mol cell exam 1 .pdf

cell -animal...

cell -animal Lysosome, S ER cutoskeletal Microvilli Vs ↑ s tru , gy al n, c. membrane Cell Wall, X microfilament D tio.S Za of or h.D ell -24 xa Ins MS iolo · inter filaments Vacuole, at c M · micro Chloroplast ity ss P C all · Te of., B tubules rs fe ar, lar F s Cell double membrane ve ro rc u 2_ > - la The Golgi structure - complex is ni P a ec 0 U nt S ol.0 overview divided into e ta N M 02 three main Th is a tic -34 compartments/ - cisternae:as tempty As ub ry OL I close L cis, medial, - - and trans > close B TER - - cisternae to o O Plasma nucleus membran k a h Eu S s r. D Please know the functions of the organelles  Eu D r. k a B I S As ub ry OL s h o X Th is a tic -34 e ta N M 02 U nt S ol.0 ni P a ec 0 ve ro rc u 2_ Endoplasmic rs fe ar, lar F ity ss P C all of or h.D ell -24 reficolum Te of., B ELECTRON MICROGRAPH of an animal cell xa Ins MS iolo s tru , gy Subcellular organization of eukaryotic cells at c M D tio.S al n, c. la s ATP villi increases is Surface area Synthesize required  Summary of the Difference Between Plant and Animal Cells cell membrane present present s tru , gy al n, c. Feature Plant Cells Animal Cells D tio.S of or h.D ell -24 xa Ins MS iolo Present (Cellulose), prevent excessive water uptake at c M Cell Wall Absent ity ss P C all and gives shape Te of., B rs fe ar, lar F s Chloroplasts Present Absent ve ro rc u 2_ la Intermediate Filaments Rare Mostly present ni P a ec 0 U nt S ol.0 Generally Absent (have alternative mechanisms for e ta N M 02 Centrioles Present spindle formation) Th is a tic -34 Large Central Vacuole (stores nutrients, waste products, Vacuoles Smaller Multiple Ones and helps in maintaining turgor pressure) Lysosomes As ub ry OL Rare (similar structures present called lytic vacuoles) I Common B o Ribosomes Cytoplasmic and Chloroplastic Cytoplasmic Only k a h Eu Communication Channels Plasmodesmata Gap Junctions S s r. Glyoxysomes Present (lipid conversion to carbohydrates) Absent D  Arrangement of specific proteins · E The three types of cytoskeletalau FIGURE: filaments 3 have characteristic proteins s tru , gy door al n, c. a bu fir D tio.S distributions within mammalian cells of or h.D ell -24 xa Ins MS iolo of To ↳ 19 at c M Y ↑ ity ss P C all Te of., B rs fe ar, lar F s ve ro rc u 2_ la ni P a ec 0 U nt S ol.0 e ta N M 02 Th is a tic -34 As ub ry OL I B o k a h Eu Cytoskeletal Microtubules (Blue) are polymers made of alpha and beta-tubulin dimers. S filaments Microfilaments (Red) are polymers of the globular protein, actin. s Intermediate filaments (Green) are made of a number of proteins. r. D  s tru , gy al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo at c M ity ss P C all Te of., B rs fe ar, lar F s ve ro rc u 2_ la ni P a ec 0 The nuclear U nt S ol.0 pore complex e ta N M 02 is Th is a tic -34 made of several As ub ry OL I proteins: B o k a h Eu The Lamina (nuclear) is composed of proteins and is present The outer membrane of the S near the inner membrane. nucleus is continuous with s It is involved in structural integrity maintenance and is also r. the Rough ER. D involved in cellular functions such as DNA repair and replication  Lumen is the inside of any membrane- s tru , gy al n, c. enclosed compartment. D tio.S of or h.D ell -24 xa Ins MS iolo Shown here is the at c M ity ss P C all Te of., B lumen of the Golgi and rs fe ar, lar F s the RER ve ro rc u 2_ la Ribosomes - - ni P a ec 0 U nt S ol.0 e ta N M 02 Vesicles are single membrane-enclosed Th is a tic -34 compartments. They As ub ry OL I carry cargo (proteins etc) in their lumen or B o embedded in their membranes as well. k a h The vesicles pictured Eu here are carrying S cargo from RER to the s r. cis- Golgi. D  LYSOSOME Break down s tru , gy worn out al n, c. FUNCTIONS: cell parts. D tio.S of or h.D ell -24 xa Ins MS iolo > misfolded exi- proteins at c M ↑ ity ss P C all Te of., B ENDOCYTOSIS: RME rs fe ar, lar F s receptor ve ro rc u 2_ la mediator ingestion -Phagocytosis: Endocytosis ni P a ec 0 of U nt S ol.0 , largersubstances (ingestion of. same ranamrane e ta N M 02 the -has insoluble material) Th is a tic -34 invagination As ub ry OL I Pinocytosis: (ingestion of soluble material) B o a AUTOPHAGY (cell