cellbiologi (Avsnitt 1,2,3) Flashcards
De olika spottkörtlar och deras egenskaper
1- glandula parotis - öronspotkötln= -största spottkörtlen
- körtlent utförsgång -belägen på utsidan av m. masseter
- mynnar på kinndens insiden
- seriöst sekret (unstimulated: 30% /stimulated: 35% )
2- glandula submandibularis - underkäksspotkötln = - ligger i vinkel mellan underkäken och munbotten
- den ligger djupt under munbottenmusklnut
- mynningen syns vid sidan av tungbandets bas
-blandat, både seriöst och muköst (unstimulated: 50%/stimulated: 50% )
3- glandula sublingualis - undertungaspotkötln= - den minsta
- belägen under menbottens slemhinna och övan för munbottensmuskel
- mynnar på ett slemhinnveck under tungan
-blandat, seriöst och muköst (unstimulated: 10% /stimulated: 10% )
Normal - Resting saliva/Stimulated saliva
Resting saliva: Flow 0.3 mL/min
Stimulated saliva: Flow 1.8 mL/min
Resting (Hypotonic) Saliva
- Low HCO3-
- Poor acid buffer
Stimulated (Isotonic) Saliva
- High HCO3- (> 25 mM)
- Good acid buffer
Protection against by Saliva function
-Abrasion (due to foreign element) Lubricant Pellicle
-Attrition (due to opposing teeth) Lubricant Pellicle
-Erosion: Pellicle barrier, Saliva dilution and clearance of acid
- Caries: Clearance of acid and sugars
Buffering (phosphate, bicarbonate, proteins) Remineralization (super saturation) pH < 5.5
Ca10(PO4)6(OH)2 —-> 10 Ca2+ + 6 PO43- + 2 OH-
- Soft tissue damage Infections: Mucus lubrication, wound healing Anti-microbial proteins
vad är saliv gjord av
-Ions = Ca2+ : Supersaturation PO43- : Supersaturation, Buffering HCO3- : Buffering F- : SCN- : Peroxidase system Urea : Biofilm buffring
-Proteins = Amylas : Digestive (starch) Lysozym : Anti-microbial Lactoferrin : Anti-microbial Salivary peroxidase : Enzym Myeloperoxidase : Enzym Agglutinins : Salivary agglutinin/gp-340 Mucins - MUC7 & MUC5B S-IgA: Pattern recognition Mucus, lubrication Anti-microbial
nutrians of Saliva
- gloucose
- lactate
- puryvate
- urea
- Vitamine & minerals
3 Types av bakterier
- patogena: obigata —> alltid otrevliga
- oppurtuniska patogen: kroppens immunförsvar är nedsat. som kan då leda till sjukdomar
- mixed species infektions: Bland infektioner
microbiol community
Microbiol community är grupper av mikroorganismer som delar ett gemensamt boendeutrymme. De mikrobiella populationerna som bildar samhället kan interagera på olika sätt, till exempel som rovdjur och rov eller som symbionter.
biofilm
- Biofilm uppstår när bakterier fäster till en yta i fuktig miljö genom att utsöndra en tjock, slem- och klisteraktig substans.
- faktorer som gör det lättare att mikroorganismer fastar på ytan och bild biofil är: kapsel, fimbrier,olime
pelikel
proteiner, lager över tänder, skyddar mott omgivning
buffering
Saliven är också en sak som fungerar som buffert eftersom munnen ofta utsätts för olika syraattacker från mat eller från magen, och då behöver stabiliseras igen.
försvar mekanismer
peroxides/enzym skyddas maltsyra i munhålan. hämmar syra prodektion hos streptokocker
platsen där batterier kan samlas
- Munhålan –> pelikel –> comelsalema ( vanlig/ofarliga bakterier) –> sekundära (orsakar problem/ patogena)–> dålig omsättning –> “platserna är (sub/supra) gingivalt och crypts” –> aerobabakterier: växter utan syre.
PLUS- Bakterier blir aeroba som då leder till dålig andedräkt. olika bakterier har olika syror
EMALJ!
hydroxyapatit: Ca10(PO4)6(OH)2
fluorapatit: Ca5(PO4)3F
Homeostas
- reglering av interna miljön
(betyder: måste avgränsa interna miljön från omvärlden) - för att upprätthålla homeostas behövs energi utifrån (betyder: ämnesomsättning/metabolism)
Reproduktion
Reproduktion, förmågan att skapa nya “enheter” (organismer) genom delning eller befruktning
Två fundamentalt olika designkoncept för celler: eukaryoter och prokaryoter
- prokaryoter: (domäner: Bacteria och Archaea) =
- ingen cellkärna
- inga/få uppdelningar av cellens interiör
- en gen har sammanhängande sekvens
- ofta (inte alltid) unicellulära - eukaryoter: (domän: Eukarya) =
- cellkärna som innesluter DNAt
- relaterade fuktioner ofta avgränsade i organeller
- gener innehåller ofta introner
- ofta (inte alltid) multicellulära
Vad är lika mellan prokaryoter och eukaryoter?
