Lecture 16: DNA Replication

Lecture 16: DNA Replication

Semiconservative mechanism

experiment: E. coli cells initially grown in 15N, then transferred into medium of 14N; through density-gradient centrifugation, pattern suggests semiconservative model

DNA Polymerases Require a Primer to Initiate Replication

DNA synthesis, like RNA synthesis, proceeds 5' 3' (formation of phosphoester bond between 3' oxygen of growing strand & alpha phosphate of dNTP

primer (preexisting RNA or DNA strand) required to begin chain growth

Duplex DNA is unwound + Daughter strands are formed at DNA Replication fork

At replication origins ORI: usually A-T rich; replication fork

helicase unwinds parental DNA strands

primase, a specialized RNA polymerase, forms a short RNA primer complementary to unwound template strands

DNA pol then elongates primer, forming new daughter strand

topoisomerase I: relieves torsional stress produced by local unwinding of duplex DNA

leading strand: 5' 3', can proceed continuously from single RNA primer, in same direction as movement of replication fork

lagging strand: cell synthesizes a new primer every few hundred bases; primers elongated in 5' 3' direction, forming Okazaki fragments; RNA primer of each Okazaki fragment is removed & replaced by DNA chain growth from neighboring Okazaki fragment; ligase joins adjacent fragments; primers are added randomly (not at specific site);

after DNA synthesis begins at primer, primer is removed by RNAse (this is why RNA is used as primer, to recognize easily amidst DNA)

when next Okasaki fragment reaches primer 3' to it, DNA pol adds in nulceotides that were removed by RNAse

Several proteins participate in DNA Replication

Replication fork protein A (RPA): ssDNA binding protein that helps stabilize unwound DNA so that it doesn't form secondary structures that would prevent DNA pol from binding to it; dislodged by Pol alpha and pol delta

Proliferating cell nuclear antigen (PCNA): stops synthesis of primer by displacing primase + polymerase alpha (synthesizes primer); keeps DNA polymerase delta (elongates daughter strand) complex connected to DNA; falls off when it hits a primer, along with DNA polymerase

DNA Replication occurs Bidirectionally from each origin

experiment: SV40 DNA's replication bubble shows bidirectional replication; edges of bubble are symmetrical to ORI and advance in either direction

two replication forks assemble at a single origin, then move in opposite directions; thus each DNA strand is leading & lagging

Origins of Replication

in bacteria: one circular DNA chromosome, one single origin of replication

in eukaryote: multiple linear chromosomes, multiple ORI = solution to replicate such a long DNA in a reasonable time

Lecture 17: Chromatin Structure

chromosome chromatin nucleosome octamer of histones

Histone: most abundant proteins in chromatin.Structure of Nucleosomes

DNA component of nucleosomes is much less susceptible to nuclease digestion than is the linker DNA between them

nucleosome consists of a protein core w/ DNA wound around its surface

core is an octamer with 2 copies of histones H2A, H2B, H3, H4 (very conserved structure)

linker has H1 histones; linker DNA is variable; roughly 200 bp for each one with nucleosome + linker region

during cell replication, DNA is assembled into nucleosomes shortly after replication fork passes: specific chaperones that bind to histones & assemble them together with newly replicated DNA into nucleosomes

Structure of 30-nm fiber

most chromatin appear as fibers ~ 30 nm in diameter

Interaction between DNA and histones

nucleosomes bind all chromatin: not sequence-specific

phosphate: property unique to DNA, also bp independent; spaced evenly, negatively charged

thus histones can bind to phosphate (not exclusively): basic amino acids lysine + arginine histone is a basic protein; binds acidic DNA, positively charged

Modifications of histone tails control chromatin condensation + function

flexible N-terminus + C-terminus histone tails: extend from globular histone octameric core; required for chromatin to condense from bead-on-a-string conformation into 30-nm fiber

histone tails subject to post-translational modifications: thus they interact between nucleosomes (protein to protein and protein to DNA) based on lysine

lysine acetylation: amine at tip of lysine residue modified by acetate to make neutral product that cannot bind to DNA or negative charges; less-condensed beads-on-a-string conformation conducive for transcription + replication

lysine deacetylation: reversible reaction, enzymes deacetylate histones to induce negative charge; chromatin becomes condensed

Detection of specific DNA fragments by Southern blot (hybridization technique)

cleavage of DNA with a restriction enzyme

mixture subject to gel electrophoresis, then restriction fragment present in gel are denatured with alkaline solution + through capillary action transferred onto a nitrocellulose membrane (blot used because probes do not readily diffuse into original gel)

filter incubated under hybridization conditions with a specific radiolabeled DNA probe; DNA restriction fragment that is complementary to the probe hybridizes, & its location on the filter can be revealed by autoradiographyNontranscribed genes are less susceptible to DNase digestion than active genes

chick embryo erythroblasts actively synthesize globin, whereas cultured undifferentiated MSB cells do not nuclei from each cell type isolated + exposed to increasing concentrations of DNase I nuclear DNA then extracted + treated with restriction enzyme BamHI, which cleaved DNA around globin sequence & normally releases 4.6-kb globin fragment DNase and BamHI-digested DNA subjected to Southern blot analysis with probe of labeled cloned adult globin DNA, which hybridizes to 4.6-kb BamHI fragment If globin gene is susceptible to initial DNase digestion, it would be cleaved repeatedly & would not show this fragment, thus the transcriptionally active DNA does not have 4.6-kb band on Southern blot

Inactive DNA from MSB cells was resistant to digestion, thus inactive DNA is in a more condensed form of chromatin that shields globin genes from DNase digestion (unacetylated histone lysine tails)Nonhistone proteins provide a structural scaffold for long chromatin loops

in situ hybridization: DNA denatured, then fluorescent probe used to label and incubate a cell in vitro, then where it binds can be scene under fluorescent microscope in situ hybridization: with several different fluorescent-labeled probes (anti-sense oligonucleotide) to DNA of one chromosome during interphase chromatin is arranged into large loops (30-nm chromatin fiber) some probe sequences separated by millions of bp in linear DNA appeared reproducibly very close to one another ( anchored to scaffold-associated regions SARs conclusion: chromatin organized with some points that are anchored and the rest are much more flexible (various conformations)

drawers: loops have drawers that allow retrieval of genes that one needs insulators: some scaffold-associated regions functions as insulators, which are DNA sequences of tens to hundreds of bp that insulate transcription units from each other; proteins regulating transcription of one gene cannot influence transcription of neighboring gene that is separated by an insulator