k h Eu eating itself) S s r. D  ↓ certain organelles D Eu Scalcium r. k B 2) Gasphages S As ub ry OLa I s h o Autophagy vs apoptosis A cell Th is a tic -34 the e ta N M 02 - involves barrier U nt S ol.0 death ↑ destroyed ni P a ec 0. the ve ro rc u 2_ rs fe ar, lar F ity ss P C all entire of or h.D ell -24 cell Te of., B xa Ins MS iolo s tru , gy at c M D tio.S al n, c. la s entireleath  D Eu r. k a B I S As ub ry OL s h Th is a tic -34 o e ta N M 02 U nt S ol.0 ni P a ec 0 ve ro rc u 2_ - rs fe ar, lar F ity ss P C all Both of or h.D ell -24 the Te of., B follow xa Ins MS iolo s tru , gy symbiotic at c M CHLOROPLAST and MITOCHONDRIA theory D tio.S al n, c. la s - Both > Both - are > Both power the Own double cell. Egenetic membrane DNA house of material have their  within the cell. changes Gosconditional. >The - proteins are CDK and > Proteins - help > Phase cyclin in cell - cucle C. differen. >temperature The Cell Cycle - s tru , gy al n, c. > lack - of resources. -toxins/ D tio.S of or h.D ell -24 xa Ins MS iolo Interphase = G0/G1+S+G2 virus/bacteria. at c M ity ss P C all Te of., B checkpoint T I Kinase > Add phosphate rs fe ar, lar F - · s ve ro rc u 2_ > there are la Growth - phosphoryldation > helps - 344 & activate the ni P a ec 0 checkpoints U nt S ol.0 Protein , e ta N M 02 ⑧ mepoint Interphase Th is a tic -34 3 As ub ry OL I Gunthesis B o Replication Duplication,A k a h cells prepare Eu T for M Phase S · ↓ s r. checkpoint D 2  Structure Primary - linear Polypeptide Protein Structure and Function: 3.1. s tru , gy al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo 3.1 Hierarchical Structure of Proteins at c M ity ss P C all Protein sequence specifies folding into secondary and tertiary Te of., B rs fe ar, lar F s structures that either are functional units or can interact with other ve ro rc u 2_ la peptides to form quaternary structure functional units. ni P a ec 0 U nt S ol.0 Exceptional conformational flexibilities of disordered proteins e ta N M 02 Th is a tic -34 contribute to their multiple functions. As ub ry OL Some polypeptides with dissimilar sequences fold into similar three- I dimensional structures. B o Homologous proteins evolved from a common ancestor; have similar k a h sequences, structures, and functions; and can be classified into families Eu and superfamilies. S s r. D  * railroad Protein functions: depend on specific tracks are cytoskeletals. binding interactions and conformational s tru , gy changes in the structure of a properly al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo folded protein. cutoskeletal almost > works like at c M - Structure — organizing the genome, Skeleta ity ss P C all Te of., B organelles, cytoplasm, protein rs fe ar, lar F s ve ro rc u 2_ la complexes, and membranes in three- Proteins have ni P a ec 0 diverse function dimensional space.. U nt S ol.0 Regulation — controlling protein e ta N M 02 secreting proteins activity of enzyme impact the - activity. > protein Th is a tic -34 - - the Proteins are Synthesized Signaling — monitoring the As ub ry OL as they are essentially environment and transmitting & secreted & information. I in > RTK GPCR- helper B - o signaling Transport — moving small molecules and ions across membranes. k a microtubule/microfilament h · Enzyme activity — catalyzing Eu chemical reactions. S > - function w/o proper Motors — generating force for s No r. D folding movement.  Four levels of protein hierarchy s tru , gy al n, c. (a) Primary structure — linear sequence of D tio.S of or h.D ell -24 xa Ins MS iolo amino acids linked together by peptide at c M bonds. > single ity ss P C all Te of., B - rs fe ar, lar F s ve ro rc u 2_ la (b) Secondary structure — folding of the ni P a ec 0 polypeptide chain into local α helices or β U nt S ol.0 - - sheets. e ta N M 02 - Domain > specific - area Th is a tic -34 (c) Tertiary structure: Structure of a peptide composed of As ub ry OL I secondary structural elements and various B o loops and turns. May form distinct, independently stable a domains. k h Eu -several domain (d) Quaternary structure — some functional S s proteins are composed of more than one r. D polypeptide.  