Proteins make up about 15% of cells and have many important functions like enzymes, structure, transport, motors, storage, signaling, and gene regulation. A protein's shape and function are determined by its amino acid sequence. The peptide backbone forms secondary structures like alpha helices and beta sheets held together by hydrogen bonds and other weak interactions. Chaperone proteins help guide proper protein folding. Proteins fold into complex 3D structures and can have multiple subunits that interact.

1. Chapter 4Chapter 4
Protein StructureProtein Structure
and Functionand Function

2. ProteinsProteins
 Make up about 15% of the cellMake up about 15% of the cell
 Have many functions in the cellHave many functions in the cell
 EnzymesEnzymes
 StructuralStructural
 TransportTransport
 MotorMotor
 StorageStorage
 SignalingSignaling
 ReceptorsReceptors
 Gene regulationGene regulation
 Special functionsSpecial functions

3. Shape = Amino Acid SequenceShape = Amino Acid Sequence
 Proteins are made of 20 amino acidsProteins are made of 20 amino acids
linked by peptide bondslinked by peptide bonds
 Polypeptide backbone is the repeatingPolypeptide backbone is the repeating
sequence of the N-C-C-N-C-C… in thesequence of the N-C-C-N-C-C… in the
peptide bondpeptide bond
 The side chain or R group is not part ofThe side chain or R group is not part of
the backbone or the peptide bondthe backbone or the peptide bond

4. PolypeptidePolypeptide
BackboneBackbone

5. Amino AcidsAmino Acids
NOTE: You need to know this tableNOTE: You need to know this table
Hydrophilic Hydrophobic

6. Protein FoldingProtein Folding
 The peptide bond allows for rotationThe peptide bond allows for rotation
around it and therefore the protein can foldaround it and therefore the protein can fold
and orient the R groups in favorableand orient the R groups in favorable
positionspositions
 Weak non-covalent interactions will holdWeak non-covalent interactions will hold
the protein in its functional shape – thesethe protein in its functional shape – these
are weak and will take many to hold theare weak and will take many to hold the
shapeshape

7. Non-covalent Bonds in ProteinsNon-covalent Bonds in Proteins

8. Globular ProteinsGlobular Proteins
 The side chains will help determine theThe side chains will help determine the
conformation in an aqueous solutionconformation in an aqueous solution

9. Hydrogen Bonds in ProteinsHydrogen Bonds in Proteins
 H-bonds form between 1) atoms involved in theH-bonds form between 1) atoms involved in the
peptide bond; 2) peptide bond atoms and Rpeptide bond; 2) peptide bond atoms and R
groups; 3) R groupsgroups; 3) R groups

10. Protein FoldingProtein Folding
 Proteins shape is determined by theProteins shape is determined by the
sequence of the amino acidssequence of the amino acids
 The final shape is called theThe final shape is called the
conformation and has the lowest freeconformation and has the lowest free
energy possibleenergy possible
 Denaturation is the process of unfoldingDenaturation is the process of unfolding
the proteinthe protein
Can be down with heat, pH or chemicalCan be down with heat, pH or chemical
compoundscompounds
In the chemical compound, can removeIn the chemical compound, can remove
and have the protein renature or refoldand have the protein renature or refold

11. Folding@homeFolding@home
 The Stanford Folding@home research goal is toThe Stanford Folding@home research goal is to
understand protein folding, misfolding, andunderstand protein folding, misfolding, and
related diseases.related diseases.
 Calculations to create models requires aCalculations to create models requires a
supercomputer OR many smaller computerssupercomputer OR many smaller computers
(distributed computing).(distributed computing).
 You can participate by visiting:You can participate by visiting:
 Fold@home web site:Fold@home web site: http://folding.stanford.edu/http://folding.stanford.edu/
 Article on Folding@home:Article on Folding@home:
http://www.sciencedaily.com/releases/2002/10/021022070http://www.sciencedaily.com/releases/2002/10/021022070

12. RefoldingRefolding
 Molecular chaperones are small proteins thatMolecular chaperones are small proteins that
help guide the folding and can help keep thehelp guide the folding and can help keep the
new protein from associating with the wrongnew protein from associating with the wrong
partnerpartner