Layers of chromatin packing in chromosomes

metaphase chromosome: condensed scaffold-associated chromatin ( interphase: extended scaffold-associated chromatin ( 30-nm chromatin fiber of packed nucleosomes (beads-on-a-string form of chromatin ( short region of DNA double helix

Lecture 18: Chromosomes

A Chromosome is heterogeneous (even in interphase)

Heterochromatin: dark regions, contains more condensed material

Euchromatin: lighter regions, de-condensed material

Metaphase Chromosome

condensation of metaphase chromosomes results from several orders of folding of 30-nm chromatin fibers

chromosomes that become visible during metaphase are duplicated structures; each metaphase chromosome consists of 2 sister chromatids, which are linked at centromere (constricted region)

telomeres: end of chromatid

centromere involved in mitosis: specific protein complexes build kinetochore, which recruits microtubules; microtubules separate chromatin in mitosis

karyotype: the number, size, + shapes of the metaphase chromosomes, distinctive for each species

In Metaphase, Chromosomes distinguished by Banding Patterns + Chromosome Painting

certain dyes selectively stain some regions of metaphase chromosomes more intensely, producing characteristic banding patterns, specific for individual chromosomes

G bands: produced when metaphase chromosomes are subjected briefly to mild heat or proteolysis, then stained with Giemsa reagent (permanent DNA dye)

G-bands correspond to unusually low G+C content; creates a very reproducible pattern Fluorescence in situ hybridization FISH: method of spectral karyotyping or chromosome painting, uses probes specific for sites scattered along length of each chromosome Probes has different fluorescent dyes; 1 chromosome detected w/ 1 color

Can detect translocation/breaks via multicolor fluorescence (chromosomes break, then refuse)

Chromosome Painting + DNA Sequencing Reveal Evolution of Chromosomes

when comparing sequence of human chromosomes with those of monkeys, the genes are 98-99% conserved ( pure DNA is very conserved

however, the way genes are spread on the chromosome are very different (chromosome breaking and repairing

even if genes are completely conserved, crossing them will still not be fertile; explains speciationPolytene Chromosome: The Drosophila Chromosome

polytene chromosome starts to replicate many times without separating asymmetrical chromosome: centromere is very close to one end; half of the chromosome is centromere and the rest is other arm of chromosome

centromere always stays united, while the rest is replicated many times; result is an enlarged chromosome composed of many parallel copies of itself, greatly increases gene copy number to supply sufficient mRNA for protein synthesis these bands are meaningful because they correspond to condensed chromatin

localization of specific gene by in situ hybridizationInterphase: heterochromatin + euchromatin

Heterochromatin: dark regions, contains more condensed material; not active or expressed; regions of heterochromatin can retract or spray; functions:

gene silencing (regions where transcription is shut off)

regions that are not changing: centromere + telomere = always heterochromatic

protection against mobile movements: keep parts of chromosomes inactive to diminish exchanges

Euchromatin: lighter regions, de-condensed material; function:

active transcription

Elements required for replication + stable inheritance of chromosomes

origin of replication ORI: at which DNA polymerase + other proteins initiate synthesis of DNA

centromere: constricted region required for proper segregation of daughter chromosomes

2 telomeres: ends; experimentally demonstrated by transfection of yeast leucine

Yeast Leucine Transfection Experiment

yeast accepts plasmids (circular DNA), which replicates along with yeasts own division; to ensure all surviving yeasts have plasmid, yeast cell must be dependant on plasmid for survival + proliferation selection: yeast is mutated so that it no longer produces leucine; plasmid has gene coding for enzyme needed for leucine production

autonomously replicating sequence ARS: origins of replication in yeast genome; short sequence ~100 bp, generates progeny + allows inheritance of plasmid; problem is that there is no mitotic segregration

centromeric sequence CEN: derived from centromeres of yeast chromosomes, segregate equally to both mother + daughter cells during mitosis; A-T rich, has specialized histones that recognize particular DNA sequences, and functions to generate proteins + stick together

telomeric sequences TEL: ligated to ends of plasmids, stabilizes linear plasmid to produce LEU colonies a primer is needed to start replication; primer lands randomly & could land far from end of chromosome, so sequence between primer & 3 end of template wont replicate; solved by addition of telomere

an assay can be used to identify origins of replication, centromere + telomeric sequences: test random fragments of DNA inserted in the plasmid

Addition of Telomeric Sequences by Telomerase Prevents Shortening of Chromosomes

telomere: repetitive sequence, in humans and other vertebrates is TTAGGG; sequence can be recognized using FISH; many thousands of bp long in humans & vertebrates DNA pol elongate DNA chains at 3 end, which requires an RNA or DNA primer; daughter DNA lagging strand shortens at each cell division

Telomerase: protein-RNA complex, serves as template for addition of deoxyribonucleotides to the ends of telomeres; thus telomerase is a reverse transcriptase that carries it own internal RNA template to direct DNA synthesis

Telomerase is active only during replication, especially in stem cells, embryo & germ cells; also often reactivated in cancer cells

Mechanism of Telomerase action

3 end of G-rich strand extends 12-16 nucleotides beyond 5 end of complementary C-rich strand

single-stranded 3 terminus of telomere is extended by telomerase: telomerase contains an RNA template that base-pairs to 3 end of lagging-strand template

reverse transcription: telomerase catalytic site then adds deoxyribonucleotides to position 35 using RNA molecule as a template

strands of resulting DNA-RNA duplex then slip relative to each other, leading to displacement of a single-stranded region of telomeric DNA strand and to uncovering of part of RNA template sequence

lagging-strand telomeric sequence then extended to position 35 by telomerase again, and DNA-RNA duplex undergoes translocation + hybridization as before; repeat etc

Lecture 19: Chromatin Modification and Epigenetic Mechanisms

Condensation of Chromatin heterochromatin: when DNA is less accessible, most genes are not transcribed; ie centromere, telomeres, specific regions (regions where chromatin is condensed is often cell-type specific)

in non-differentiated cells, lots of euchromatin present for flexibility in gene expression

in differentiated cells, lots of heterochromatin present because cells are highly specialized and do not require many genes; unneeded genes are silenced within heterochromatin

Silencing Mechanism at Yeast Telomeres: telomeres are heteromeric sequences multiple copies of RAP1 recognize and bind to simple repeated sequence at each telomere region that lacks nucleosomes

SIR3 + SIR4 bind to RAP1, SIR2 binds to SIR4; thus RAP1 recruites SIR2

SIR2 is a histone deacetylase that deacetylates the tails on histones neighboring the repeated RAP1 binding cite

Hypoacetylated histone tails are also binding sites for SIR3 + SIR4, which in turn binds additional SIR2, deacetylating neighboring histones

Result is chromatin condensation and association of several telomeres; end of telomere is embedded inside big chromatin structure

