chapter 4: protein structure and folding. life … is a relationship between molecules. linus...
TRANSCRIPT
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Chapter 4:
Protein Structure and Folding
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Life … is a relationship between molecules.
Linus Pauling, as quoted in T. Hager, Force of Nature: The Life of Linus Pauling (1997), p. 542
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4.1 Introduction
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• Proteins are found in all living systems, ranging from bacteria and archaea through the unicellular eukaryotes, to plants, fungi, and animals.
• In all life forms, proteins are made up of the same building blocks―amino acids.
• Each cell contains thousands of different genes and makes thousands of different proteins.
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What is a gene?
• In the late 1930s…
“A molecule of living stuff made up of many atoms held together.”
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What is a gene?
• A specific stretch of nucleotides in DNA (or in some viruses, RNA) that contains information for making a particular RNA molecule that in most cases is used to make a particular protein.
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4.2 Primary structure: amino acids and the genetic code
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The 22 amino acids found in proteins
• Proteins are chain-like polymers of amino acids specified by the genetic code.
• Each amino acid has an amino group (NH3+)
and a carboxyl group (COO) attached to a central carbon called the -carbon.
• The only difference between two amino acids is in their different side chain or “R group.”
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• At pH 7 the amino and carboxyl groups of amino acids are charged.
• Over a pH range from 1 to 14 these groups exhibit binding and dissociation of a proton.
• The weak acid-base behavior of amino acids provides the basis for many techniques for amino acid identification and protein separations.
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Protein primary structure
• Amino acids joined together by peptide bonds form the primary structure of a protein.
• The amino group of one molecule reacts with the carboxyl group of the other in a condensation reaction.
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• When joined in a series of peptide bonds, amino acids are called residues.
• A short sequence of amino acids is called a peptide; the term polypeptide applies to longer chains of amino acids.
• The arrangement of amino acids, with their distinct side chains, gives each protein its characteristic structure and function.
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• The peptide bond has a partial double bond character as a result of resonance.
• Free rotation occurs only between the -carbon and the peptide unit.
• Trans and cis-configurations are possible about the rigid peptide bond.
• The peptide chain is flexible, but it is more rigid than it would be if there were free rotation about all of the bonds.
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• How is the genetic code translated into a specific sequence of amino acids?
• The mechanism of translation is described in detail in Chapter 14.
Translating the genetic code
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• A DNA sequence is read in triplets using the antisense (non-coding) strand as a template that directs synthesis of RNA via complementary base pairing.
• An open reading frame (ORF) in the mRNA indicates the presence of a start codon followed by codons for a series of amino acids and ending with a termination codon.
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The genetic code
• Each “codon box” is composed of four three-letter codes, 64 in all.
• 61 codons are recognized by tRNAs for the incorporation of the 20 common amino acids.
• 3 codons signal termination, or code for selenocysteine and pyrrolysine.
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The genetic code is degenerate
• tRNAs specific to a particular amino acid recognize multiple codon triplets that differ only in the third letter.
e.g. leucine is coded for by 6 different codons, while methionine has only one codon
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The “wobble hypothesis”
• Pairing between codon and anticodon at the first two codon positions always follows the usual rule of complementary base pairing.
• Exceptional “wobbles” (non-Watson-Crick base pairing) can occur at the third position.
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The genetic code is not universal
• In certain organisms and organelles the meaning of select codons has been changed.
e.g. Tetrahymena reads UAA and UAG as glutamine (Gln)
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The 21st and 22nd genetically encoded amino acids
The UGA code for selenocysteine is found in:• >15 genes in prokaryotes that are involved in
redox reactions.• >40 genes in eukaryotes that code for various
antioxidants and the type I iodothyronine deiodinase.
The UAG code for pyrrolysine has been found in:• a few archaebacteria and eubacteria.
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Modified nucleotides and codon bias
• “Wobbles” can occur at the third position.
• When bases in the anticodon are modified, further pairing patterns are possible.
• Examples:
Inosine can pair with U, C, and A.
2-thiouracil restricts pairing to A alone.
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Implications of codon bias for molecular biologists
• The frequencies with which different codons are used vary significantly between different organisms and between proteins expressed at high or low levels within the same organism.
• Expression of functional proteins in heterologous hosts is a cornerstone of molecular biology research.
• Codon bias can have a major impact on the efficiency of expression of proteins if they contain codons that are rarely used in the desired host.
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• What might happen if you tried to express a Tetrahymena gene that encodes a glutamine-rich protein in E. coli?
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D- and L-amino acids in nature
• D- and L-amino acids are enantiomers (sterioisomers that are mirror images of each other).
• Living organisms are composed predominantly of L-amino acids.
• Ribosomes only use L-amino acids to make proteins.
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Exceptions:
• D-amino acids are found in some peptides in microorganisms, but are synthesized by pathways that do not involve the ribosome.
• D-amino acids are present in some peptides in other organisms, but are made from the genetically encoded L-amino acids by a post-translational process.
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Examples:
• D-amino acids are present in the venom of some bivalves, snails, spiders, amphibians, and the duck-bill platypus.
• The presence of D-amino acids is linked to more potent venom.