Trots att celler kan vara extremt olika till utseende och storlek är alla levande celler baserade på samma kemi intracellulärt
Alla celler innehåller genetisk information som kodar för proteiner
Livets kemi: byggstenar: nukleinsyra
-DNA dubbelhelix
Adenin (A) basparar alltid med Tymin (T). Guanin (G) basparar alltid med Cytosin (C).
Detta är den kemiska principen bakom DNAs replikation, och därför för livets fortplantning på jorden.
-DNA dubbelhelix i mer detajl
“Sidorna” av dubbelhelixen består av sockermolekyler (deoxyribos) ihoplänkade med fosfatgrupper (i figuren kallat “phosphate-deoxyribose backbone”)
A, C, T och G kallas kvävebaser.
Eftersom A och G (“puriner”) som är större alltid basparar med de mindre C och T (“pyrimidiner”) blir helixens diameter alltid lika stor
Vätebindningar håller ihop de två strängarna strängarna (2 mellan A-T, 3 mellan G-C)
-DNA dubbelhelix i 3D
Varje varv innehåller 10 baspar
Det finns en major groove och en minor groove i helixen (“stor fåra” resp “liten fåra”)
DNA-helixens utsida är negativt laddat pga fosfatgrupperna och gör att katjoner eller positivt laddade proteiner gärna binder till DNA
Vanligaste 3D-konformationen av DNA kallas “B-DNA”
Centrala dogmat
-Replikation: den cellulära processen då DNA:t kopieras.
Byggstenarna är dNTP (dATP, dGTP, dCTP och dTTP) sätts ihop till deoxyribonukleinsyra (DNA). Dessutom krävs enzymet DNA-polymeras. Hela processen kräver många ytterligare proteiner och enzymer
-Transkription: den process som översätter DNA till mRNA med enzymet RNA- polymeras och nukleotiderna AUCG till ribonukleinsyra (RNA).
mRNA kallas meddelande-RNA och fungerar som en mall för proteinsyntes.
Alla gener översätts inte till mRNA i alla celler och ibland gör cellen olika mängder
-Translation: den process då mRNA med hjälp av tRNA översätts till aminosyrasekvens (polypeptid/protein). Sker i ribosomerna (rRNA)
Livets kemi: byggstenar: kolhydrater (CH2O)n
- Isomerer: Ex monosackariderna glukos och galaktos har samma kemiska formel C6H12O6 men bindningarna mellan atomerna har olika riktning vilket kan ha stor biologisk betydelse.
- Monosackarider kopplas ihop med varandra, och sätts på andra molekyler.
- Proteoglykaner: (kolhydrater kopplade till proteiner)
- Polysackarider: stärkelse, cellulosa (liten kemisk skillnad mellan stärkelse och cellulosa gör stor skillnad)
- Disackarider: skars, laktos, maltos
Livets kemi: byggstenar: lipider
Lipider är ett samlingsnamn på relativs stora, kolväten som är svårlösliga i vatten.
T ex fetter, fettsyror (karboxylsyror), fosfolipider, steroider.
Fosfolipider
Fosfolipider är en grupp av fetter som tillsammans med en mindre mängd kolesterol bygger upp cellernas membran. Fosfolipider är uppbyggda av ett polärt, hydrofilt, huvud och en opolär, hydrofob, svans. Det betyder förenklat att huvudet är vattenlösligt och svansen fettlöslig.
Livets kemi: byggstenar: proteiner
Proteiner är organiska ämnen med hög molekylvikt. Tillsammans med polysackarider, fetter och nukleinsyror utgör proteinerna huvudbeståndsdelen i allt levande. Ett äldre namn är äggviteämnen. Kemiskt består proteinerna av långa kedjor av aminosyror hopbundna genom peptidbindningar. 20 olika aminosyror (olika sidokedjor). Aminosyror länkas samman till polypeptider genom en peptidbindning.
Why should we learn about amino acids?
• Amino acids are protein building blocks. If we know their properties, we can understand and even predict how proteins will fold and function.
• Amino acids are important metabolites:
- because they are needed for protein synthesis
- they are starting materials for other metabolites, e.g. nucleic acid bases
- they are intermediates in metabolic pathways
- several are “essential” - human body can’t make them
From Amino Acid Sequence to Protein Function
- You should know the amino acids general structure and properties.
- Understand the characteristics of the peptide bond.
- What is protein primary, secondary and tertiary/quaternary structures.
- The factors governing their formation (“protein folding”).
- Relation structure - function?