translation > synthesis of polypeptide from - amine carboxul Structure of a polypeptide MRNA ↑ T terminal Amino acid side chains determine the distinct properties end s tru , gy al n, c. of individual proteins. D tio.S of or h.D ell -24 xa Ins MS iolo bu at c M > Forming - multiple (a) Peptide bond — is formed by a dehydration ity ss P C all Te of., B Peptide reaction linking one amino acid C-terminus to rs fe ar, lar F s form ve ro rc u 2_ la 12 condensation" another amino acid N-terminus. ni P a ec 0 U nt S ol.0 (b) Polypeptide — linear polymer has a free amino e ta N M 02 end (N-terminus) and a free carboxyl end (C- Th is a tic -34 terminus). As ub ry OL I (c) Peptide bond (yellow) — links the amino nitrogen B o atom (blue) of one amino acid (aa) with the carbonyl carbon atom (gray) of an adjacent amino a acid in the chain. k h Eu Protein function — derived from three-dimensional S s structure, which is determined by the amino acid r. D sequence and intramolecular noncovalent interactions  The 𝛂𝛂 helix, a common secondary structure in proteins s tru , gy al n, c. X always D tio.S Secondary structures — stable spatial arrangements of of or h.D ell -24 xa Ins MS iolo projected at c M outside. polypeptide chain segments held together by hydrogen ity ss P C all Te of., B bonds between backbone amide and carbonyl groups. rs fe ar, lar F s - - ve ro rc u 2_ la 𝛂𝛂 Helix: ni P a ec 0 T U nt S ol.0 Polypeptide backbone (ribbon) folds into a spiral/helix e ta N M 02 with 3.6 amino acids per turn (0.54 nm). Th is a tic -34 - ages Helix is stabilized by hydrogen bonds between arrage As ub ry OL backbone oxygen and hydrogen atoms (more bonds, X Compared avere more stable). B I to 3.4 nm o pitch in R groups project outward from the surface of the helix dsDNA helix and determine the chemical nature of helix faces. k a h Prolines (usually) cannot participate in hydrogen Eu ↓ bonding and usually are excluded from an 𝛂𝛂 helix. S s r. excluded from a helix D  The 𝛃𝛃 sheet, another common secondary structure in proteins s tru , gy al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo β Sheet — laterally packed β strands (5-8 residues), each at c M ity ss P C all of which is a nearly fully extended polypeptide segment. Te of., B rs fe ar, lar F s ve ro rc u 2_ la (a) Three-stranded β sheet — antiparallel β strands with ni P a ec 0 connecting loops (top view): U nt S ol.0 Stabilized by hydrogen bonds between backbone e ta N M 02 oxygen and hydrogen atoms in amino acids on Th is a tic -34 ·minus B Pens different strands. As ub ry OL(b) Antiparallel β sheet (side view): I 2 terminus B GPCRE 𝛂𝛂 Carbon bond angles produce a pleated o polypeptide backbone contour. a Alternate R groups project above and below the k h Eu plane of the sheet. S s (c) Parallel β strand sheet: same N-to-C strand r. D orientations with connecting loops.  Structure of a 𝛃𝛃 turn Composed of four residues. s tru , gy al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo at c M Reverses the direction of a ity ss P C all Te of., B rs fe ar, lar F polypeptide chain (180° U-turn). s ve ro rc u 2_ la ni P a ec 0 Cα carbons of the first and fourth U nt S ol.0 e ta N M 02 residues are usually less than 0.7 nm Th is a tic -34 boud apart and linked by a hydrogen bond. > - Hydrogen As ub ry OL I β Turns facilitate the folding of long B o polypeptides into compact structures. a ARgroup just has I hudrogen k h Glycine (smallest R group) and proline Eu (built in bend) are commonly found in S s r. β turns. D  - Motifs of protein secondary structure Especialized s tru , gy al n, c. 2 a helixes sections D tio.S that of or h.D ell -24 xa Ins MS iolo have Have calcium at c M specialized X w/the loop ity ss P C all Te of., B function rs fe ar, lar F s ve ro rc u 2_ la ni P a ec 0 U nt S ol.0 e ta N M 02 Th is a tic -34 W > - B As ub ry OL ahelix pleated sheets L B I o k a h Eu Examples: S Coiled-coil motif: Oncoproteins c-Jun, c-Fos s E-F hand/Helix-Loop-Helix: c-Myc, Calmodulin r. D Zn-Finger domain: Blimp-1, EBF-1 Two α helices-heptad repeat sequences  Motifs of protein secondary structure Structural motifs: s tru , gy al n, c. Regular combinations of secondary structures usually with a specific type of function. D tio.S of or h.D ell -24 xa Ins MS iolo Can be encoded by a highly conserved sequence motif. at