Role of Chromatin in gene silencing: mating phenotype in Yeast Central mating-type locus MAT determines whether cell has a or alpha phenotype

silent mating-type genes are located at HML locus; opposite mating-type genes are present at silent HMR locus; when alpha or a sequences are present at MAT locus, they can be transcribed into mRNAs whose encoded proteins specify mating-type phenotype of cell

silencer sequences near HML and HMR bind proteins that are critical for repression of these silent loci; when mating occurs, one of the 2 genes translocate to MAT

silencer sequence: not specific for a particular gene or chromosome region; can repress any nearby gene; if silencer removed, both mating types are expressed simultaneously

deacetylation is involved in silencing

mutate histone tail by replacing lysine with arginine (which cannot be acetylated) ( silencing

mutate histone tail by removing positive charges and put glycine instead; this mimics acetylation ( no silencing; HML and HMRa were expressed, inhibition of binding to SIR protein RAP1 can also bind silencing sequences: also triggers chain reaction with SIR 2,3,4 proteins; leads to deacetylation of lysine tails + condensation of chromatin

In multicellular eukaryotes, condensation is more complicated: methylation + Polycomb complexes silence whole regions of the genome; these silenced regions can be inherited

Chromatin Immunoprecipitation: identifying chromatin regions containing acetylated histones

one cannot predict which region of genome is heterochromatin an antibody can specifically recognize the epitope that is specific to acetylated or deacetylated histones; difficulty is that antibody is hard to produce

isolate and shear chromatin mechanically: breaks down DNA into 2-3 nucleosome frags

add antibody against a particular acetylated histone tail sequence; bound nucleosomes are immunoprecipitated (centrifugation, interaction with antibody antigen)

separate DNA from proteins, then determine DNA sequences bound to the histones (PCR)Localized regulation of acetylation/deacetylation at the level of promoters, similar processes of acetylation + deacetylation occurs

repression/deacetylation

TATA box (or any sequence) has specific regions activating or repressing sequences that can bind to activators and repressors, recruit polymerase, but it can also have effects on histones

DNA binding domain recruits many proteins; in this case, it is a repressing sequence that recruits deacetylase which reaches nearby histones b/c of 3D conformation of DNA ( URS1, upstream repressing sequence 1 Deacetylation also inhibits binding of TFIID

activation/acetylation

an activating sequence can recruit reactive component of polymerase & proteins that acetylate histones, which locally de-condensate chromatin ( UAS, upstream activating sequence TFIID has evolved to bind acetylated histones (direct effect on polymerase; effect on histones, which also seem to affect polymerase)

Forcing acetylation will also produce chain reaction

Modification of Histones

acetylation: lysine

phosphorylation: serine/threonine

mono-ubiquitination: lysine, add 1 small peptide to end of a tail

methylation/trimethylation: lysine/arginine; irreversible because once you methylate, you cannot acetylate; to prevent acetylation, heterochromatin protein induces condensation by binding methyl

enzyme: histone methyl transferase HMT

3MeK9-histones recruit heterochromatin protein 1 HP1; HP1 bind to each other ( chromatin compacted; Hp1 recruits HMT ( spreading of methylation more than one modification can occur on the same residue

Chromatin-Remodeling Factors Help Activate or Repress Transcription

in addition to histone acetylase complexes, multiprotein chromatin-remodeling complexes are also required for activation at many promotors

ie yeast SWI/SNF chromatin-remodeling complex: in vitro, the complex pushes DNA into the nucleosome so that DNA bound to surface of histone octomer transiently dissociates from the surface and translocates, causing the nucleosomes to slide along the DNA

net result of such chromatin remodeling is to facilitate binding of transcription factors to specific DNA sequences in chromatin; reversible process, as when proteins dissociate, DNA takes back its normal configureation around the histones

many activation domains bind to chromatin-remodeling complexes, and this binding stimulates in vitro transcription from chromatin templates (DNA bound to nucleosomes)

chromatin-remodeling complexes also represses transcription by binding to transcription repression domains of repressors & fold chromatin into condensed structures

Lecture 20: The Genome and General Gene Structure

Genes gene: region of DNA controlling a distinct hereditary character; includes entire DNA sequence necessary for synthesis of functional gene product (ie protein, tRNA, rRNA, miRNA); has promoter, untranslated regions UTR, and regulatory elements mutations can occur at any position in gene region and have very different effects on gene

bacteria: compact genome, operons; genes produce polycistronic RNA (one promoter, one RNA, produces several polypeptides) eukaryote: genes produce monocistronic RNA (one promoter for each coding sequence; each mRNA produces a single polypeptide)

exception: microRNA is polycistronic because the gene is very small

eukaryotic promoters are regulated at a distance through multiple regulatory sequences

eukaryotic transcripts are spliced: transcribed RNA (pre-mRNA) contains exons (pieces of coding sequence) alternating with introns (non-coding sequences) that are removed to produce mature mRNA; also alternative splicing takes place in 60% of human genesHuman Genome

size of genome independent of number of genes &/or complexity of organism

human genome contains much non-coding DNA: transposable elements, repetitive sequences, unclassified spacer DNA, tandem repeats TR, genes, protein-coding regions of genes (< 2%)

genes can be split into two groups

solitary genes: one gene with a particular function; the next gene is remote/far

gene families: group of genes that are closely related, but show divergence and have specialized functions (ie ECM proteins like collagen, cytoskeletal proteins like keratin in skin, transcription factors and growth factors)

gene family: gene is duplicated; only one copy is required, the second copy can diverge through a series of small mutations to acquire new function

Exon Duplication via Unequal Crossing Over leads to creation of new Gene Products ie in globin gene, repeat sequence L1 is spread throughout the genome; during meiosis, recombination between two chromosomes may lead to unequal cross-over

result of unequal crossing over is that one recombinant chromosome has 2 exons, the other chromosome has none

Exons are cassettes that can be shuffled and used in various numbers and combinations

example: receptor family of tyrosine kinases these transmembrane proteins bind hormones; each receptor has a tyrosine kinase domain, but receptors at the surface differ (explained by reshuffling)