How proteins are build up
- Proteins are polymers of amino acids. Amino acids are their building blocks or alphabet. This alphabet contains 20 ”letters”-amino acids
- Shorter than ca. 50 amino acids are peptides, unstructured
- Proteins are polypeptides with very variable length, from kDa to MDa in molecular weight.
What are amino acids?
(BILD) there are different groups of amigo acids: • Carboxyl group • Amino group • Side-chain group - R • a-carbon Ca • Chirality of Ca
-Chirality: With the hydrogen atom away from the viewer, if the arrangement
of the CO→R→N groups around Ca as a center is clockwise, then it is
the D form. If anti-clockwise - the L form. Amino acids in proteins are in the L-form. Ca in Gly is not chiral.
-Humans can produce 11 of 20 protein amino acids. The others must be supplied in the food - essential amino acids: phenylalanine, valine, lysine, threonine, tryptophan, methionine, leucine, isoleucine, histidine. Shortage of even 1 essential amino acid results in degradation of body protein —muscle and so forth—to obtain the one amino acid that is needed. Unlike fat and starch, the human body does not store excess amino acids for later use.
Amino acid side chains
• The properties of side-chains determine protein structure and function.
SIZE FLEXIBILITY, DYNAMICS POLARITY CHARGE pKa HYDROPHOBICITY OXIDATION/REDUCTION CHEMICAL MODIFICATIONS
Chemical properties of amino acids Titration of amino acid without charged side chain:
amino acids act as buffers!
amino acids with additional ionizable groups -COOH / -NH2 are called acidic / basic amino acids
Proteinogenic and non-proteinogenic amino acids
• 20 a amino acids are incorporated into proteins (seleno-Cys can be counted as 21)
• Some of these subject to post-translational modification
• These are all L amino acids
• D amino acids are very rare, occur in antibiotic gramicidin
which is formed by soil bacteria for defense
• Other amino acids are not inserted into proteins, examples:
- -aminobutyric acid: neurotransmitter
QuickTimeTM and a decompressor
are needed to see this picture.
Side-chains of amino acids determine role in protein. Therefore: address their chemical properties and which bonds / forces they can generate in protein structure
- Ionic interactions
- Dipole interactions
- Ion-dipole interactions • Hydrogen bonding
- van der Waals force
- Hydrofobic effect
Acidic Amino Acids and their Amides
Acidic amino acids are polar and negatively charged at physiological pH. Both acidic amino acids have a second carboxyl group.
Amides are polar and uncharged.
Basic Amino Acids
Basic amino acids are positively charged at pH values below their pKa.
Lys has also a long hydrophobic side-chain, it is often located close to protein surface, with the amino group of the side chain in contact with solvent.
Histidine has pKa close to neutral pH important for H+ transfer in enzymes!
Charge interactions
- Salt bridge between side-chain COO- of Glu and
side-chain NH + of Lys
• Electrostatic contribution is long-range
• Interaction between equal charges repulsive
• H bonding contribution strongly orientation- dependent!!! - Charged amino acids can interact with water by: -hydrogen bonds
-dipole binding
-ion dipole binding
They are solvent exposed.
Protein charges are important for:
- Interactions with water, buffer, ions etc.
- Interactions with other proteins: Protein-protein binding, quaternary structure, protein aggregation
- Interaction with other biological molecules and structures (cell membranes)
Hydroxyl (R-OH) Amino Acids
Hydroxyl amino acids are polar and uncharged at physiological pH.
The phenolic hydroxyl ionizes with a pKa of 10 to yield the phenolate anion. The pKa of hydroxyl groups of serine and threonine are so high that they are generally regarded as nonionizing.
Dipole interactions
- A polar molecule has a net dipole (i.e. Having EQUAL partial positive and partial negative charges).
- Dipole-interactions are formed between delocalised electrons. Electrostatic attraction.
- H-bonds are electrostatic attractions between H atoms bound to highly electronegative atoms such as N, O or F and other highly electronegative atoms.
- Van der Waals forces–the residual forces that arise from induced dipoles, e.g. Temporary dipoles of non-polar side chains. Weaker than H-bonds.
Aliphatic amino acids
Hydrophobicity increases with increasing number of C atoms in the hydrocarbon chain.
Glycine has such a small side chain that it does not have much effect on the hydrophobic interactions.
Hydrophobic amino acids form hydrophobic core of the proteins. They are buried inside.
Hydrophobic effect
Proposed explanation: non-polar solute disrupts H bonding network of water, this disruption is smallest when solute is spherically enclosed
Sulfur-containing amino acids
The thiol group of cysteine can react with other thiol groups in an oxidation reaction that yields a disulfide bond.
Cys-Cys bonds are often formed in proteins
-SH group is a weak polar group, but its dipole moment is very small
- Methionine is one of the most hydrophobic amino acids and is almost always found in the interior of proteins.Methionine helps with chelation, which is the removal of heavy metals from the body to ensure that the liver, kidneys, and bladder remain healthy.