13. Protein FoldingProtein Folding
 2 regular folding patterns2 regular folding patterns
have been identified –have been identified –
formed between theformed between the
bonds of the peptidebonds of the peptide
backbonebackbone
 αα-helix – protein turns like-helix – protein turns like
a spiral – fibrous proteinsa spiral – fibrous proteins
(hair, nails, horns)(hair, nails, horns)
 ββ-sheet – protein folds-sheet – protein folds
back on itself as in aback on itself as in a
ribbon –globular proteinribbon –globular protein

14. ββ SheetsSheets  Core of many proteins isCore of many proteins is
thethe ββ sheetsheet
 Form rigid structuresForm rigid structures
with the H-bondwith the H-bond
 Can be of 2 typesCan be of 2 types
 Anti-parallel – run in anAnti-parallel – run in an
opposite direction of itsopposite direction of its
neighbor (A)neighbor (A)
 Parallel – run in the sameParallel – run in the same
direction with longerdirection with longer
looping sections betweenlooping sections between
them (B)them (B)

15. αα HelixHelix
 Formed by a H-bondFormed by a H-bond
between every 4between every 4thth
peptidepeptide
bond – C=O to N-Hbond – C=O to N-H
 Usually in proteins thatUsually in proteins that
span a membranespan a membrane
 TheThe αα helix can either coilhelix can either coil
to the right or the leftto the right or the left
 Can also coil around eachCan also coil around each
other – coiled-coil shapeother – coiled-coil shape
– a framework for– a framework for
structural proteins suchstructural proteins such
as nails and skinas nails and skin

16. CD from TextCD from Text
 The CD that is included on your textbookThe CD that is included on your textbook
back cover has some video clips that willback cover has some video clips that will
show theshow the αα helix andhelix and ββ sheets as well assheets as well as
other things in this chapter. You will wantother things in this chapter. You will want
to look at them.to look at them.

17. Levels of OrganizationLevels of Organization
 PrimaryPrimary structurestructure
Amino acid sequence of the proteinAmino acid sequence of the protein
 SecondarySecondary structurestructure
H bonds in the peptide chain backboneH bonds in the peptide chain backbone
αα-helix and-helix and ββ-sheets-sheets
 TertiaryTertiary structurestructure
Non-covalent interactions between the RNon-covalent interactions between the R
groups within the proteingroups within the protein
 QuanternaryQuanternary structurestructure
Interaction between 2 polypeptide chainsInteraction between 2 polypeptide chains

18. Protein StructureProtein Structure

19. DomainsDomains
 AA domaindomain is a basic structural unit of ais a basic structural unit of a
protein structure – distinct from thoseprotein structure – distinct from those
that make up the conformationsthat make up the conformations
 Part of protein that can fold into a stablePart of protein that can fold into a stable
structure independentlystructure independently
 Different domains can impart differentDifferent domains can impart different
functions to proteinsfunctions to proteins
 Proteins can have one to manyProteins can have one to many
domains depending on protein sizedomains depending on protein size

20. DomainsDomains

21. Useful ProteinsUseful Proteins
 There are thousands and thousands ofThere are thousands and thousands of
different combinations of amino acids thatdifferent combinations of amino acids that
can make up proteins and that wouldcan make up proteins and that would
increase if each one had multiple shapesincrease if each one had multiple shapes
 Proteins usually have only one usefulProteins usually have only one useful
conformation because otherwise it would notconformation because otherwise it would not
be efficient use of the energy available to thebe efficient use of the energy available to the
systemsystem
 Natural selection has eliminated proteins thatNatural selection has eliminated proteins that
do not perform a specific function in the celldo not perform a specific function in the cell

22. ProteinProtein
FamiliesFamilies
 Have similarities in amino acid sequence andHave similarities in amino acid sequence and
3-D structure3-D structure
 Have similar functions such as breakdownHave similar functions such as breakdown
proteins but do it differentlyproteins but do it differently

23. Proteins – Multiple PeptidesProteins – Multiple Peptides
 Non-covalent bonds can form interactionsNon-covalent bonds can form interactions
between individual polypeptide chainsbetween individual polypeptide chains
Binding site – where proteins interact with oneBinding site – where proteins interact with one
anotheranother
Subunit – each polypeptide chain of largeSubunit – each polypeptide chain of large
proteinprotein
Dimer – protein made of 2 subunitsDimer – protein made of 2 subunits
Can be same subunit or different subunitsCan be same subunit or different subunits