c M ity ss P C all Te of., B (a) Coiled-coil motif/Molecular Zippers: rs fe ar, lar F doi: 10.3390/ijms21103584 doi.org/10.1016/S0962-8924(00)01898-5 s ve ro rc u 2_ Two α helices wound around each other. la Each α helix-heptad repeat sequence with a hydrophobic residue at positions 1 and 4. ni P a ec 0 U nt S ol.0 Example: oncoproteins such as c-Fos and c-Jun e ta N M 02 (b) EF-hand/helix-loop-helix motif : doi.org/10.1110/ps.33302 Th is a tic -34 A type of helix-loop-helix motif in many proteins, including many calcium-binding and DNA-binding regulatory As ub ry OL proteins. Calcium-binding proteins (calmodulin) — oxygen atoms from five residues in the acidic glutamate- and aspartate- B I rich loop and one water molecule form ionic bonds to coordinate a Ca2+ ion. o a (c) Zinc-finger motif: k doi: 10.1007/s00775-023-01991-6 h Present in many DNA-binding proteins that help regulate transcription. Eu 25-Residue motif with two invariant cysteine residues usually at positions 3 and 6 and two invariant histidine D S residues at positions- 20 and 24. - s r. Zn ion binds between a pair of β strands (blue) and a single α helix (red) to the conserved cysteine histidine 2+ D residues.  Evolution of protein families s tru , gy al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo at c M ity ss P C all Te of., B rs fe ar, lar F s ve ro rc u 2_ la ni P a ec 0 U nt S ol.0 e ta N M 02 Th is a tic -34 (a) Hemoglobin: As ub ry OL Tetramer of two α and two β subunits. I B o Each subunit is structurally similar to leghemoglobin and myoglobin monomers. O2 binds directly to heme molecule (red) noncovalently associated with each globin polypeptide. k a h (b) Primitive monomeric oxygen-binding globin: thought to be the ancestor of modern-day globins. Eu Evolution of globin proteins parallels evolution of animals and plants. Major changes are introduced with divergence of plant globins from animal globins and of myoglobin from S s hemoglobin. r. Gene duplication gave rise to α and β subunits of hemoglobin. > mutation D - Homologs — proteins with common ancestors.  Polymerase used to bind Protein Structure and Function 3.2 to genome s tru , gy al n, c. ↓ You D tio.S F of or h.D ell -24 xa Ins MS iolo DNA at c M Polymerase Polymerase ity ss P C all Te of., B 3.2 Protein Folding rs fe ar, lar F s ve ro rc u 2_ la Protein amino acid sequence determines its three-dimensional ni P a ec 0 U nt S ol.0 structure and function. e ta N M 02 ATP-dependent molecular chaperones and chaperonins assist protein Th is a tic -34 - folding in vivo. As ub ry OL ↳ within the cell Misfolded/denatured proteins can form well-organized amyloid fibril I B o aggregates that can cause diseases, including Alzheimer’s disease and a Parkinson’s disease. k h Eu S s r. D  PIP posite - trans 4/447 next to each Other - Rotation between s tru , gy as planar peptide groups al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo in proteins. at c M ity ss P C all Te of., B Polypeptide rs fe ar, lar F s ve ro rc u 2_ la backbone steric Planar peptide bonds limit the shapes into which ni P a ec 0 restraints and amino U nt S ol.0 proteins can fold. acid side chain e ta N M 02 properties severely Th is a tic -34 Polypeptide chain on either side of the peptide restrict Cα–amino bond (P1 and P2) can be oriented in either a trans As ub ry OL (center) or a cis (right) configuration relative to I nitrogen bond (the Φ/Phi angle) and Cα– B the peptide bond. o carbonyl carbon bond a (the Ψ/Psi angle) Approximately 99.97 percent of the peptide k h A rotations. Eu bonds that have any residue other than proline can determine how 50 S ↓ at P2 are in the trans configuration. well the chain can s r. - rotate. D  s tru , gy al n, c. D tio.S Hypothetical protein-folding pathway. of or h.D ell -24 xa Ins MS iolo at c M ity ss P C all Te of., B Monomeric protein folding hierarchy: rs fe ar, lar F s ve ro rc u 2_ la primary (a) → secondary (b–d) → ni P a ec 0 tertiary (e) structure. U nt S ol.0 e ta N M 02 Formation of small structural motifs Th is a tic -34 Formation of small structural motifs (c) appears to precede the formation As ub ry OL I of domains (d) and the final tertiary B o structure (e). k a h Native state — usually the Eu formation conformation