Proline
When proline is in a peptide bond, it does not have a hydrogen on the α amino group, so it cannot donate a hydrogen bond to stabilize an α helix or a β sheet.
Due to its unique structure, proline occurs in proteins frequently in turns or bends, which are often on the surface.
Can be converted into hydroxyproline (common in collagen) by post- translational modification.
Aromatic amino acids
All aromatic amino acids absorb ultraviolet light to different degrees, used for protein analysis.
Functional role of amino acid side-chains
• Lysine, Arganin: nucleicacid and proteinbinding
• Glutamin, Aspartic Acid: proteinbinding, acid-basecatalysis, metal
binding – Ca2+, Mg2+, Cu2+ , Na+ , K+ ets
• Proline, Glycin, : bends, flexibility.
• Cystein: stability via disulfide bonds, redox chemistry.
• Cystein: Tyrosin: radical chemistry.
• Aromatic amino acids: photo-sensitivity, hydrophobic stacking.
• Histidin: Acid-base catalysis, metal binding.
• Serine, Thrreonine, Tyrosine: Regulation via phosphorylation.
• Asparagin, Serine, Thrreonine: glycosylation, antigen recognition.
Scales of hydrophobicity
Used e.g. to predict transmembran helices: stretch of strongly hydrophobic amino acids.
Isoelectric point (PI)
• The isoelectric point (pI) of a protein is the pH at which that protein has no net charge.
Positively charged < pI < Negatively charged
• Characteristic of the protein, gien by balance between acidic
and basic amino acids.
• At pH = PI the solubility of a protein lowest (avoid this pH in order to increase the solubility)
Polypeptider bildas på ribosomen,
Polypeptidens struktur
A polypeptide is a chain of amino acids. Amino acids bond together with peptide bonds in order to form a polypeptide. The n-terminal (amino terminal) is located at one end of the polypeptide while the c-terminal (carboxyl terminal) is located at its other end.
Peptidbindningen är platt – jämför med en dubbelbindning
- En resonans mellan syre och kväves fria elektronpar gör bindningen svår att vrida
- Detta begränsar dess rörelsefrihet
Proteins – from Structure to Function
• Proteins can be soluble, sit in a membrane, or form large structures.
• Most proteins “fold” into defined structures in their functional native states.
• In vitro, small proteins fold spontaneously.
• In vivo, complex processes control that proteins adopt their native states,
their activities,
and their degradation.
• There are about 30000 proteins in the human body.
• Proteins are involved in virtually every function life depends on.
• Proteins change conformation and interact to carry out their function.
Examples of protein function
- Enzyme: Lysozyme
- Structural Protein: Collagen
- Transport Protein: Hemoglobin
- Motor Protein: Myosin
- Storage Protein: Ferritin
- Signaling Protein: Calmodulin
- Receptor Protein: Rhodopsin
From Amino Acid Sequence to Protein Function
• What holds proteins together? • Levels of protein structure: primary, secondary, tertiary, quaternary • Classes of protein structures • How do proteins work? • Examples: e.g. Membrane proteins
Organization Layers in protein structure
- Primary structure: Amino acid sequence
- Secondary structure: Alpha-helices, beta-sheets and coils
- Tertiary structure: Three-dimensional protein structure
- Quaternary structure: Dimer or larger complex formation
- Domains: protein parts that independently fold, 50-200 amino acids long, small peptides are unstructured.
- Protein Family: evolutionary related, similar sequence & structure.
Common structure of amino acids
• common structure H2N-CH(R)-COOH
• all amino acids in proteins are L forms
• side chain R determines amino acid’s properties CH3 = Ala
CH2COO- = Aspartate
H = Gly is special
Bildning av en peptidbindning
Karboxylgruppen och aminogruppen försvinner och bildar en amidbindning – eliminering av vatten!
The Peptide Bond
- Explains why there is no rotation around peptide bond.
- Explains why peptide bonds are normally trans.
Polypeptidens struktur
• Polymeravamidbindningenkallas”backbone”
• Aminosyror numreras från ”N-terminalen till C-
terminalen
• SidokedjankallasR-grupp
R differs in properties along the chain, sequence of R defines protein structure & function
Secondary Structure Elements: a helix and b sheet
ALL protein structures contain helices (red), and/or b sheets (yellow) and connecting loops.
1. Why are there only these secondary structure elements? 2. Why do these repetitive structural elements exist?
The backbone angles f, y,w determine local structure.
Rotation around polypetide backbone bonds: Ramachandran plot
w = almost always trans, no rotation
Secondary structure is defined by two angles around the α-carbon: φ (phi) and ψ (psi). Secondary structure elements are determined by repetitive sterically allowed combinations of f, y.
Backbone Conformation: Ramachandran plot
Only certain combinations of f,yoccur–inALLproteins.
Only these combinations of f, y avoid clashes in repetitive structures.
