubiquitin protein ligase; catalyzes an isopeptide bond. Transfers 76G to lysine residue on target protein.

E1 [ubiquitin]

Ubiquitin-activatin enzyme; covalently attaches to the UB via ATP hydrolysis.

Proteasomes

Very large protein-degrading macromolecular machines that influence many different cell functions. -Have the cylindrical 20S catalytic core. -two 19S caps bind to either side of the 20Sc core. -the two 19Slids+20S core= 26S proteasome. -important to cell life, yeast cells shown to not being able to survive at all without them present in cell.

Molecular Machines

When proteins assemble into larger complexes to accomplish their more diverse tasks efficiently.

Rate-Zonal centrifugation

a mixture is spun just long enough to separate molecules that differ in mass but may be similar in shape and density into discrete zones within a density gradient commonly formed by a concentrated sucrose solution. (large proteins on bottom, smaller on top)

Antibody-Affinity Chromatography

a mixture of proteins thats passed though a column packed with beads to which specific antibody is covalently attached. -only protein with high affinity for the antibody is retained by the column; all the nonbonding proteins flow through.

Structural Motif

a particular combination of two or more secondary structures that form a distinct 3D structure and appears in multiple proteins (as a regular combination of secondary structures) that are usually associated with a specific function.

Tissue Plasminogen Activator (TPA)

a protease that is used to dissolve blood clots in heart attack victims.

Solvent-Accessible surface

a protein model that reveals the numerous lumps, bumps, and crevices on the protein surface. Regions of + charge are purple and - charge are red. -This one is of more interest since it details the surface of the protein and that is what interacts with other molecules that bind to it.

Notch protein

a receptor protein in the plasma membrane that functions in developmentally important in signaling.

structural domains

a region of 40+ AA in length arranged in a single stable and distinct structure often comprising one or more secondary structures. -often fold into their characteristic structures independently of the rest of the protein in which they are embedded. -consequently, distinct domains can be linked together to form larger multi domain proteins. -are usually incorporated as modules into different proteins; allows them to confer distinct activities that perform different functions simultaneously and are independent of the rest of the protein. -domains can be ID be x-ray crystallography, NMR, and electron microscopy.

Oligopeptide (or just peptide)

a short chain of amino acids linked by peptide bonds and having a defined sequence. -longer chains referred to as polypeptides.

Epidermal Growth Factor (EGF) domain

a small, soluble peptide hormone that binds to cells in the embryo and in skin and connective tissue in adults, causing them to divide. -generated by proteolytic cleavage (breaking of a peptide bond)

Electrophoresis

a technique for separating molecules in a mixture under the influence of an applied electric filed and is one of the most frequently used techniques to study proteins and nucleic acids. -Dissolved molecules in Efield migrate at a speed determined by their charge to mass ratio and physical properties of the medium through which they migrate. ->so if two molecules have the same MASS and SHAPE, the one with the GREATER net charge would move faster to an electrode of the opposite polarity.

Mass Spectrometry

a very sensitive and highly precise method of detecting, identifying, and characterizing proteins and peptides. -based on M:Z ratio.

molecular complementarity

allows molecules to form multiple noncovalent interactions at close range and thus stick together.

GTPase superfamily

another group of intracellular switch proteins; these are enzymes that can hydrolyze GTP to GDP. [include RAS]

Environment for enzymes to properly function in

aq environment at 37*C at 1atm and physiological pH values (6.5-7.5)

antigens

are macromolecules present in infectious agents that have specific epitope binding sites from antibodies to bind too.

Substrate binding site

are on active site that recognizes and binds the substrate or substrates. -Site is responsible for the specificity of the enzymes- and their ability to act selectively on one substrate or a small number of chemically similar substrates.

Integral Membrane Proteins

are proteins embedded within the phospholipid bilayer of the membranes that enclose cells and organelles.

Leucine Zippers

because leucine frequently appears in the 4th position and the hydrophobic side chains merge together like the teeth of a zipper these structural motifs have this name.

kinases

catalyzes phosphorylation in proteins.

Cell Functions influenced by proteasomes

cell cycle, transcription, DNA repair, programmed cell death [apoptosis], recognition and response to infections, and removal of misfiled proteins.

Molecular Chaperones

chaperones that bind to a short segment of a protein substrate and stabilize or partly unfold proteins, thereby preventing these proteins from aggregating and being degraded.

Chaperonins

chaperons which form small folding chambers into which all or part of an unfolded protein can be sequestered, giving it time and an appropriate environment to fold properly. -Group 1 chaperones are found in prokaryotes, chloroplasts, and mitochondria and interact with co-chaperone "lid" -Group 2 chaperones are found in the cytosol of eukaryotic cells (TriC)and in archaea

Immunoblotting

combines the resolving power of gel electrophoresis and the specificity of antibodies. Used to separate proteins and then identify a specific protein of interest. -WESTERN BLOTS

Active Site

consists of the substrate binding site and the catalytic site. On an enzyme

EFhand/Helix-loop-helix motif

contains two helices connected by a short loop in a specific conformation. prevalent in calcium binding and DNA binding regulatory proteins. -used for sensing calcium levels in cells. Ca2+ concentration directly controls the proteins structure and function by finding to oxygen atoms in the residues in the loops and can induce a conformational change.