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4.3 The three-dimensional structure of proteins
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• There is tremendous variation in the size and complexity of proteins.
• Dalton (Da) units are typically used to describe the molecular weight of proteins.
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• Typical polypeptide chains have molecular weights of 20 to 70 kDa (20,000 to 70,000 Da).
• The average molecular weight of an amino acid is 110.
• A typical polypeptide chain thus contains 181 to 636 amino acids.
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Secondary structure
• Interactions of amino acids with their neighbors gives a protein its secondary structure.
• Primarily stabilized by hydrogen bonds.
• Also depends on disulfide bridges, van der Waals interactions, hydrophobic contacts, and electrostatic interactions.
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The three basic elements of protein secondary structure
-helix
-pleated sheet
• Unstructured turns
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-helix
• Most common structural motif in proteins.
• Tight helical structure stabilized by hydrogen bonding among near-neighbor amino acids.
• Proline, the “helix-breaking residue”, cannot participate as a donor in hydrogen bonding.
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-pleated sheet
• Extended amino acids chains packed side by side to create a pleated, accordian-like appearance.
• Stabilized by hydrogen bonding.
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Parallel structure• Two segments of a polypeptide chain (or two
individual polypeptides) are aligned in the N-terminal to C-terminal direction or vice versa.
Antiparallel structure• One segment is N-terminal to C-terminal and
the other is C-terminal to N-terminal.
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Unstructured turns
• “Turns” connect the -helices and -pleated sheets in proteins.
• Relatively short loops that do not exhibit a defined secondary structure.
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Tertiary structure• The folded three-dimensional shape of a
polypeptide.
• Most interactions are stabilized by noncovalent bonds:
Hydrophobic interactions
Hydrogen bonds
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• The principle covalent bonds within and between polypeptides are disulfide (S-S) bonds or “bridges” between cysteines.
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Three main categories of tertiary structure
• Globular proteins
• Fibrous proteins
• Membrane proteins
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Globular proteins
• The overall shape of most proteins is roughly spherical.
e.g. the enzyme lysozyme folds up into a globular tertiary structure forming the active site.
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Fibrous proteins
• Long filamentous or “rod-like” structures.
• Structural components of cells and tissues.
• A number of major designs:
- triple helical arrangement
- “coiled coils”
- antiparallel -pleated sheets
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Membrane proteins
• Differ from soluble proteins in the relative distribution of hydrophobic amino acid residues.
• The seven transmembrane helix structure is a common motif in membrane proteins.
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Prediction of protein structure
• By comparing the sequences of proteins of unknown structure with those that have been determined, it is often possible to make structural predictions based on identified similarity.
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Quaternary structure
• A functional protein can be composed of one or more polypeptide subunits.
• Can be identical or nonidentical subunits.
• Stabilizing bonds are the same as those for tertiary structure.
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• Quaternary structure allows greater versatility of function.
• Catalytic or binding sites are often formed at the interface between subunits.
e.g. the two and two subunits in hemoglobin form a binding site for a heme group
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4.4 Protein function and regulation of activity
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• Proteins larger than about 20 kDa are often formed from two or more domains with specific functions.
• A single domain is usually formed from a continuous amino acid sequence.
e.g. DNA-binding domain
• Domains can contain common structural-functional motifs.
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• Proteins have a diversity of functions in cells.
• One vital role of proteins is to serve as enzymes that catalyze the hundreds of chemical reactions necessary for life.
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Enzymes are biological catalysts
• Enzymes lower the activation energies of the chemical groups that participate in a reaction and thereby speed up the reaction.
• The substrate forms a tight complex with the enzyme by binding to a region called the active site.
• Most enzymes act through an induced-fit mechanism.
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Example:• Lysozyme catalyzes the breakdown of
polysaccharides from the E. coli peptidoglycan layer.
• The active site is a long, deep cleft that can bind six N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) units.
• Lysozyme brings the reacting species together in a geometry that favors reaction.
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• For the fourth NAG-NAM unit to fit in the active site, it must be distorted, and forms a less stable conformation.
• Asp 52 and Glu35 residues of lysozyme interact with the fourth and fifth NAG-NAM units, breaking the C-O bond between them by hydrolysis.
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Regulation of protein activity by post-translational modifications
The functional activity of proteins can be regulated at several different levels:
•Transcription
•RNA processing
•Translation
•Post-translational modifications, such as phosphorylation and allosteric effectors
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• After translation, proteins are joined covalently and noncovalently to other molecules.
e.g. lipoproteins, glycoproteins, metalloproteins
• The most common regulatory mechanism is the reversible phosphorylation of amino acid side chains.
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Protein phosphorylation
• May cause a protein to change shape and unmask or mask a catalytic or functional domain.
• Phosphorylated side chain may be part of a binding motif to facilitate formation of a multiprotein complex.
• Phosphorylated side chain may promote dissociation of a multiprotein complex.
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Kinases
• Catalyze the addition of phosphate groups.
• Tend to be very specific, acting on very few substrates.
• Two protein kinase groups have been widely studied in eukaryotes:
1. Those that phosphorylate serine or threonine side chains.
2. Those that phosphorylate tyrosine side chains.
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Phosphatases
• Remove phosphates.