24. Single Subunit ProteinsSingle Subunit Proteins

25. Different Subunit ProteinsDifferent Subunit Proteins
HemoglobinHemoglobin
22 αα globinglobin
subunitssubunits
22 ββ globinglobin
subunitssubunits

26. Protein AssembliesProtein Assemblies
 Proteins can form veryProteins can form very
large assemblieslarge assemblies
 Can form long chains ifCan form long chains if
the protein has 2the protein has 2
binding sites – linkbinding sites – link
together as a helix or atogether as a helix or a
ringring
 Actin fibers in musclesActin fibers in muscles
and cytoskeleton – isand cytoskeleton – is
made from thousandsmade from thousands
of actin molecules as aof actin molecules as a
helical fiberhelical fiber

27. Types of ProteinsTypes of Proteins
 Globular ProteinsGlobular Proteins – most of what we– most of what we
have dealt with so farhave dealt with so far
Compact shape like a ball with irregularCompact shape like a ball with irregular
surfacessurfaces
Enzymes are globularEnzymes are globular
 Fibrous ProteinsFibrous Proteins – usually span a long– usually span a long
distance in the celldistance in the cell
3-D structure is usually long and rod3-D structure is usually long and rod
shapedshaped

28. Important Fibrous ProteinsImportant Fibrous Proteins
 Intermediate filaments of theIntermediate filaments of the
cytoskeletoncytoskeleton
Structural scaffold inside the cellStructural scaffold inside the cell
Keratin in hair, horns and nailsKeratin in hair, horns and nails
 Extracellular matrixExtracellular matrix
Bind cells together to make tissuesBind cells together to make tissues
Secreted from cells and assemble in longSecreted from cells and assemble in long
fibersfibers
Collagen – fiber with a glycine every third aminoCollagen – fiber with a glycine every third amino
acid in the proteinacid in the protein
Elastin – unstructured fibers that gives tissue anElastin – unstructured fibers that gives tissue an
elastic characteristicelastic characteristic

29. Collagen and ElastinCollagen and Elastin

30. Stabilizing Cross-LinksStabilizing Cross-Links
 Cross linkages can be between 2 parts of aCross linkages can be between 2 parts of a
protein or between 2 subunitsprotein or between 2 subunits
 Disulfide bonds (S-S) form between adjacent -SHDisulfide bonds (S-S) form between adjacent -SH
groups on the amino acid cysteinegroups on the amino acid cysteine

31. Proteins at WorkProteins at Work
 The conformation of a protein gives it aThe conformation of a protein gives it a
unique functionunique function
 To work proteins must interact with otherTo work proteins must interact with other
molecules, usually 1 or a few molecules frommolecules, usually 1 or a few molecules from
the thousands to 1 proteinthe thousands to 1 protein
 Ligand – the molecule that a protein can bindLigand – the molecule that a protein can bind
 Binding site – part of the protein that interactsBinding site – part of the protein that interacts
with the ligandwith the ligand
 Consists of a cavity formed by a specificConsists of a cavity formed by a specific
arrangement of amino acidsarrangement of amino acids

32. Ligand BindingLigand Binding

33. Formation of Binding SiteFormation of Binding Site
 The binding site forms when amino acids fromThe binding site forms when amino acids from
within the protein come together in the foldingwithin the protein come together in the folding
 The remaining sequences may play a role inThe remaining sequences may play a role in
regulating the protein’s activityregulating the protein’s activity

34. Antibody FamilyAntibody Family
 A family of proteins that can be createdA family of proteins that can be created
to bind to almost any moleculeto bind to almost any molecule
 AntibodiesAntibodies (immunoglobulins) are made(immunoglobulins) are made
in response to a foreign molecule ie.in response to a foreign molecule ie.
bacteria, virus, pollen… called thebacteria, virus, pollen… called the
antigenantigen
 Bind together tightly and thereforeBind together tightly and therefore
inactivates the antigen or marks it forinactivates the antigen or marks it for
destructiondestruction