Secondary structure: a helix
α-helix bildas av att en polypeptidkedja viras runt sig själv och bildar en cylinder.
Always right-handed rotation!
N-H donerar en vätebindning till C=O från peptidbindningen 4 aminosyror tidigare, dvs i+4i.
18 aminosyror bildar 5 hela varv på helixen 3.6 aminosyror för ett komplett varv.
Aminosyrornas sidokedjor pekar ut från helixen.
Hydrogen bonds
- H bond is a strong dipole bond, where a H is bound between two electronegative atoms.
- X-H group is “donor”, other electronegative atom “acceptor”
- Directional: strongest when X, H, Y are in one line
- Max. 5 kcal/mol (covalent bond ≈ 100 kcal/mol)
α-helix
Side chains are outside.
Densely packed structure.
Side-chains determine the hydrophobic or hydrophilic surfaces of α-helix and its interactions with other elements of the secondary structure.
Secondary Structure: b-sheet
• a helix, b sheet exist because their H bonds stabilize the structure.
Olika sätt att representera proteinstrukturer
Alla representationer visar samma struktur, men med olika nivåer av detaljer
Buried and exposed amino acids
- amino acids prefer to be exposed / buried – linked to tertiary structure
- A, C, I, L, M, V, F, W are often buried,
- R, K, are almost never 100% buried,
- other buried or on surface.
Tertiary Structure: Hydrophobic effect
Hydrophobic side chains (white) are hidden in the “hydrophobic core”
Charged and polar side chains on the outside.
Burial of hydrophobic side chains ”hydrophobic effect“ stabilizes tertiary structure.
What determines 3D structure?
- Backbone conformation defines a helix and b sheet, but not tertiary structure.
- The side chains fill out the hydrophobic core, make soluble, prevent aggregation.
- Long-range interactions, mainly between side chains, stabilize the tertiary structure!
- Each amino acid sequence adopts ONE structure, encodes secondary- and tertiary structure.
- Each fold (arrangement of secondary structure elements) possible with many sequences.
Domains of proteins
- Lysozyme: One domain,
a helix and b sheet - Calmodulin: Two domains, almost only a helix,
central helix flexible - MyoD: Quaternary Structure of two domains:
- Structural definition: Can fold independently.
-Functional definition: Does a certain job.
Protein folds
- Proteinveckning är den process genom vilken ett protein får sin specifika tredimensionella form, i vilken det kan fylla sin funktion. Man skiljer mellan globulära proteiner samt fiber-proteiner. De globulära proteinerna förmedlar cellens funktionalitet, medan ett fibröst protein ofta är ett strukturelement.
• Protein structures can be classified according to:
- Which secondary structures occur?
- How are secondary structures aligned in primary sequence? - How are secondary structures arranged in space?
• Some “folds” occur very often, others are very rare. Tens of thousands of protein structures, a few hundred folds.
• the same protein function can be done by proteins with completely different folds.
• Example from SCOP data base of folds: a+b class:
121 “folds” = different arrangments of a helix & b sheet
Triose phosphate isomerase superfamily: triose phosphate isomerase
RuBisCo (C-terminal domain) superfamily: CO2 fixation in plants
TIM barrel fold
- parallel beta-sheet barrel, closed (first and last b strand H bonded) • 8 b strands in same order in primary sequence and structure)
- b strands connected by a helices
Kofaktor - Prostetisk grupp
Ibland kräver ett protein extra ”verktyg” för att utföra en speciell funktion. De binder då kofaktorer, molekyler som inte är proteiner.
En typ av kofaktorer är prostetiska grupper.
En prostetisk grupp är starkt, ibland
t.o.m. kovalent bundet till proteinet.
Är nödvändig för proteinets funktion OCH är del av proteinets struktur!
Den prostetiska gruppen kan vara organisk (vitaminer, sockerarter, lipider, mm) eller oorgansik (metalljoner, järn- svavel kluster).
Om en prostetisk grupp förändras vid t.ex. en enzymatisk katalys måste den regenereras på plats.
Myoglobin: heme group for O2 binding
Kvartärstruktur
Många proteiner uppnår inte någon funktion förrän de bildar komplex med andra proteiner.
Kvartärstrukturen beskriver hur de olika polypeptiderna (proteinerna) förhåller sig till varandra i komplexet.
Proteinerna i ett kvartärt komplex kallas ofta för ”subunits” (subenheter).
Konformationsförändringar i ett kvartärt komplex kan ske via; (1) konformations- förändring i en eller flera subenheter,
(2) reorientering av subenheterna
Konformationsförändringen i ett kvartärt komplex är grunden till kooperativitet och i vissa fall allosteri.
hemoglobin, a2b2 quaternary structure with 4 heme
Triple helix of collagen – quaternary structure
Right-handed bundle of three parallel, left-handed helices of individual collagen peptides.