Phosphatases

dephosphorylation; or the removal of a phosphate group

2D gel electrophoresis

electrophoresis that separates proteins on basis of charge and mass; -Proteins are FIRST separated into bands on the basis of their charges by Isoelectric focusing. -SECONDLY, proteins are separated into spots by mass. -third, dye proteins to ID.

autophagy

enzymes with lysosomes; the interior fills with hydrolytic enzymes. directed at ages or defective organelles or towards extracellular proteins taken up by the cell.

Pulse chase

experiments that are useful for tracing changes int he intracellular location of proteins or the modification of proteins or metabolite over time. -in it, a cell sample is exposed to a radiolabled compound that cab be incorporated or otherwise attached to a cellular molecule of interest- the "pulse"- for a brief period. -Ends when the unicorportated radioactive molecules are washed away and the cells are exposed to a vast excess of the identical but unlabeled molecules.

high order regulation

form of regulation that includes comparmentation of proteins and control of protein concentration.

Tertiary structural categories

globular, fibrous, and integral membrane proteins. Not all mutually exclusive, some proteins are made up of combinations of two or even all three categories.

GEF

guanine exchange factors; specific proteins of cells that modulate the baseline rate of GTPase activity for any given GTPase switch and so can control how long the switch remains on/off. -mediate exchange from GDP/GTP

Signaling Proteins

hormones and cell-surface receptors that transmit extracellular signals to the cell interior.

Protein-Ligands Bonding

hormones binding to receptors; regulatory molecules binding to DNA promoters; CAM binding to extracellular matrix. -ALL are examples of what?

zymogens

inactive precursor enzymes.

competitive inhibitors

inhibitors that bind directly to the enzymes binding site and compete with the normal substrates ability to bind.

noncompetitive inhibitors

inhibitors that bind to some other site on an enzyme causing it to change its conformation.

plaque

insoluble, disordered aggregates of twisted-together proteins in various organs including the liver and brain. can cause neurological diseases if they are stably folded in an alternative conformation.

Neu protein

involved in embryonic differentiation

GTPase switch proteins 2 forms

involved in intracellular signaling. exist in either: 1. active form with bound GTP to influence activity of a specific target proteins to which they bind to. 2. an inactive form with bound GDP. —switch is turned "on" when it goes from inactive to active, when a bound GDP is exchanged for another GTP.

Negative allostery

involves the end product of a multistep biochemical pathway binding to and reducing the activity of an enzyme that catalyzes an early rate-controlled step for that pathway. -Prevents the excessive build up of product. -Also called end-product inhibition or FEEDBACK inhibition.

Functional Domain

is a region of a protein that exhibits a particular activity characteristic of that protein, usually even when isolated from the rest of the protein. -a region of catalytic activity (Kinase->adds phosphates to molecules); or binding ability (DNA or membrane binding domain); -ARE often identified by whittling down protein into its smallest active fragment with the help of 'proteases' -It is possible to modify the DNA encoding the protein so that when its used to generate that protein only a particular region or domain is made. -You can determine if specific parts of a protein are responsible for particular activities as exhibited by the protein. -associated with structural domains.

Cooperativity

is a term often used synonymously with allostery and refers to the influence ( positive or negative ) that the binding of a ligand at one site has on the binding of another molecule of the same type of ligand at a different site. -HEMOGLOBIN is an example of positive binding in that the binding of a single ligand (molecular oxygen) [O2] increases the affinity of the binding for the next oxygen molecule. The binding of the first induces a change thats effect spreads to the other subunits and increases the affinity for binding of other O2 molecules.

Proteolytic cleavage

nonreversible form of regulation; protein self-splicing.

Nuclear Pore

one of the most complex multi protein assemblies; a structure that allows communication and passage of macromolecules between nucleoplasm and cytoplasm.

Enzyme cofactor or (co-enzymes)

or 'prosthetic group' is a helper group; a non polypeptide small molecule or ion (Fe2+, Zn2+, Cu2+, Mg2+) that is bound in the active site and plays an essential role in the reaction mechanism.

Epitope

part of the antigen that antibodies have ability to recognize and bind too based on their characteristics (topography) that induced the production of antibodies to begin with.

Irregular Structures

parts of the polypeptide that don't form these structures but never the less have a well-defined, stable shape.

allowed by noncovalent binding

permits allosteric or cooperative regulation of proteins

chemiluminescence

process that produces light that can be readily recorded by film or a sensitive detector.

Gel-Filtration Chromatography

proteins that differ in mass are separated on a column composed of porous beads made from Polyacrylamide. small proteins travel through the column more slowly than larger proteins. Thus different proteins emerging out at different times are called fractions.