with the lowest free S of domains s energy (G). r. final tertiary D structure  certain Protein folding mediated by molecular chaperones - - helps fold Parts ATP is - not attached only towards s tru , gy al n, c. Polypeptide conformation immediately. HSP90 sito D tio.S of or h.D ell -24 xa Ins MS iolo - sequence Active ↑ at c M ity ss P C all Te of., B Step li reversible rs fe ar, lar F s BINDS ve ro rc u 2_ la ni P a ec 0 Example 2: need U nt S ol.0 - ZATP - e ta N M 02 A the Example ATP energyaing Th is a tic -34 As ub ry OL no achange intermediate B I o Step cochaperone a changesATP k h - - helps Eu from hydrolyze To S ATP ADP s r. > incase PNAJ mutated - D AtP won't hydrolyze BAG-1: Bcl-2-associated athanogene-1 GrpE: GroP-like gene E  Protein folding mediated by molecular chaperones Molecular chaperones: bind to a short segment of a protein substrate and stabilize unfolded or partly folded proteins, s tru , gy al n, c. preventing aggregation or degradation. D tio.S of or h.D ell -24 xa Ins MS iolo (a) Hsp70 chaperone protein cycle: at c M Binds transiently to a nascent polypeptide as it emerges from a ribosome or to a protein that has unfolded. ity ss P C all Te of., B Step 1: Hsp70 binds unfolded protein in rapid equilibrium to the open conformation of the substrate-binding domain rs fe ar, lar F s (orange) and ATP (purple) in the nucleotide-binding domain (light blue). ve ro rc u 2_ la Step 2: Co-chaperone accessory proteins (DnaJ/Hsp40) stimulate ATP hydrolysis inducing a large conformational ni P a ec 0 change in the substrate-binding domain that locks the unfolded protein region into the substrate-binding U nt S ol.0 domain — proper folding is facilitated. e ta N M 02 Step 3: Exchange of ATP for the bound ADP stimulated by other accessory co-chaperone proteins (GrpE/BAG1). Th is a tic -34 Step 4: Releases the properly folded substrate, regenerating the open conformation. As ub ry OL (b) Hsp90 molecular chaperone cycle — three conformational states: Step 1: No nucleotide bound to the nucleotide-binding domain (light blue) — dimer in a very flexible, open I B o configuration that can bind a partially-folded client protein. Step 2: Rapid ATP binding causes conformational change — the nucleotide-binding domains dimerize and the a substrate-binding domains move together. k h Eu Step 3: Intermediate state. Step 4: Closed conformation. S Step 5: ATP hydrolysis — conformational change in Hsp90 that may include a highly compact form, folding of the client, s r. D and client protein release (not shown). Step 6: Release of ADP regenerates the initial flexible open state.  eukaryotic Prokaryotic group I Protein folding mediated by chaperonins okamotic chaperonin GroEL/GroES s tru , gy al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo at c M ity ss P C all Te of., B rs fe ar, lar F s ve ro rc u 2_ ATP la ni P a ec 0 U nt S ol.0 e ta N M 02 Th is a tic -34 As ub ry OL I B o k a h Eu ADP S s r. D Eukaryotic Group 2 chaperonin: TRiC/CCT (chaperonin-containing TCP-1) Group-1 chaperonin GroEL/GroES in bacteria  group It prokaryotes - Protein folding mediated by chaperonins group 2- eukaryotesrcnae Chaperonins: folding chambers into which all or part of an unfolded protein can be bound in an appropriate environment, s tru , gy al n, c. giving it time to fold properly. D tio.S of or h.D ell -24 xa Ins MS iolo Prokaryotic group I chaperonin GroEL: at c M Barrel-shaped complex of 14 identical ∼60,000-MW subunits, arranged in two stacked rings (blue) of seven subunits ity ss P C all Te of., B each that form two distinct internal polypeptide folding chambers. rs fe ar, lar F s Homoheptameric GroES lids (10,000-MW subunits) seal either end of the barrel. ve ro rc u 2_ la ni P a ec 0 GroEL–GroES folding cycle: U nt S ol.0 e ta N M 02 Step 1: A partly folded or misfolded polypeptide (captured by hydrophobic residues) enters one of the folding chambers. Th is a tic -34 Step 2: The second chamber is blocked by a GroES lid. Each ring of seven GroEL subunits binds seven ATPs, hydrolyzes As ub ry OL them, and then releases the ADPs in a set order coordinated with GroES binding and release and polypeptide binding, folding, and release. The major conformational changes that take place