Tandem Repeats: rRNA, tRNA and snRNA (small nuclear)

these genes are highly duplicated and have the tendency to repeat

the duplication is not evolutional; the only function is to help produce RNA

rRNA produces identical to near identical copies, because the function must be preserved to ensure the RNA sequence is conserved; proliferating cells need synthesis of millions of ribosomes per day

rRNA + tRNA required for protein synthesis, snRNAs for mRNA splicing

Single Sequence DNA

single-sequence DNA: repeats of very short sequences, 3-6% of genome satellite (20-100kb), minisatellite (1-5kb), microsatellite ( peptides

proteins = natural polypeptide or a complex of polypeptides with a well-defined structure

reminder: one polypeptide = one RNA translation

Secondary Structure

random coil: no intramolecular noncovalent interaction between amino acids

stabilized structures: polypeptides are stabilized by noncovalent interactions, usually hydrogen bonds

alpha helix and beta sheet: together make up to 60% of polypeptides; also U-turns

alpha helix: spiral, has a backbone (peptide bonds of polypeptide);

central spiral is made only of peptide bonds and is held by hydrogen bonds that cement the spiral in stable structure; H bonds are between carboxyl and amine functions

Periodicity of 3.6 residues per helical turn; straight rod

R residues all stick outside, making a sheet around the backbone; interactions with environment are exclusively dependent on composition of side chains

alpha helix: in transmembrane proteins, the part of protein crossing the lipid bilayer is always an alpha helix; the R residues are hydrophobic

if alpha helix is at the surface of a protein, one face will often display hydrophobic residues that will interact with other parts of the protein, while hydrophilic residues will be exposed at the surface (interacting with water and maintaining protein in solution)

typical periodicity: i, i + 3, i + 4, i + 7 that are hydrophobic

beta sheet: second most abundant structure, good at making surfaces

consists of laterally packed beta strands (5-8 residue) stability of structure is due to hydrogen bonds within backbone of polypeptide

residues are either above or below the plain

residues can be organized so that one property is on one side and another property on the other side

conformation at the surface of a protein has a periodicity of 2; hydrophobic residues at positions i, i + 2, i + 4, i + 8, & polar residues at positions i, i + 3, i + 5

Tertiary Structure

4 representations of tertiary structure

C backbone trace: looks only at backbone, demonstrates how the polypeptide is tightly packed into a small volume

Ball and Stick: reveals location of all atoms

Ribbons: more analytical, distinguishes the different structures (helixes, sheets, turns, loops) Solvent-accessible surface: map of chemical properties of surface, ie where we find hydrophilic molecules (+,,0 charge)

different types of proteins: long fibrous proteins (ECM proteins), globular proteins (hemoglobin, -globin), transmembrane proteins (2 independent sides linked by a helix)

Tertiary Structure: Motifs

structural motifs: particular combinations of secondary or tertiary structures; contribute to global structure of the entire protein, and often performs a common function in different proteins (ie binding to a particular small molecule or ion)

helix-loop-helix motif/calcium-binding motif: so effective that more than 100 calcium-binding proteins have the same motif

zinc-finger motif: found in DNA and RNA-binding proteins; Zn holds alpha helix and beta strands together; zinc-finger provides negative charges on the sides to bind zinc

coiled coil motif: motif often involved in self association; present in fiber proteins; two alpha helices that bind tightly together and make a second-degree spiral; lots of the residues on this motif are hydrophobic (hydrophobic bonds are short, holding the structure very tightly = dense)

Tertiary Structure: Domains

structural domain: 100-150 aa-long region, compactly folded, can be made of various motifs; large proteins are built from several domains

functional domain: region of a protein that exhibits a particular activity characteristic of the protein; ie catalytic domain of an enzyme, or regulatory domains, DNA binding domains, and EGF domain

structural domain: region that often can fold into its characteristic structure independently of the rest of the protein; ie hemagglutinin HA has two domains ( fibrous and globular

proline-rich domain: because of its aa composition, it functions to bind other proteins

SH3 domain: motif of several amino acids, found in many proteins; has sequence conservation

Quaternary Structure: Multimeric Proteins

multimeric proteins can contain any number of identical or different polypeptides

quaternary structure: the number and relative positions of the subunits in multimeric domains

ie: hemagglutinin HA is a homotrimer made of three identical polypeptides associated together

dimmer, trimer, tetramer; homomultimer (subunits are identical), heteromultimer (subunits are different)

macromolecular assemblies: association at even higher levels; up to a megadalton in mass; contains tens to hundreds of polypeptide chains, sometimes also other biopolymers like nucleic acids (ie ribosomes have inside proteins made of polypeptides, all assembled) examples of macromolecular assemblies: transcription initiation machinery, replisome, spliceosome, nuclear pore complex, components of replication fork (topoisomerase, polymerase, primase, helicase), proton pump

Evolution of the Globin Gene Family

Evolution of proteins conserves structure and function, but not primary sequence

One can change amino acids without changing the structure; ie change leucine for isoleucine (both are hydrophobic)

If a point mutation is made in a place that breaks alpha helix or beta sheets, the probability of having a functional protein is very low because whole structure will break

Example: hemoglobin (2 alpha, 2 beta), myoglobin, and leghemoglobin (roots of plants, binds oxygen); primary sequence is very different, but structure + functions are similar

Lecture 25: Protein Structure: Sequence Analysis and Structure Prediction

Sequence Comparison

read and interpret sequences: some amino acids are perfectly conserved; others have made conservative amino acid exchanges conservative amino acid exchanges: function of residue is maintained

arginine-lysine: both are positively charged amino acids; differ in shape leucine-isoleucine: slightly different in shape; both are hydrophobic residues and of the same length

aspartate-glutament: both negatively charged and acidic

not-so-obvious exchanges

histidine-aspartate: one is positive, the other negative; if charge is negligent, then one can replace the other

serine-threonine: both have OH in their structure; function of OH is phosphorylation, so in phosphorylation enzymes (kinase) there is generally no diff

hydrophilic aa: basic amino acids lysine and arginine, and acidic amino acids aspartate and glutamate have strong personalities, since there not few positively and negatively charged amino acids

proline: hydrophobic residue, makes rigid kink in the backbone of structure

phenylalanine + leucine have different structures, but both are hydrophobic and thus are considered conservative exchanges

Structure of a Transmembrane Protein: Ephrin (receptor for hormones or other ligands) ephrin has intracellular, transmembrane, and extracellular components intracellular: kinase domain, has an enzymatic activity that triggers a signaling cascade; phosphorylates residues; regulation by phosphorylation (receptors bind together and one phosphorylates another, which causes cascade)

transmembrane domain: found by computer, the transmembrane domain makes an alpha helix in the lipid bilayer (it has the right length and is hydrophobic) to determine phosphorylation sites, create a computer program that looks for residues that could potentially be phosphorylated by a kinase

the protein folds and makes a scaffold: the surface is what interacts with other molecules; this surface is dictated by the primary sequence

mutations in terms of evolution: genetic diseases caused by replacement of one amino acid by another one with a different property; mutations are selected by evolution and cancerLecture 25B: Protein Folding, Modification and DegradationProtein Folding

a protein is usually divided into domains, which are basic folding units

an unfolded polypeptide has polar and nonpolar side chains; the chain folds to hide the hydrophobic residues from the surface of the molecule and expose residues

