Flashcards
nonenzymatic protein function
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- 1cell adhesion moleculesproteins found on the surface of msot cells and aid in binding the cell to the extracellular matrix or other cells, are integral membrane proteins, there are three major families of adhesion molecules,
Cadherins, integrins and selectins - 2cadherinsare a group of glycoproteins that mediate calcium-dependent cell adhesion, cadherins often hold similar cell types together such as epithelial cells, different cells have type specific cadherins, for example, epithelial cells use E cadherin while nerve cells use N cadherin
- 3integrinsare a group of proteins that all ahve two membrane spanning chains called alpha and beta, these chains are very important in BINDING AND COMMUNICATING WITH THE EXTRACELLULAR MATRIX, play an important role in cellular signaling and can greatly impact cellular function by promoting cell division, apoptosis or other processes
for example one type of integrin allows platelets to stick to fibrinogen, a clotting factor which causes activation of platelets to stabilize the clot - 4selectinsare unique because they bind to carbohydrate molecules that project from other cell surfaces, these bonds are the weakest formed by the CAMS, selectins are expressed on white blood cells and the endothelial cells that line blood vessels, like integrins they play a role in host defense including inflammation and white blood cell migration
- 5antibodies (immunoglobulins) (Ig) what holds the light and heavy chains together?disulfide linkages and noncovalent interactions hold the heavy and light chain together, each antibody has an ANTIGEN BINDING REGION at the tips of the Y
-> within this region there are specific polypeptide sequences that will bind one and only one specific antigenic sequence
the remaining part of hte antibody is known as teh constant region that is involved in recruitment and binding of other cells of the immune system such as macrophages - 6what are the three outcomes that happen when an antibody bind to their target?neutralizes the antigen, making the pathogen or toxin unable to exert its effect on the body
marking the pathogen for destruction by other white blood cells immediately,
- this marking function is called OPSONIZATION
clumping together (agglutinating) the antigen and antibody into large insoluble protein complexes that can be phagocytized and digested by macrophages - 7cytoskeletal proteins vs motor proteinscytoskeletons tend to be fibrous with repeating domains, while motor proteins tend to have ATPase activity and binding heads, both types of protein function in cellular motility
- 8biosignalingis the process where cells receive and act on signals
- 9ion channelsare proteins that create specific pathways for charged molecules, classified into three main groups however they all permit facilitated diffusion of charged particles
three main types of ion channels are ungated, voltage gated, and ligand gated
facilitated diffusion is a type of passive transport, is the diffusion of molecules down a concentration gradient through a pore in the membrane created by this transmembrane protein, it is used for molecules that are impermeable to the membrane (large, polar, or charged), facilitated diffusion allows integral membrane proteins to serve as channels for these substrates to avoid the hydrophobic fatty acid tails of the phospholipid bilayer - 10ungated channelshave no gates and therefore unregulated, for example all cells possess ungated potassium channels, this means there will be a net efflux of potassium ions through these channels unless potassium is at equilbrium
- 11voltage gated channelthe gate is regulated by the membrane potential change near the channel, many excitable cells such as neurons possess voltage gated sodium channels, the channels are closed under resting conditions but membrane depolarization causes a protein conformation change that allows them to quickly open and then quickly close as the voltage increases
voltage gated nonspecific sodium potassium channels are found in cells of sinoatrial node of the heart, they serve as the pacemaker current, as the voltage drops, these channels open to bring the cell back to threshold and fire another action potential - 12ligand gated channelsbinding of a specific substance or ligand to the channel causes it to open or close, neurotransmitters act at ligand-gated channels at the postynaptic membrane, GABA binds to a chloride channel to open it
- 13Km and V max parameters apply to enzymescan be applied to transporters such as ion channels in membranes,
Km refers to the solute concentration at which the transporter is functioning at half of its maximum activity - 14enzyme linked receptorsmembrane receptors may also display catalytic activity in response to ligand binding, have three primary protein domains: a membrane spanning domain, a ligand binding domain and a catalytic domain
membrane spanning domain anchors the receptor in the cell membrane
ligand binding domain is stimulated by the appropriate ligand and induces a conformational change that activiates the CATALYTIC DOMAIN
often results in the initiation of a second messenger cascade - 15example of enzyme linked receptorreceptor tyrosine kinase (RTK), RTKs are composed of a monomer that dimerizes upon ligand binding, the dimer is the active form that phosphorylates additional cellular enzymes, including the receptor itself