Gly-X-Y repetitive motif
Structure is stabilised by H- bonds between collagen peptides
Protein function: “Induced Fit” Binding
Induced fit kan beskriva hur olika domäner i ett protein reorienteras när det binder sin interaktionspartner.
Induced fit kan beskriva hur en ostrukturerad del (eller helt protein) veckas när den träffar på sin interaktionspartner
Enzyme active site - mechanism
Chymotrypsin, hydrolyses peptides
1. active site makes Ser195 more reactive
(pK of side chains often shifted in active site) 2. Good arrangement of all catalytic side chains 3. Substrate gets bound in fitting orientation.
Mutations can influence binding or activity <> mechanism
Integral Membrane Protein: Bacterial Porin
…..
Porin Structure
Porins are beta barrel proteins that cross a cellular membrane and act as a pore, through which molecules can diffuse. Unlike other membrane transport proteins, porins are large enough to allow passive diffusion, i.e., they act as channels that are specific to different types of molecules.
Membrane Protein: K+ Ion Channel
….
K+ channel: Mechanism
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Muscle proteins: Myosin moves on Actin
- Actin quaternary structure: polymer of monomers pointing one way:
- –> directional, “polar”
-Myosin: 2 repetitive 1300 aa a-helices (yellow, orange) with headgroups, assembled a helical coiled coil
regular repeat for packing in thick filaments
- Myosin headgroups bind ATP - change conformation, leave actin - hydrolyze ATP - grab actin, release Pi - ADP-ATP cycle starts new
Natively unfolded proteins
- Unstructured in free state
- Fold upon binding
- Large fraction of human genome!
Summary of terms
• Active site: Part of protein where functional amino acids are arranged
• Coil: unstructured part of a folded protein
• Coiled coil: special tertiary structure of collagen
• Random coil: unfolded state of a protein
• Domain: protein part that folds independently, ~50-200 aa
• Family: proteins related in evolution, similar sequence/structure;
can have structural or functional meaning.
• Fold: 3D arrangement of secondary structures
• Globular structure: ball-like structure of soluble proteins
• Native structure: Functional in vivo structure
• residue: amino acid in protein
• Subunit: Part of a quarternary structure
• turn: (structured) protein part connecting secondary structure elements
• conserved amino acid, conservative mutation, WT
Native structure and misfolded structure - prion protein
- Native structure, based on NMR results
- Fibril structure, MODEL based on low- resolution structural studies
- Native structure seems to be most stable structure.
Relevance of Protein Folding
- Most proteins must fold to function, why do mostly these occur?
- Stability of folded structure determines protein’s lifetime
- Small changes in structure can strongly influence function: - Single Glu -> Val mutation causes sickle cell anaemia
- misfolding diseases: Alzheimers’, Prion disease, ALS …
- Astronomical number of possible sequences:
Around 1 million billion times more 50-amino-acid sequences than atoms on Earth, but: - Few sequences occur,
- many have same “fold” - why?
- Protein production: will it fold?
Folding Problem
Small proteins fold spontaneously – amino acid sequence encodes structure!
• What structure does a polypeptide fold into?
H bonds in secondary structures are not sequence-specific!!!
• How does a peptide chain find its fold???
• Unfolded states expose hydrophobic side chains, these can aggregate / precipitation, consequence can be misfolding diseases.
Which structure?
• There are 20100 100-amino-acid sequences
probably only a TINY fraction folds, or:
“How will a random sequence fold?” Is wrong question: VERY unlikely it folds
• Amino acid sequences of natural proteins are VERY SPECIAL: SELECTED TO FOLD
• The amino acid sequence encodes the structure, nobody knows the code
• Determine structure experimentally: X-ray crystallography, NMR
• Tens of thousands of structures, just a few hundred folds – secondary structure patterns
Energetics: Folding as Chemical Equilibrium
• Many small proteins can fold and unfold reversibly: equilibrium
• In the transition state (TS) bonds form or break:
Here the non-covalent bonds of secondary & tertiary structure
• DG=DH–TDS DG: related to stability DH: heat of reaction, related to bond energy DS: change of entropy (”disorder”) • So what determines DH and DS?
Entropy & Enthalpy of folding: The protein
-Thermodynamics of folding: DG = DH – TDS
•DH and TDS have contributions from protein AND water
- DH contains: energy of H bonds in secondary structures!! van-der Waals bonds in hydrophobic core: —> stabilizes N
• folding of random coil into ordered structure reduces
entropy: -TDS OPPOSES folding
• Protein would not fold (without water‘s contribution)
Water‘s contribution to folding
• Main effect: Exposed hydrophobic side chains of U molecules
reduce motional freedom of nearby water molecules.
• Folding burries hydrophibic side chains, motional freedom =
entropy of water INCREASES during folding!