Protein Folding: in vitro conversion between native and denatured conformations

In vitro, place purified protein in urea, denaturing the protein; urea keeps protein unfolded in suspension Remove urea slowly, dilute by dialysis; proteins fold up in correct way and goes back to native conformation; if unsuccessful, then proteins become misfolded

Misfolded proteins aggregate

Chaperones/chaperonins help protein folding

chaperone: binds to exposed hydrophobic residues of nascent polypeptide, helps folding, & protects from aggregation until properly folded

example: Hsp70-ATP (Hsp = heat shock protein, accumulate during heat shocks) binds to exposed hydrophobic residues to maintain polypeptide in motion; Hsp70 protects everything that cannot be in an aqueous environment

chaperones are directed by ATP cycle: ATP hydrolyzed into ADP, then replaced by new ATP; makes chaperones bind and unbind polypeptide

once chaperone is released from polypeptide, polypeptide can fold properly; if misfolded, chaperones bind again

back-up system for misfolded or denatured proteins: Hsp60

Hsp60: barrel-like structure; recovers proteins that are misfolded before they aggregate together; recognizes hydrophobic residues that shouldnt be exposed; these misfolded polypeptides (hydrophobic residues on the outside) are inserted into the cavity of Hsp60

Once inside Hsp60 barrel, polypeptide unfolds again; with the help of ATP and GroES, the protein can fold properly; in this case, ATP is used to regulate the function of the protein itself

Post-Transcriptional Modifications: Covalent Modifications

acetylation of amine of N-terminus: the free NH2 at the end of the polypeptide is protected from attack by exopeotidases proteolytic enzymes: exopeptidases + endopeptidases

exopeptidase: cuts at end of polypeptide; ie N-terminus exopeptidase cuts N-terminus of polypeptide

endopeptidase: cuts middle of polypeptide; these are enzymes important in many mechanisms, ie lysosomes of cells (degrade proteins) + ECM (for some molecules to cross membrane, like cancer cells)

dipeptidase cuts 2 amino acids, tripeptidase cuts 3, polypeptidase cut peptides + oligopeptides

presinilin: enzyme that cuts proteins in neurons; if not working properly ( Alzheimer

some proteins, like insulin, must be cleaved to function normally

Post-Translational Modifications: Covalent Modifications

acetylation: acetyl lysine on histones

phosphorylation: serine, thereonine, tyrosine

hydroxylation (collagen), methylation, carboxylation glycosylation: add sugar to proteins; occurs massively at cell surface or in ECM

ex. In the cartilage, long polymers of sugars surround protein core; the huge molecule thus acts like a sponge

Ubiquitination and Degradation

denatured or misfolded proteins must be rid of

Ubiquitin: short polypeptide thats present in all cells in high abundance; attaches to and flags misfolded protein; Ubiquitin flag is recognized by a proteasome

Proteasome: degradation machinery; barrel-like structure that denatures the polypeptide into different small peptides

This process is used to degrade proteins that are targeted for particular reasons

Neurodegenerative Diseases Caused by Accumulation of Misfolded Proteins

misfolding: Alzheimers, Parkinsons, and Mad Cow disease

principle: a precursor is cleaves and makes a mature protein that is properly folded, but also has a tendency to switch from alpha helix to beta sheet conformation; these beta sheet proteins aggregate into filaments resistant to proteolysis in Mad Cow disease, the change in conformation is infectious: when one protein switches to beta sheet, it induces surrounding cells to change conformation as well; extremely protease resistant

Lecture 26: Protein Function

Specific Binding of Ligands Underlies the Functions of Most Proteins ligand-binding has 2 principles: selectivity and affinity

selectivity: spectrum of possible interactions for a given protein; determines whether a protein can recognize only a particular molecule or sequence or whether it can recognize a spectrum of sequences

affinity: how strongly the molecules bind together; Kd is affinity constant

ie histones has high affinity for binding DNA, but no selectivity in terms of sequence

Molecular Complementarity: Antibodies

complementarity determining region CDR: on tip of antibody, the hypervariable region that recognizes the antigen

epitope: region of antigen recognized by one specific antibody antigen: induces immune response; a single antigen can have many epitopes

interaction between antibody and antigen may involve many loops and turns on different subunits of proteins

Enzymes are Efficient and Specific Catalysts

in a biochemical reaction, one needs to add energy to surpass the threshold of the transition state and generate product, even if reaction total is favorable

enzymes accomplish this by lowering energy required for the transition state

the simplest way to expedite a reaction is to add heat; however, cells are limited to ~37 degrees, as excessive heat denatures proteins

example: Protein Kinase A

enzyme that catalyzes reaction that removes third phosphate of ATP and links it to polypeptide; ie Serine or Threonine in polypeptide + ATP ( P-Ser + ADP this catalytic enzyme has two domains, linked by a flexible region; each domain has a specialized region with specific amino acid composition (small domain, large domain) glycine lid: a glycine-rich region, specifically used to block, bind and trap the nucleotide (ATP); charged residues in the enzymes pocket stabilize phosphate groups of ATP

active site: region between the two subunits; forms the groove where ATP will be localized the kinase has a surface devoted to recognizing a specific peptide sequence; it phosphorylates the particular sequence, ie serine

specificity of recognition site is distinct from that of enzymatic site: in order for enzyme to function it is crucial for there to be 2 arginines on the N-terminal side (determines enzymes specificity for its substrate); the 2 arginines and Serine bind to hollow groove

proteins are dynamic: each step in reaction induces change in both substrate & enzyme

open enzyme complex allows ATP to enter and substrate to bind ( binding induces change in conformation in enzyme, closing it ( peptide + ATP brought in close proximity ( reaction where ATP is hydrolyzed and phosphopeptide is formed ( products cause reopening of enzyme, thus releasing product

enzymes delocalize electrons to break the strong covalent bond (lysine and positive ions lock the molecule and attract e-, which destabilizes the molecule)

Evolution of Multifunctional Enzymes

reactions in cells usually occur as chain reactions, not as single reactions; to increase chain reaction efficiency ( scaffold protein

scaffold protein: enzymes are grouped together and stabilized by scaffold protein; scaffold protein only have protein-protein interaction domains, and so can recruit all components required for a reaction to take place; also localizes reaction

example: MAP-kinase pathway: an enzyme is activated that phosphorylates a kinase, which phosphorylates another kinase and so on; MAP-kinase-kinase-kinase phosphorylates MAP-kinase-kinase activates Map-kinase