in the GroEL rings control B I the binding of the GroES lid that seals the chamber. o Step 3: The polypeptide remains encased in the chamber capped by the lid, where it can undergo folding until ATP a hydrolysis — the slowest, rate-limiting step in the cycle (t1/2~10 s) — induces binding of ATP and a different k h Y 10 seconds GroES to the other ring (transient intermediate shown in brackets). Eu Step 4: This binding then causes the GroES lid and ADP bound to the peptide-containing ring to be released, opening S the chamber and permitting the folded protein to diffuse out of the chamber. s r. If the polypeptide has folded properly, it can proceed to function in the cell. If it remains partially folded or D misfolded, it can rebind to an unoccupied GroEL and the cycle can be repeated.  Basic differences between Chaperone and Chaperonin can also help fold ↑ s tru , gy certain parts al n, c. D tio.S Aspect Chaperone Chaperonin of or h.D ell -24 xa Ins MS iolo at c M Chaperones are typically small proteins or Chaperonins are large, multi-subunit protein complexes ity ss P C all Te of., B protein complexes that assist in protein folding that provide a protected environment for protein Structure rs fe ar, lar F s by preventing misfolding and aggregation. folding within their central cavity. much ve ro rc u 2_ la Monomer with Mol wt. 70-100 kDa. Oligomer with mol wt. 800 kDa. heavier ni P a ec 0 U nt S ol.0 Chaperonins use an "inside-out" folding mechanism. Chaperones work by binding to partially folded e ta N M 02 They encapsulate misfolded or partially folded proteins Mechanism or unfolded proteins, stabilizing them, and Th is a tic -34 within their central cavity and provide a controlled facilitating correct folding. environment for folding to occur. As ub ry OL Chaperones primarily assist in the folding of I Chaperonins are often involved in folding all or part of B individual proteins (nascent chain of a protein o an unfolded protein or in situations where folding is Function as it is synthesized and as it exits the ribosome) particularly challenging, such as with certain types of a and prevent their aggregation, helping them proteins or under stress conditions. k h reach their native functional conformation. Eu Hsp60s, Group-1 chaperonin GroEL/GroES in bacteria S Hsp70, Hsp90, small heat shock proteins Examples and the Group-2 chaperonin TRiC/CCT (chaperonin- s r. (sHsps) containing TCP-1) complex in eukaryotes and Archaea. D  Misfolded proteins can form ordered amyloid aggregates based on a cross-𝛃𝛃 sheet structure. s tru , gy al n, c. Misfolded proteins/proteolytic fragments can accumulate D tio.S of or h.D ell -24 xa Ins MS iolo at c M as aggregates or plaques, inside or outside of cells in ity ss P C all Te of., B various organs including joints between bones, the liver, rs fe ar, lar F s and the brain (prions, plaques). ve ro rc u 2_ la ni P a ec 0 Many diverse proteins can aggregate into amyloid (well- U nt S ol.0 e ta N M 02 ordered) fibrils that have a common structure and can welender cause amyloidoses diseases, including neurodegenerative Th is a tic -34 diseases such as human Alzheimer’s disease and As ub ry OL Parkinson’s disease and transmissible spongiform encephalopathy (“mad cow” disease) in cows and sheep. I B o (a) Cross-β sheet: Protofilaments assemble into thicker fibrils. a Fibrils can aggregate into macroscopic plaques k h Eu and fibrillary tangles in tissues. S s (b) Alzheimer’s patient brain tissue section with multiple amyloid r. D plaques and fibrillary tangles. Protein Structure and Function 3.4 s tru , gy al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo at c M 3.4 Regulating Protein Function ity ss P C all Te of., B rs fe ar, lar F s ve ro rc u 2_ la Proteins may be regulated at the level of protein synthesis or protein ni P a ec 0 U nt S ol.0 degradation or through noncovalent or covalent interactions. adds ubiquitin e ta N M 02 T proteins to Proteins marked for destruction with a polyubiquitin tag by ubiquitin supposed Th is a tic -34 that are ligases are degraded in proteasomes. be As ub ry OL to degraded. Several allosteric mechanisms act as switches, reversibly turning protein B I o activity on and off. k a h Higher-order regulation