• water is involved in folding, its entropy increase stabilizes N
• Stabilization of N due to water’s entropy gain hinges on burial of hydrophobic amino acid side chains.
• Smallest proteins are ~50 amino acids, else no hydrophobic core.
HOW do proteins fold? Simple models for folding paths
-Diffusion-collison: secondary structure forms first, then tertiary structure
-Hydrophobic collapse: protein becomes compact first, then secondary structures form
-Nucleation / growth: one secondary structure element forms first, rest attaches to it
- Different intermediates should be observable! Intermediates rarely observed
Natural proteins have evolved against - dangerous - intermediates.
Folding funnel
Native-like secondary, tertiary interactions formed from many starting structures.
Most “early” interactions lead towards folded state:
No “traps”
- intermediates - in funnel.
Folding in the Cell
- Fundamental interest: Do proteins fold spontaneously in the cell like in vitro?
- If you produce a human protein in bacteria, why does it often not fold?
- Proteins are synthesised in the cytosol. Where does a protein fold that works in a different compartment?
- There are proteins that get translated, fold, get processed. The processed protein will then typically not fold after denaturation, classical example: insulin.
The cellular environment:
CROWDED: many possible interactions
Co-translational Folding on Ribosome
• Do proteins also fold
spontaneously in the cell?
• ”Trigger factor protects ”nascent chain”
• Domains fold independently
Chaperones
- Some proteins don’t fold fast enough to their folded state, although it is more stable.
- Chaperones are proteins that help such proteins fold.
- disulfide isomerases, proline cis-trans isomerases
- Bind unfolded states and prevent them from precipitating.
- Can unfold misfolded states using energy, so that they can fold.
Function of GroEL chaperone
GroEL is a protein which belongs to the chaperonin family of molecular chaperones, and is found in many bacteria. It is required for the proper folding of many proteins. To function properly, GroEL requires the lid-like cochaperonin protein complex GroES.
Natively unfolded proteins
Natively unfolded or intrinsically unstructured proteins constitute a unique group of the protein kingdom. … These proteins show a low level of ordered secondary structure and no tightly packed core. They are very flexible, but may adopt relatively rigid conformations in the presence of natural ligands.
Native and Amyloid Structures
Amyloids are aggregates of proteins characterised by a fibrillar morphology of 7–13 nm in diameter, a β-sheet secondary structure (known as cross-β) and ability to be stained by particular dyes, such as Congo red. In the human body, amyloids have been linked to the development of various diseases.
Why can most proteins form amyloid fibrils?
- Amyloid is stabilized by b sheet & interactions between chains - interactions are not sequence-specific
- Native structure must / cannot be too stable.
Prediction of protein structure?
• Structures of similar sequences can be predicted
• Extremely difficult to predict structure of new sequence
• Structure determination routine, prediction tricky!
Computer simulation of small protein structure, here
- local structure statistics for
~5 amino acid stretches
> assemble local structures - select structures with good
packing, secondary structures.
Computer analysis of proteins????
Data bases:
- genomes of organisms: deduce amino acid sequences
- pdb: biomolecular structures
- nucleic acid data base
- BMRB: NMR data & structures
Computer programs:
• sequence comparison
• fold recognition: sequence → structure
• prediction of transmembrane helices
• structure determination: X-ray / NMR / EM
• Structure prediction, folding@home: use YOUR computer
• visualization: structures, surface properties, docking
• motions: molecular dynamics simulations
Det centrala dogmat—–> - Ritning
Den centrala dogmen inom molekylärbiologin innebär att den genetiska informationen överförs från DNA via RNA till protein. De virus som enbart har RNA och inget DNA var tidigt kända, men även hos dem vandrar informationen åt rätt håll enligt dogmen, från RNA till protein. För att dela sig måste celler duplicera alla sina beståndsdelar
Människans DNA-mängd och organisation
- MänniskansDNAäruppdelatikromosomer,tvåav varje sort (diploid)
- 3.2 x109 nukleotidpar
- 2% av totala DNA-sekvensen kodande
- 21.000 proteinkodande-gener
- En cells totalt 2m DNA finns inne i kärnan (6 μm) organiserat som kromatin (DNA+ protein)
- DNA är uppdelat i kromosomer, två kopior (homologer) av varje kromosom (diploid)
- Kromosomerna har nr 1 till 22, + 2 könskromosomer (XX eller XY)= totalt 46 kromosomer
Eukaryota celler har även ett cirkulärt mitokondriellt genom
Bakterien E. coli:s genom
• Ingenkärna(prokaryot) • Enkopiaavgenom (haploid) • CirkulärDNA-molekyl • 4.6 x106 nukleotidpar med 4.300 gener • 90%avDNA-sekvensen kodande
DNA - uppbyggnad och struktur
Byggstenarna till DNA och RNA syntetiseras i cytoplasman eller tas upp från omgivningen
(se senare föreläsning av Paulina Wanrooij)
DNA består av två stycken antiparallella kedjor av deoxyribonukleotider som bildar en dubbelhelix
DNA - uppbyggnad och struktur
-Tre fosfatgrupper
-Fyra olika kvävebaser
I DNA: Adenine Thymine Cytosine Guanine
-N-glukosidbindning
- OBS!! Numrering av kolatomer
-En sockerdel: deoxyribos i DNA
-DNAs baser binder varandra (basparar) via vätebindningar A parar alltid med T och C parar alltid med G
- pyrimidin och purin
-• DNA-syntes sker i 5’- 3’-riktning
• Vid syntes agerar DNAs två strängar mall (templat) för varsin ny sträng
Packning av DNA
DNA-tråden är ca 2 meter lång – För att få plats i kärnan (diam ~10 μm) måste tråden packas
Nukleosom: ~140 bp DNA + histon-oktamer 2 st var av (H2A, H2B, H3, H4)
1 histon H1 binder på utsidan av varje nukleosom. Fungerar som brygga mellan nukleosomerna
Leder till bildande av ”30 nm fiber” - Resultat: 40 ggr packning av DNA
C:a 1.000 ggr packning krävs för att humant DNA ska få plats i kärnan.