Molecular Motors: Linear Motors + Rotary Motors

proteins can be motors: actin and myosin motor in which myosin slides filaments, producing work; protein motor causes bacteria flagellum to rotate

a motor is something that displaces an object relative to another object: DNA and RNA polymerases or ribosomes are motors because they produce force and displacement ATP synthase: enzyme that produces ATP in mitochondria; the complex machine acts like a motor; uses proton gradient to synthesize ATP from ADP; enzyme has axis that turns ATPase subunits, this rotation is required for ATP synthesis

ATP synthase has 12 rotating A subunits, with identical composition, and a large subunit with 2 channels for protons to travel through

An acidic residue (aspartate) must be exposed at surface of each A subunit

Proton (+) is attracted to aspartate (-) and neutralizes its charged; this changes the conformation of that particular A subunit; in order to stabilize itself, it turns slightly to the side

Process is repeated on each individual residue, in each subunit; thus protons force the subunits to rotate

Lecture 27: Regulation of Protein Function regulation by changes in conformation: allostery and cooperativity

common regulators: calcium, GTP, phosphorylation/dephosphorylation

Allostery

Allostery: binding of one ligand influences binding of another ligand in a different region of the protein; because a protein is a network of intereactions involving covalent bonds of backbone and non-covalent bonds between various amino acids, change implies formation of new non-covalent bonds Positive allostery: binding of one ligand induces change in conformation of protein, which favors binding of second ligand

Negative allostery: modification decreases affinity of second ligand

Cooperativity

cooperativity: binding of the first ligand induces changes in conformation that influence binding of B, but alters conformation of the second subunit, favoring binding of the second A (first binding induces allostery, involves several binding sites for the same ligand); cooperativity can also be negative

cooperativity makes the system more sensitive to small changes in concentration of ligand

Allostery and Cooperativity: Regulation of Protein Kinase A

the catalytic part of PKA is made of two domains, which phosphorylates particular peptides; regulated by pseudo-substrate that binds strongly to the catalytic site and cannot get phosphorylated to release pseudo-substrate, bind cAMP; there are two binding sites per regulatory unit, so binding of cAMP induces allostery that results in release of catalytic sites of the two regulatory units; biochemically, inhibition of inhibition

ligand can now bind; binding of one ligand makes binding of second ligand easier

Cooperativity of Oxygen binding to Hemoglobin

hemoglobin is a tetramer that binds 4 O2 molecules; interaction between hemoglobin subunits favors binding of the next oxygen moleculeCalmodulin: Calcium-dependent regulator

calmodulin: modifies activity of proteins; regulation dependent on calcium

contains 4 calcium binding domains in protein; once activated (loop form), this protein interacts with other peptides (globular) and modify their activity

trick of regulation: Kd is low, and a small increase in low conc. of calcium in the cell is sufficient in activating calmodulin loop

calcium is at a low concentration in the cell because cell pumps out calcium as a messenger; calcium concentration is higher in blood

Cycling GTP/GDP as a Common Switch Mechanism

GTPase: series of enzyme that hydrolyzes GTP to regulate itself in order to regulate other proteins in the cell; lousy enzyme because of slow hydrolysis and exchange Thus GEFs, GTP exchange factors, activate GDP ( GTP

GAPs, RGS, and GDI hydrolyze GTP ( GDP

Example: trimeric G protein has an alpha unit, which is a GTPase

Interact with receptors sensible to hormones or light, changing the subunit to its active form; causes dissociation of alpha subunit with beta and gamma subunits; the new freed surfaces can now interact with target proteins

Phosphorylation: another Common Switch Mechanism

phosphorylation: reversible cycle of an amino acid with a phosphate (kinase, active form) or no phosphate (phosphatase); very dynamic

example: receptor kinase: once phosphorylated, receptor kinase can bind adaptor proteins, and this triggers cascade

Other Common Mechanisms

cleavage: activation or inactivation; proteases are destructive machines, can be very specific; cleave at specific sites that give a function to the products; ie insulin (proinsulin is cut at 2 sites by protease; the two final fragments, forming insulin, are linked by disulfide bonds) subcellular location: the cell has compartments; some interactions occur at a specific location, so activity can very from location to location; ie membrane anchoring and nuclear/cytoplasmic localization of factors regulating transcription (modified by signals)

Introduction to Signaling Pathways

cells respond to external and internal stimuli (growth factors, hormones, ions, extracellular substrate, mechanical stress, other cells etc); binding of ligands to receptors trigger cascades in the cytoplasm (network of interactions)

signaling cascades regulate metabolism, ion channels, cytoskeleton, and nuclear gene expression

intracellular signaling cascades: protein-protein interactions, GTPases, Phosphorylation, proteolytic cleavage, regulation of protein stability etc

gene activation/ repression via regulation of transcription factors

Lecture 28: Purification, Detection, and Characterization of Proteins I

Protein Purification

protein are much more diverse than DNA, thus several methods are combined for purification

separation of proteins according to physical and chemical criteria: mass (and shape), density, charge, binding affinity

3 techniques in protein purification: centrifugation, electrophoresis, chromatography

Centrifugation: Mass and Density

differential centrifugation: separation according to mass (=size)

sample is poured into tube ( centrifuge; particles settle according to mass ( stop centrifuge and decant liquid into container

obtain 2 fractions: pellet and supernatant; crude method for particles of largely differing sizes

separation optimized by changing centrifugation time and speed to separate bigger or smaller fragments

rate-zonal centrifugation: separation according to size + density sample is layered on top of gradient (increasing dense medium, such as sucrose; viscous medium = slower sedimentation)

centrifuge: particles settle according to mass; thus separation by size is better

flaw: if some particles are less dense than the medium they will stop and float

equilibrium density-gradient centrifugation: separation by density

if rate-zonal centrifugation is spinned long enough and the density of the medium is high enough, all the particles will eventually settle to density equal to themselves

density gradient yields the purest DNA, since DNA is very dense

Eletrophoresis: Charge to Mass Ratio

DNA is negative, attracted towards the cathode (+); thus separation is straightforward Proteins have variable composition, charge results from addition of all the charges of the protein; most proteins have a negative charge and goes toward the cathode

Modify pH to deprotonate proteins; negative charge moves faster, less mass moves faster

This method retains protein structure; proteins are separated in this native state, properly folded; can be retrieved to test enzymatic activity via assays

SDS-Page: Mass (Size)

denature proteins completely with a strong detergent, SDS; now shape has no influence

SDS: negatively charged detergent that binds hydrophobic residues of the polypeptide (which is inside the structure), causing its unfolding + denaturation ( all proteins become negatively charged