- 16characteristics of GPCRG protein coupled receptor are a large family of integral membrane proteins involved in signal transduction, characterized by their 7 membrane spanning alpha helices, the receptor differ in specificy of the ligand binding area found on the extracellular surface of the cell, in order for GPCRs to transmit signals to an effector in the cell, they use a HETEROTRIMERIC G protein
- 17how do GPCR's work?use a heterotrimeric G protein (G proteins are named for their intracellular link to guanine nucleotides (GDP and GTP), the binding of a ligand increases the affinity of the receptor for the G protein, the binding of the G protein represents a switch to the active state and affects the intracellular signaling pathway, there are several different G proteins that can result in either stimulation or inhibition of the signaling pathway
- 18what are the three main types of G proteins?G_s stimulates adenylate cyclase, which increases levels of cAMP in the cell
G_i inhibits adenylate cyclase which decreases levels of cAMP in the cell
Mind your p's and q's: G_q activates
P hospholipase C which cleaves a phospholipid from the membrane to form PIP_2. PIP_2 is then cleaved into DAG and IP3, IP3 can open calcium channels in the endoplasmic reticulum increasing calcium levels in the cell - 19How does the trimeric G protein cycle work for G_s and G_i?the heterotrimeric G protein is made up of three subunits, alpha, beta and gamma, in the inactive form alpha is bound to a GDP and to the other two subunits beta and gamma, when the GPCR is activated the G protein is engaged and the GDP is replaced with GTP, the alpha subunit is then able to dissociate from the beta and gamma subunits (both beta and gamma are still attached to each other), the alpha subunit alpha_s or alpha_i is able to either stimulate or inhibit adenylate cyclase, once GTP on the activated alpha subunit is dephosphoyrlated the G protein is once again deactivated
- 20homogenizationcrushing, grinding or blending the tissue of interest into an evenly mixed solution, how you isolate biomolecules e.g protein
- 21electrophoresisworks by subjecting compound to an electric field which moves them according to their net charge and size, negatively charged compounds will migrate toward the negatively charged cathode, the velocity of this migration known as the migration velocity of molecule, V, is directly proportional to the electric field strength, E, and to the net charge on the molecule, z, and is inversely proportional to a frictional coefficient, f which depends on the mass and shape of the migrating molecules
equation v=(Ez)/f - 22PAGEpolyacrylamide gel electrophoresis - method for analyzing proteins in their native states, limited by the varying mass-to-charge and mass-to-size ratio of cellular proteins because multiple different proteins may experience the same level of migration, protein can be recovered from gel if the gel is not stained, most stains denature proteins,
PAGE is most useful to compare the molecular size or the charge of proteins known to be similar in size from other analytic methods like SDS-PAGE or size-exclusion chromatography - 23SDS PAGEsodium dodecyl sulfate (SDS) PAGE is useful because it separates proteins based on mass alone, technique starts with the premise of PAGE but adds SDS, a detergent that disrupts all noncovalent interactions, it binds to proteins and creates large chains with net negative charges thereby neutrailizing the proteins original charge and denatures it, the only two variables that affect protein velocity is E the strength of the electric field and f the frictional coefficient which depends on mass
- 24isoelectric focusingproteins can be separated on the basis of their isoelectric point (pI), mixture of protein is placed in a gel with a pH gradient (acidic gel at the positive anode and basic gel at the cathode), an electric field is then generated across the gel, proteins that are positively charged will begin migrating toward the cathode and proteins that are negatively charged will begin migrating toward the anode, as the protein reaches the portion of gel where the pH is equal to the protein's pI the protein takes on a neutral charge and will stop moving
protein stops moving when pH=pI - 25chromatography general infouses physical and chemical properties to separate and identify compounds from a complete mixture, refers to a variety of techniques that require the homogenized protein mixture to be fractionated through a porous matrix, one of the reasons chromatography is valuable is that the isolated proteins are immediately available for identification and quantification
concept is that the more similar the compound is to its surroundings (by polarity, charge and so on) the more it will stick to and move slowly through its surroundings
chromatography is prefered over electrophoresis when large amounts of proteins are being separated - 26chromotography step by step procedure1) process begins by placing a sample onto a solid medium called the stationary phase or adsorbent
2) next step is to run the mobile phase through the stationary phase, this will allow the sample to run through the stationary phase or ELUTE.