Vidare packning sker via H1:H1 interaktioner samt mindre utforskade mekanismer
Nukleosomens struktur
Nukleosomerna är uppbyggda av ca 147 baspar långt DNA uppvindat runt nukleosomkärnan som består av fyra par (8 st) histonproteiner. … Den metylerade nukleosomen kan binda till proteiner (HPI) som medierar övergång till den kondenserade, icke aktiva formen: heterokromatin.
Hur mycket DNA syntetiserar humana celler?
Sammanfattning 2
DNA:s uppbyggnad och struktur
• Dubbelsträngat: Består av två deoxy-nukleotidkedjor
• De två DNA-strängarna hålls ihop genom vätebindningar mellan de ingående baserna
• A (adenin) basparar med T (tymin)
G (guanin) basparar med C (cytosine)
• DNAs två strängar är antiparallella med varandra och bildar en dubbelspiral (helix)
• Vid DNA-replikation används DNA-strängarna som templat för att bilda varsin ny komplementär sträng
• DNA i eukaryoter packas i kromatin m h a bl a histonproteiner för att få plats i kärnan
Centrala dogmat är gemensamt för allt liv —> - Arbetskopia
Transkription (RNA-syntes) sker genom avläsning av DNA till flera RNA-kopior
RNA - uppbyggnad och struktur
Molekylen är uppbyggd som en kedja av sammankopplade nukleotider. Varje nukleotid består av en sockermolekyl, ribos, en fosfatgrupp och en kvävebas som kan vara av fyra olika slag. I RNA betecknas kvävebaserna med första bokstaven i namnen: A för adenin, U för uracil, G för guanin och C för cytosin.
-Sockret i RNA är ribos:
RNA är (oftast) enkeltrådigt
ribose (used in rebionucleic acid, RNA ), deoxyribose (used in deoxyribonucleic acid DNA ),
-RNA använder basen Uracil istället för Thymin:
uracil (used in RNA), themyne (used in DNA)
- Reglering av transkription påverkar mängderna av olika proteiner i celler
- Reglering av genuttryck styrs av proteiner som binder specifika DNA-sekvenser uppströms om gener
Centrala dogmat är gemensamt för allt liv —> product
Proteinsyntes (translation) katalyseras av ribosomen…
Utanför cellkärnan finns cytoplasman och där översätts den genetiska informationen i mRNA:t till ett protein. Denna process kallas proteinsyntes eller translation. Översättningen sker med hjälp av två andra nukleinsyror, ribosomalt-RNA (rRNA) och transport-RNA (tRNA), och flera proteiner i den så kallade ribosomen.
…ett stort komplex bestående av både RNA och protein
- Proteinsyntes sker genom ihopsättning av aminosyror
Sammanfattning 2
Uppbyggnad och struktur av RNA och proteiner
- RNA är oftast en enkelsträngad kedja bestående av ribonukleotider
- Sockret i RNA är ribos
- RNA använder basen Uracil istället för Thymin
- Reglering av transkription påverkar mängderna av olika proteiner i celler
- Reglering av genuttryck styrs av proteiner som binder specifika DNA-sekvenser uppströms om gener
- Proteinsyntes innebär ihopsättning av aminosyror och katalyseras av ribosomen
DNA replikation: Strukturen hos DNA
- Strukturen hos DNA: Dubbelspiral, antiparallell helix, baspar, major/minor groove
- DNA-syntesens kemi: Basparning, bildande av fosfodiesterbindningar
- DNA-replikation – originalsträngar agerar templat för varsin ny sträng
- Ny DNA-kedja byggs i 5’ till 3’ riktning