• Tend to be less specific, acting on many substrates.
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Allosteric regulation of protein activity
• Ligand-induced conformational change.
• An active site or another binding site is altered in a way that increases or decreases its activity.
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Example:
• Cyclin-dependent kinase (CDK) activity is regulated by both allosteric modification and phosphorylation.
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Inactive conformation of CDK
• The T loop is located at the entrance to the active site.
• Polypeptide substrates are blocked from gaining access to the ATP molecule in the active site.
• A critical glutamate residue in the PSTAIRE helix is held at a distance from the active site.
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Partial activation of CDK
• Binding of cyclin to CDK induces a conformational change.
• T loop moves away from the entrance of the active site.
• Critical glutamate in PSTAIRE helix moves into active site.
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Full activation of CDK
• Phosphorylation of Thr160 in T loop by CDK-activating kinase (CAK).
• Stabilizes active site “catalytic cleft.”
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Macromolecular assemblages
• Expression of the genetic information relies on the sequential action of large and dynamic macromolecular assemblages or “molecular machines.”
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4.5 Protein folding and misfolding
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• In some cases, protein folding is initiated before the completion of protein synthesis.
• Other proteins undergo major folding after release into the cytoplasm or a specific organelle.
• Most proteins require “molecular chaperones” to fold properly in vivo.
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Molecular chaperones• Increase the efficiency of protein folding.
• Reduce the probability of competing reactions such as aggregation.
• Aid in the destruction of misfolded proteins.
• Typically ATP-dependent.
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• Heat-shock proteins promote protein folding and aid in the destruction of misfolded protein.
e.g. Hsp40, Hsp70, Hsp90
• Hsp90 mediates protein folding by undergoing major shape changes upon binding and hydrolysis of ATP and interaction with p23.
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Endoplasmic reticulum “quality control”
• Secreted proteins are translocated into the endoplasmic reticulum (ER).
• Folding takes place before secretion through the Golgi apparatus.
• Folding catalysts accelerate potentially slow steps in the folding process
e.g. peptidylprolyl and protein disulfide isomerases
• Incorrectly folded proteins are detected by the “unfolded protein response” and targeted for degradation.
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Ubiquitin-mediated protein degradation
• Ubiquitin (a 76 amino acid polypeptide) is attached to a protein by a series of enzyme-mediated reactions.
• The ubiquitin-conjugated protein is then targeted to the 26S proteasome.
• Ubiquitin is released and the target protein is degraded by proteases.
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Protein misfolding diseases
• Formation of protein aggregates is linked to at least 20 different human diseases.
• Normally soluble proteins accumulate as insoluble deposits known as amyloid or amyloid-like fibrils.
• Proteins in amyloid-like fibrils fold into a cross -spine.
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Prions
The primary cause of transmissible spongiform encephalopathies (TSEs).
•Progressive neurodegeneration.•Dementia.•Loss of muscle control of voluntary movements.•Once symptoms appear, death results in 6 months to 1 year. •There is no cure.
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Human forms of prion disease• Kuru• Creutzfeldt-Jakob disease• Gerstmann-Straussler syndrome• Fatal familial insomnia
Animal forms• Scrapie (sheep)• Bovine spongiform encephalopathy (BSE: “mad cow
disease”)• Chronic wasting disease (elk and deer)
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The “prion only” hypothesis of infection
• Stanley Prusiner: Nobel Prize in 1997.
• Lack of immune response characteristic of infectious diseases.
• Long incubation time (up to 40 years for kuru).
• Resistance of the infectious agent to radiation that destroys living microorganisms (e.g. viruses, bacteria).
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• The infectious agent is not a living organism but a protein called PrPSc with the unusual ability to replicate itself within the body.
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• The prion PrPSc has the same amino acid sequence as the normal host protein PrPC.
• But, the prion is misfolded into a different 3-D structure.
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After misfolding the prion protein becomes…
• Aggregated (brain plaques).
• Protease resistant.
• Infectious.
• Able to survive standard sterilization techniques.
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Normal cell
• The normal cellular protein PrPC is a cell surface protein expressed in neurons.
Infected cell
• Host protein PrPC is misfolded to form new prions called PrPSc.
• Formation of fibrils, aggregates, and amyloid plaques.
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Human sporadic transmissible spongiform encephalopathies
• PrPC misfolds spontaneously and generates more prions by “autoinfection”
Creutzfeldt-Jakob disease (CJD)
• Preventative action?
None – frequency of one in a million.
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Human inherited transmissible spongiform encephalopathies
• Mutated PrPC gene with greater tendency to spontaneously misfold to prion form.
Gerstmann-Straussler syndrome
Fatal familial insomnia
• Preventative action?
None – 100% likelihood of disease progression.
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Human infectious transmissible spongiform encephalopathies
• Eating brains or infected meat products
Kuru: former ritualistic cannibalism in Papua New Guinea
New variant CJD: consumption of tainted beef
• Preventative action?
Don’t eat contaminated meat products.
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Pathway from infection to disease
• Penetration
• Translocation
• Multiplication
• Pathogenesis