includes the intracellular compartmentation of Eu proteins. S s r. D Protein Structure and Function 3.5 s tru , gy al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo at c M ity ss P C all 3.5 Purifying, Detecting, and Characterizing Proteins Te of., B rs fe ar, lar F s Proteins can be isolated from other cell components on the basis of a ve ro rc u 2_ la ni P a ec 0 variety of physical and chemical properties. U nt S ol.0 e ta N M 02 Proteins can be detected and quantified by various assays and specific Th is a tic -34 antibody recognition. As ub ry OL Tagging with various types of markers can be used to investigate protein I B synthesis, location, processing, and stability. o X-ray crystallography, cryoelectron microscopy, and nuclear magnetic k a h Eu resonance (NMR) spectroscopy reveal three-dimensional structures of S proteins. s r. D  Centrifugation techniques separate particles that differ in mass or density. s tru , gy al n, c. (a) Differential centrifugation: D tio.S of or h.D ell -24 xa Ins MS iolo Step 1: Prepare a cell homogenate or other at c M mixture. ity ss P C all Te of., B Step 2: Centrifuge long enough to sediment rs fe ar, lar F s ve ro rc u 2_ la larger particles (e.g., cell organelles, cells) ni P a ec 0 into a pellet at the bottom of the tube. U nt S ol.0 Step 3: Separate supernatant containing e ta N M 02 smaller particles and soluble molecules from Th is a tic -34 pellet. solution that a take has As ub ry OL differen Revels T (b) Rate-zonal centrifugation: of density B I o Step 1: Layer mixture on top of density gradient made with sucrose or another a highly soluble molecule (CsCl). k h Eu Step 2: Centrifuge long enough to band molecules that differ in density in discrete S s zones within a density gradient. r. D Step 3: Collect fractions from regions of the gradient.  Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS- PAGE) separates proteins primarily on the basis of their masses. (a) SDS-PAGE: s tru , gy Step 1: Denature proteins with SDS, a negatively charged al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo detergent that interacts with hydrophobic amino acids to at c M dissociate multimeric proteins, denature protein structures, and ity ss P C all Te of., B coat each protein with negative charges in proportion to the rs fe ar, lar F mass of the protein. s Step 2: Electrophoresis: thelps denature ein ve ro rc u 2_ la Cathode ni P a ec 0 - Electric field drives SDS-protein complexes through the U nt S ol.0 polyacrylamide gel — small complexes move through the e ta N M 02 of DNA majority pores faster than larger complexes. move Th is a tic -34 will Proteins separate into bands according to their sizes as to the Plus side they migrate. As ub ry OL Anode I Step 3: The separated protein bands are stained with a dye for visualization. B o (b) SDS-PAGE separation of proteins in a whole-cell lysate (detergent- solubilized cells): a (Left) All cell proteins in the lysate. k h Eu (Right) A single protein purified from the lysate by antibody- affinity chromatography. S Comparison of migration distance to that of molecular weight s r. D standard proteins (kDa numbers) reveals unknown protein molecular weight.  Ampholytes with an excess of negative charges will migrate toward the anode (upward)(( +) upward s tru , gy Two-dimensional gel electrophoresis separates proteins al n, c. on the basis of charge and mass (especially, for proteins D tio.S of or h.D ell -24 xa Ins MS iolo with similar masses). at c M ity ss P C all Te of., B Ampholytes with an excess of (a) Two-dimensional electrophoresis: rs fe ar, lar F positive charges will migrate s ve ro rc u 2_ Step 1: Isoelectric focusing — separates proteins la toward the cathode (downward)(() (downward) in gel based on isoelectric point ni P a ec 0 U nt S ol.0 (pI; net charge). e ta N M 02 Charged ampholytes in gel establish a pH gradient in the electric field. Th is a tic -34 ⑦ Each protein migrates until it reaches the pH As ub ry OL in the gel at which the protein net charge is zero (pI). B I o Step 2: Apply gel strip to SDS-polyacrylamide gel. Step 3: SDS-PAGE separates proteins in gel based a on the molecular weight. k h Eu ⑦ (b) Two-dimensional gel of protein extract from cultured S Ampholytes and zwitterions: are molecules with at least two pKa values, cells — each spot represents a single polypeptide. s r. at least one of which is acidic and at least one is basic. D The isoelectric point: of an amino acid is the pH at which the amino acid has a neutral charge.  