3) depending on the relative affinity of the sample for the stationary phase and mobile phases, different substances will migrate through at different speeds
components with high affinity for the stationary phase will move with slow speeds/much less, components with high affinity for the mobile phase will move fast
the amount of time a compound spends in the stationary phase is referred to as the retention time, varying retention times of each compound in the solution results in separation of the componenets within the stationary phase or partitioning, each component can then be isolated individually - 27column chromatographycolumn is filled with silica (often polar silica) or alumina beads as an adsorbent, and gravity moves the solvent and compounds down the column, the less polar the faster it can move through the column (short retention time), different fractions of the eluted compounds can be collected based on the time that they elute,
column chromatography is particularly useful in biochemistry because it can be used to separate and collect other macromolecules besides proteins such as nucleic acids - 28ion exchange chromatographythe beads in the column are coated with charged substances so they attract or bind compounds that have an opposite charge, a positively charged column will attract and hold a negatively charged protein as it passes through the column, either increasing its retention time or retaining it completely
- 29size exclusion chromatographybeads used in the column contain tiny pores of varying sizes, these pores allow small compounds to enter the beads thus slowing them down, large compounds can't fit into the pores so they will move around them and travel through the column faster, it is important to remember that in this type of chromatography the small compounds are slowed down and retained longer which may sound counter intuitive, a common approach in protein purification is to use ion exchange column followed by a size exclusion column
- 30affinity chromatographywe can customize columns to bind any protein of interest by creating a column with high affinity for that protein, can coat beads with receptor that binds the protein or a specific antibody to the protein,
common stationary phase molecules include nickel which is used in separation of genetically engineered proteins with histidine tags, antibodies or antigens, and enzyme substrate anologues which mimic the natural substrate for an enzyme of interest, once the protein is retained in the column it can be eluted by washing the column with a free receptor or antibody which will compete with the bead bound receptor and ultimately free the protein from the column'
eluents can also have specific pH or salnity level that disripts the bonds between the ligand and the protein of interest, the only drawback of the elution step is that the recovered substance can be bound to the eluent, if for example the eluent was an inhibitor of an enzyme it could be difficult to remove - 31how can protein structure be determined?X ray crystallography and nuclear magnetic resonance (NMR) spectroscopy
- 32X ray crystallographybefore crystallographic analysis the protein must be isolated and crystalized, X ray crystallography is the most reliable and common method 75% of protein structures known today were analyzed through this method
crystallography measures electron density on a high resolution scale and can be used for nucleic acids - 33how to analyze small protein for amino acid composition?best analyzed with Edman degradation which uses cleavage to sequence proteins of up to 50 - 70 amino acids, the Edman degradation selectively and sequentially removes the N-terminal amino acid of the protein which can be analyzed via mass spectroscopy
- 34how to analyze amino acid composition of large proteins?for larger proteins digestion with chymotrypsin, trypsin, and cyanogen bromide a synthetic reagent may be used
this digestion selectively cleaves proteins at specific amino acid residues creating smaller fragments that can then be analyzed via electrophoresis or the Edman degradation, because disulfide links and salt bridges are broken to reduce the protein to its primary structure, their positions cannot be determined by these methods - 35how is protein activity monitored?protein activity is generally determined by monitoring a known reaction with a given concentration of substrate and comparing it to a standard, activity is correlated with conc but is also affected by the purification methods used and the conditions of the assay, reactions with a color change have particular applicability because microarrays can rapidly identify the samples from a chromatographic analysis that contains the compound of interest
- 36how is protein concentration determined?concentration is determined almost exclusively through spectroscopy, because proteins contain aromatic side chains they can be analyzed with UV spectroscopy without any treatment, however this type of analysis if particualarly sensitive to sample contaminants
proteins also cause colorimetric changes with particular reactions, particularly the BCA assay (bicinochoninic acid), the lowry reagent assay and Bradford protein assay, the Bradford method is most common because of its reliability and simplicity - 37Bradford protein assaymixes a protein in a solution with coomassie brillant blue dye, in its protonated form the dye exists as a brown green color, the dye gives up proteons to the ionizable groups in the protein turning blue in the process, noncovalent attractions between the deprotonated dye and the protein then stabilize this blue form of the dye, thus increased protein conc correspond to a larger concentration of blue dye in the solution, samples of known protein conc are reacted with the bradford reagent and then absorbance is measured to create a standard curve, this is less accurate when more than one amino acid is present, is also limited by the presence of detergent in the sample or by excessive buffer
- 38what ions are most likely to be found bound to a protein in the body?calcium and magnesium
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- Flashcards on nonenzymatic protein function, created by Xiao Yuan on 11/02/2018.
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