Affinity

refers to the tightness or strength of binding, usually expressed as the disassociation constant, Kd. -The stronger the interaction between a protein and ligand, the lower the value of Kd.

Topological Domains

regions of proteins that are defined by their distinctive spatial relationships to the rest of the protein. -Proteins associated with a cell can have an extracellular, membrane spanning, and then cytoplasmic domains.

Ball (atoms) and Stick (bonds) model

representation of a protein that reveals the location of all atoms. The most complex model that shows the every atom. -details the interactions between side-chain atoms, including those that stabilize the proteins conformation and interact with the other molecules as well as the atoms of the backbone.

Liquid chromatography

resolves proteins based on their mass, charge, and affinity. in it, the sample is placed on top of a tightly packed column of spherical beads held within a cylinder. the sample flows down the column. —> The nature of the beads in the column determine whether the separation of proteins depends on difference in mass, charge, or affinity.

Phosphorylation and Ubiquitination

reversible covalent/noncovalent regulations

Ion-Exchange Chromatography

separates proteins that differ in net charge in columns packed with special beads that either carry a positive or negative charge. Proteins having the same net charge as the beads are repelled through the column while those with opposites bind to the beads. The Bound proteins are eluted by passing a SALT gradient through the column. As salt ions bind to beads they displace the proteins. (the more weakly bound dissociate first, the stronger bound ones last)

Michaelis and Maud Menton hypothesis (Lock and Key)

showed that the rate of enzyme reaction is proportional to the substrate concentrations at low substrate concentration, but that as the substrate concentrations increased the rate reached a Vmax and became substrate concentration independent with the value of Vmax being proportional to the amount of the enzyme present in the reaction mixture.

enzymatic inhibitors

small molecules that bind to active sites and disrupt catalytic reactions. -The basis for most drugs.

coenzymes

small organic prosthetic groups in enzymes. [NAD+, FAS, and heme groups are...?] and vitamins too.

basic helix-loop-helix

structural motifs that are used for protein binding to DNA and consequently the regulation of gene activity.

conformations

the 3d Shapes proteins fold themselves into. Coupled along with the distinctive chemical properties of its amino acid side chains determine the proteins functions.

Consequence of Peptide linkage

the backbone exhibits directionality referred to as an N- to- C orientation, because all of the amino groups are located on the same side of the alpha Carbon atoms.

Supramolecular Complexes

the highest level of hierarchy in a protein. ex: the viral genome that encases the nucleic acids of the viral genome; the cytoskeletal filaments that support and give shape to the plasma membrane; others are machines that combine multiple proteins for a specific function (I.E. transcription-> a machine is used to synthesize mRNA from DNA strand) - fatty acid synthases; polyketide synthases

"Oil Drop Model" of Protein Conformation

the hydrophobic residues of a polypeptide chain tend to cluster together, somewhat like an oil drop, on the inside, or core, of a folded protein, driven away from the aqueous surroundings by the hydrophobic effect. Charged and unchained polar side chains appear on the proteins surface where they can form stabilizing interactions interactions with surrounding water and ions.

ligand

the molecule to which a protein binds too. causes a shape of a protein to change.

Phosphorylation

the most common covalent mechanism for regulating protein activity. a reversible addition of a phosphate group to hydroxyl groups on the side chains of residues. [serine/tyrosine] -causes a change in a proteins charge that can lead to conformational changes which can affect ligand binding or lead to inc/dec in protein activity.

Differential centrifugation

the most common type of protein purification from cells or tissues by separating the water-soluble proteins from the insoluble cellular material. -pour homogenate into tube and spin at high speeds for a time to separate out insoluble cell fragments and the soluble supernatant.

Enzyme pH sensitivity

the pH sensitivity of an enzymes activity can be due to changes in the ionization of catalytic groups, groups that participate directly in substrate binding, or groups the influence the conformation of the protein.

daltons (D)

the sizes of proteins or polypeptides are expressed in either its mass in 'THIS' or as its molecular weight. (a dimensionless number equal to its mass in 'THIS')

metabolic coupling

to overcome the impediment of potentially slow inefficient movements by diffuse of enzymes- cells have evolved mechanisms for bringing enzymes in a common pathway into a closer proximity. THIS process is...?

centrifugation

two particles in suspension with different masses or densities will settle to the bottom of a tube at different rates after being subjected to a variety of speeds. -Good for: 1. preparative technique to separate one type of material from others. 2. as a analytical technique to measure the physical properties of macromolecules.

E2 [ubiquitin]

ubiquitin-conjugating enzyme; transfer of this UB molecule to covalently bind to a cysteine residue.

allosteric effector/ and allosteric protein.

when a ligand binds to one site in a protein and induces a conformational change and associated change in activity of a different site. The ligand is the WHAT of the protein...? Protein is called what? -this change can be positive or negative, either increasing or decreasing a protein activity; -