Place protein mixture on cross-linked polyacrylamide gel and apply electric field; stain to visualize the separated bonds

Protein activity and protein-protein interactions is lost; however, gain high resolution

Isoelectric Focusing: Charge isoelectric point: sum of all charges = 0; depends on amino acid composition of each protein; isoelectric point is expressed as pH, and different for every protein

pH gradient is established using special buffers (ampholytes) immobilized in acrylamide gel; proteins are subjected to electric field and migrate, lose charge, and reach equilibrium line

two-dimensional gel electrophoresis: isoelectric focusing is often followed by SDS-Page: separate in first dimension by charge ( apply first gel on top of second ( separate second dimension by size

Liquid Chromatography

liquid chromatography: useful for isolation of large amounts of proteins

principle: columns filled with polymer beads slow down all proteins down their path; size of meshwork within the beads is calculated in a way that proteins have the probability to go into the beads or bypass the beads

based on size/mass (gel filtration), charge (ion exchange), and binding affinity

Gel Filtration Chromatography

separation according to size (mass); analytical technique for small amount of proteins

layer sample on column, add buffer to wash proteins through column, collect fractions

tube is filled with polymer meshwork of beads; breads slow down molecules because path of particles become very long; beads trap proteins that are small enough; larger protein = faster elusion

any protein bigger than the pore size of polymer breads will not be separated; separation comes with smaller proteins

Ion Exchange Chromatography separates proteins according to charge

anion exchange: beads bind to negatively charged proteins; also cation exchange

layer sample on column, positively charged gel bead collects negatively charged protein; elute negatively charged protein with salt solution (NaCl)

elution with NaCl: low salt = all negatively charged particles bind; medium salt = weakly charged proteins are eluted; high salt = highly charged proteins are eluted charge of protein depends on pH, so binding of protein can be influenced by change in buffer pH

Antibody Affinity Chromatography

load protein in pH 7 buffer; antibody beads; protein recognized by antibody stays on beads, protein not recognized by antibody filters through; elute with pH 4 buffer to collect protein recognized by antibody

antibody-antigen interaction is pH sensitive ( destabilized at acidic pH

very effective purification, but never to purity

Protein Detection: Western Blotting

to recognize protein of interest, transfer molecule from SDS-polyacrylamide gel (denatured protein) to membrane, using electric current to force a faster transfer

if protein has enzymatic activity, perform an enzymatic assay for detection

antibody detection: incubate membrane with primary antibody for specificity; then incubate membrane with enzyme linked secondary antibody for detection chromogenic detection: react with substrate for secondary antibody-linked enzyme

Western Blotting provides antibody-antigen reaction AND size of protein (from gel electrophoresis SDS-PAGE)

Lecture 29: Purification, Detection, and Characterization of Proteins IIProtein Sequencing: characterization of proteins

edman method/high pressure liquid chromatography: choose peptide, label N-terminal amino acid, then cleave N-terminal amino acid; identify aa by high-pressure liquid chromatography only sequences the first few amino acids; once a piece of the sequence is known, it can be used to search in DNA sequence databases to determine entire sequence

mass spectroscopy: bombard molecule with strong energy to break molecular bonds

free radicals are produced (charged) and attracted to electrical field

positively charged molecules are attracted to the detector; thus to get a positively charged peptide, trypsin (a protease) is used to produce positively charged peptide, since it usually cuts the fragment after reaching an Arginine, or Lysine, which are positively charged basic amino acids

magnet attracts free radicals according to mass; measures atomic mass at precise units (1 mass unit)

one can tune energy of laser to weaken peptide bonds of amino acids, producing peptide fragments; each amino acid has a different molecular weight (except leucine and isoleucine), thus one can identify a protein or a mixture of proteins

Determination of 3D Structure

x-ray crystallography, cryoelectron microscopy, nuclear magnetic resonance NMR spectroscopy

x-ray crystallography: most precise technique to determine structure of protein

principle: when a beam of x-ray passes through a protein crystal, the electrons in the crystal scatter the x-ray and produce a diffraction pattern of discrete spots on the detector film; by analyzing the pattern produced, one can construct 3D structure of that protein

limitation: since only crystal can produce diffraction pattern in x-ray crystallography, protein must be crystallized; however, it is impossible to crystallize most membrane proteins, ie those containing transmembrane domains

NMR spectroscopy

Principle: protein solution is placed in strong magnetic field; measure the influence of neighboring atoms on the spin of atoms (generally H and C); frequency + strength of magnetic field influences spin state of atoms

Advantage: does not require protein crystal, more physiological

Limitation: can only be used to analyze polypeptides that have less than 200 amino acids; used to determine structure of protein domains

cryoelectron microscopy

principle: protein is rapidly frozen to preserve its structure; pictures of this protein are take at different angles, and computer can reconstruct structures of this proteins by analyzing its pictures

Proteomics: Comprehensive View of Proteins

applications of proteomics

isolate large protein complexes and assemblies and determine their composition

isolate organelles and identify their components

can also be used for known proteins: compare same protein from different cell type, determine cell type-specific patterns of proteins or protein modifications; comparison of different conditions, treatments (ie drugs)

genomics can only reveal whether a gene is expressed or not, but will not show what modifications has been done on that gene

Lecture 28B: Example of experiment using various methods: siRNA, cell fractionation, Western blot and immunofluorescence

role of RanBP3 in -catenin nuclear export: what happens when RanBP3 is depleted?

Role of RanBP3 in -catenin nuclear export RanBP3: Ran binding protein -catenin: short half-life; undergoes ubiquitination; is a co-repressor or co-activator of gene expression

Wnt pathway: Wnt binds to receptors on the plasma membrane and triggers a signaling cascade

Usually, -catenin is in the cytoplasm and is degraded rapidly due to its short half-life; when Wnt pathway is activated, -catenin ubiquitination is blocked and b-catenin is stabilized; stabilized -catenin is imported into the nucleus

Without Wnt (hormone that triggers Wnt pathway), -catenin is inactive (phosphorylated) and is degraded rapidly; Wnt signaling pathway stimulates dephosphorylation of -catenin, which permits -catenin to enter the nucleus, interact with transcription factors, and regulate transcription

The goal is to test whether there is a shuttling mechanism and whether protein RanBP3 is involved in export from nucleus

A protein stabilizes -catenin in the cytosol, but it shuttles; when we block export, the protein is now stuck in the nucleus and the -catenin left in the cytosol is degradedsiRNA

to test RanBP3, the method of choice to partially get rid of the studied protein is to use siRNA, by making a short plasmid transcribing a short precursor of the dsRNA with a loop

when writing a sequence, only write one strand of the dsRNA in direction of transcription, so that the sequence is equivalent of what will be the RNA sequence