size affinity aganose - usually Three commonly used liquid s tru , gy chromatographic techniques al n, c. - neutral D tio.S separate proteins: of or h.D ell -24 xa Ins MS iolo at c M on the basis of mass/size, charge, ity ss P C all Te of., B or affinity for a specific binding rs fe ar, lar F s partner: ve ro rc u 2_ la the -proteins larger ni P a ec 0 proteins get U nt S ol.0 eluted first Gel filtration chromatography e ta N M 02 compared to smaller Ion-exchange chromatography Th is a tic -34 proteins Antibody-affinity chromatography charge As ub ry OL - I Variation in Affinity Chromatography: Examples of fusion proteins B o Fusion protein Beads linked to: partner k a h Biotin (Vitamin B) Strepatvidin (bacterial protein) Eu Glutathione S- Glutathione gets erted S transferase (GST) s r. > - Poly-Histidine Divalent metal (Ni2+ or Zn2+) D  asize Three commonly used liquid chromatographic techniques (a) Gel filtration chromatography — separates proteins on the basis of size: s tru , gy al n, c. Column — porous beads. D tio.S of or h.D ell -24 xa Ins MS iolo A mixture of proteins is loaded on the top of the column followed by flow of solution. at c M Different proteins emerge in the eluate flowing out of the bottom of the column at different times (different ity ss P C all Te of., B elution volumes). rs fe ar, lar F s Smaller proteins are slowed by diffusing into and out of spaces in beads less accessible to larger proteins ve ro rc u 2_ la that elute sooner. ni P a ec 0 U nt S ol.0 Separated proteins can be collected in different fractions. > net charge e ta N M 02. - (b) Ion-exchange chromatography — separates proteins on the basis of net charge: Th is a tic -34 Columns — beads with either a positive charge (shown here) or a negative charge. Proteins with the same net charge as the beads are repelled and flow through the column. As ub ry OL Proteins with the opposite charge bind to the beads with different affinities, depending on their structures. I Bound proteins can be eluted by passing a salt gradient (usually NaCl or KCl) through the column. B o Salt ions binding to the beads displace the proteins; more tightly bound proteins require higher salt antibody interaction. a concentrations in order to be > released. k h - (c) Antibody-affinity chromatography — binds a specific protein on the basis of antibody interaction: Eu Column — beads to which a specific antibody is covalently attached. S Column antibodies retain only the protein to which they have high affinity. s r. Bound protein can be eluted with an acidic solution or some other solution that disrupts the antigen- D antibody complexes.  blotting Western s tru , gy al n, c. D tio.S of or h.D ell -24 xa Ins MS iolo variableregion light at c M ity ss P C all Te of., B ab region rs fe ar, lar F s ve ro rc u 2_ la neam ni P a ec 0 region U nt S ol.0 e ta N M 02 (a) Western immunoblotting: Th is a tic -34 Step 1: Transfer/blot proteins separated by SDS-PAGE or two-dimensional electrophoresis onto a porous membrane that avidly binds all proteins. As ub ry OL Step 2: Primary antibody: Incubate membrane with a solution of an antibody (Ab1) specific for the protein of interest, which binds only to I B a specific protein on the blot. o Step 3: Secondary antibody: a Incubate membrane with a solution of a second antibody (Ab2) which specifically binds to the first (Ab1). k h Eu Ab2 is covalently linked to a detectable label such as an enzyme that catalyzes a chromogenic reaction or ↳produce a color releases light (e.g., chemiluminescence), a radioactive isotope, or some other substance whose presence can S be detected with great sensitivity. s r. Step 4: Detect label on the second Ab, revealing the location of protein bound by primary antibody. D  see when cells should telemores help > - stop dividing. divide forever Culturing and Visualizing Cells 4.1 > cancer - cells can s tru , gy al n, c. ↓ term experiments care bes of longer D tio.S of or h.D ell -24