Preparation of RNA interference

search RanBP3 sequence in database, design 3-5 short interfering sequences

synthesize two complementary oligonucleotides covering these short sequences, including flanking overhands corresponding to restriction sites in vector; anneal the two oligonucleotides to produce a dsDNA insert use a special vector optimized for expression of short hairpin RNAs: cut vector at two restriction sites in polylinker, ligate insert; transform bacteria, pick clones, check presence of insert by restriction digest, sequence region to verify right sequence

transfect mammalian cells with newly constructed plasmids; verify that the clone is correct by sequencing it

test efficiency of RanBP3 depletion: transfect cells to test whether the plasmid has acted properly and managed to deplete the protein properly

western blot shows that sequences that target RanBP3 lead to its significant decrease; thus sequence that targets RanBP3 is specific

Effect of RanBP3 depletion of -catenin export

in cells that are not treated with Wnt, no activated -catenin Is present (all is phosphorylated) cells treated with Wnt hormone contain active -catenin in the nucleus

what happens when one activates -catenin but removes RanBP3?

Preparation of sample: break cells and biochemically separate nuclei (very large and heavy) from the rest of the cytoplasm (soluble proteins) by differential centrifugation; supernatant will be cytoplasm, pellet will be nuclei

Analysis: analyze supernatant fraction with Western blot (with specific antibodies)

for Wnt-treated cell that has lost RanBP3 after depletion, there is much more active -catenin in nucleus than in cytoplasm; thus RanBP3 plays a role in -catenin exportLecture 30: Protein Sorting/Targeting

a few proteins are synthesized in the mitochondria, while most are synthesized in the cytoplasm; they have to know where to go

Cell Compartments

mitochondria, nucleus, ER, Golgi, secretion, endocytosis all create compartments in the cell surrounded by a biomembrane

ER is connected to the nuclear membrane, forming a network

Two Major Pathways of Protein Targeting

targeting to the membrane of an intracellular organelle

occurs during or soon after synthesis of protein

for membrane proteins, targeting leads to insertion in the organelle membrane; for water-soluble proteins, targeting leads to translocation of the protein to the interior of the organelle

proteins go to the ER, mitochondria, chloroplasts, peroxisomes, and nucleus via this process

targeting to the ER for subsequent secretion

involves nascent proteins still being synthesized

proteins are transported by small vesicles

protein targeting involves signal sequences (20-50 aa segments that contain targeting information)

Experiment on Co-translational Translocation

this historical experiment demonstrates that very soon after synthesis, proteins are already in the lumen, inside vesicles

pulse-labeling: a few radioactively-labeled amino acids are added only to newly synthesized proteins; proteins are incubated in cells for a short period of time

homogenization: cells put in a blender, vesicle is broken into little spheres called microsomes, which still bind ribosomes

conclusion: soon after synthesis, polypeptides can already be found inside vesicles

Co-translational Translocation of Secreted Proteins

a signal sequence is present in all proteins targeted to the ER

the ER signal is composed of charged amino acids, followed by a stretch of hydrophobic aa, almost at the beginning

ER signal is recognized and bound by a protein receptor: SRP (Signal Recognition Particle)

Binding of SRP blocks translation

SRP bind the SRP-receptor; thus SRP is a soluble protein that acts like a linker between the ER signal and the SRP-receptor; the SRP-nascent peptide-ribosomes complex is now docked to the ER through the SRP receptor

The nascent polypeptide-ribosome complex is handed to a pore complex called translocon, which opens; a little synthesized part of the protein is pushed in the ER lumen

SRP is released, translation resues

Once around 70 aa are translocated, a signal peptidase cleaves the signal sequence; the rest of the polypeptide is translated and translocated

Once synthesis is terminated, ribosomes leave and pore translocon closes

Energy is not really needed for translocation: translocation uses energy from translation, which pushes the polypeptide chain through the pore

The only use of GTP hydrolysis in the SRP and SRP-receptor is for the purpose of control, like in the case of chaperones

Translocation in Yeast

in yeast, proteins are able to enter the ER after they have been synthesized

translocation is unidirectional because BiP, a molecular chaperone found in the ER, is needed for protein folding in the lumen; usually in the cytoplasm, Hsp70 takes care of folding; however, in this system, chaperons in the lumen fold proteins because proteins are transferred directly from the ribosomes to the ER

the part of the polypeptide chain that crosses the translocon is bound by BiP chaperones, which have affinity for the peptide and prevent it from sliding backwards

even though Hsp70 is found in the cytosol, it doesnt bind as strongly to the peptide as BiP; therefore, BiP wins and the protein cannot slide back outside anymore; the more the peptide goes in, the more BiPs bind

Targeting of Transmembrane Proteins

many different scenarios apply for membrane proteins; unlike secreted proteins, which end up in the lumen, transmembrane proteins are integrated in the membrane, halfway through synthesis

a stop-transfer anchor sequence (hydrophobic transfer domain) is found in approximately the middle of the protein

when half of the protein is in the lumen and the translocon sees the transfer domain (signal), the transfer domain is kicked out of the translocon and is integrated immediately in the membrane; translocation is interrupted when the protein is no longer in the pore, and translation is completed in the cytosol

one signal: once signal is cleaved, and the rest of the sequence has no transmembrane domain, the protein will be free to float in the lumen

two signals: second signal acts as a stop sequence, can stop translocation and act as a transmembreane domain

three signals: the third signal would be recognized as something to be integrated in the bilayer; thus one can have alternated sequences

Targeting to Mitochondria

mitochondria depend largely on nuclear genome for production of most of their components signal peptide for matrix proteins: 20-50 amino acid long amphipathic alpha helix (one side is hydrophobic, the other is charged); the protein has to cross two membranes to get in there

protein goes through translocons to get to the matrix; when a protein is translocated, its signal is cleaved

difference between ER and mitochodria: in mitochondria translocation, the protein has been synthesized in the cytoplasm; it has to stay unfoled to go through the translocon

proteins are maintained unfolded by constant binding of Hsp70; Hsp70 also brings protein to the mitochondria

if the protein wants to go into inner membrane or intermembrane space, it needs extra signals

inner membrane protein: signal 1 = matrix signal (amphipathic alpha helix), signal 2 = transmembrane domain that stops translocation so that protein stays in inner membrane

intermembrane space protein: signal 1 = matrix signal (amphipathic alpha helix), signal 2 = transmembrane domain that stops translocation, signal 3 = cleavage site, so the protein is released from the membrane and stays in intermembrane space

chloroplasts: similar to mitochondrial translocation