35. AntibodiesAntibodies
 Y-shaped molecules with 2 binding sites atY-shaped molecules with 2 binding sites at
the upper ends of the Ythe upper ends of the Y
 The loops of polypeptides on the end ofThe loops of polypeptides on the end of
the binding site are what imparts thethe binding site are what imparts the
recognition of the antigenrecognition of the antigen
 Changes in the sequence of the loopsChanges in the sequence of the loops
make the antibody recognize differentmake the antibody recognize different
antigens - specificityantigens - specificity

36. AntibodiesAntibodies

37. Binding StrengthBinding Strength
 Can be measured directlyCan be measured directly
 Antibodies and antigens are mixing around inAntibodies and antigens are mixing around in
a solution, eventually they will bump into eacha solution, eventually they will bump into each
other in a way that the antigen sticks to theother in a way that the antigen sticks to the
antibody, eventually they will separate due toantibody, eventually they will separate due to
the motion in the moleculesthe motion in the molecules
 This process continues until theThis process continues until the equilibriumequilibrium isis
reached – number sticking is constant andreached – number sticking is constant and
number leaving is constantnumber leaving is constant
 This can be determined for any protein and itsThis can be determined for any protein and its
ligandligand

38. EquilibriumEquilibrium
ConstantConstant
 Concentration of antigen, antibody andConcentration of antigen, antibody and
antigen/antibody complex at equilibrium can beantigen/antibody complex at equilibrium can be
measured –measured – equilibrium constant (K)equilibrium constant (K)
 Larger the K the tighter the binding or the moreLarger the K the tighter the binding or the more
non-covalent bonds that hold the 2 togethernon-covalent bonds that hold the 2 together

39. Enzymes as CatalystsEnzymes as Catalysts
 Enzymes are proteins that bind to their ligandEnzymes are proteins that bind to their ligand
as the 1as the 1stst
step in a processstep in a process
 An enzyme’s ligand is called aAn enzyme’s ligand is called a substratesubstrate
 May be 1 or more moleculesMay be 1 or more molecules
 Output of the reaction is called the productOutput of the reaction is called the product
 Enzymes can repeat these steps many timesEnzymes can repeat these steps many times
and rapidly, called catalystsand rapidly, called catalysts
 Many different kinds – see table 5-2, p 168Many different kinds – see table 5-2, p 168

40. Enzymes at WorkEnzymes at Work
 Lysozyme is an important enzyme thatLysozyme is an important enzyme that
protects us from bacteria by making holes inprotects us from bacteria by making holes in
the bacterial cell wall and causing it to breakthe bacterial cell wall and causing it to break
 Lysozyme adds HLysozyme adds H22O to the glycosidic bond inO to the glycosidic bond in
the cell wallthe cell wall
 Lysozyme holds the polysaccharide in aLysozyme holds the polysaccharide in a
position that allows the Hposition that allows the H22O to break the bondO to break the bond
– this is the– this is the transition statetransition state – state between– state between
substrate and productsubstrate and product
 Active siteActive site is a special binding site inis a special binding site in
enzymes where the chemical reaction takesenzymes where the chemical reaction takes
placeplace

41. LysozymeLysozyme
 Non-covalent bonds hold the polysaccharide inNon-covalent bonds hold the polysaccharide in
the active site until the reaction occursthe active site until the reaction occurs

42. Features of Enzyme CatalysisFeatures of Enzyme Catalysis

43. Enzyme PerformanceEnzyme Performance
E + SE + S ↔↔ ESES ↔↔ EPEP ↔↔ E + PE + P
 Step 1 – binding of the substrateStep 1 – binding of the substrate
 Limiting step depending on [S] and/or [E]Limiting step depending on [S] and/or [E]
 VVmaxmax – maximum rate of the reaction– maximum rate of the reaction
 Turnover number determines how fast theTurnover number determines how fast the
substrate can be processed = rate of rxnsubstrate can be processed = rate of rxn ÷÷ [E][E]
 Step 2 – stabilize the transition stateStep 2 – stabilize the transition state
 State of substrate prior to becoming productState of substrate prior to becoming product
 Enzymes lowers the energy of transition state andEnzymes lowers the energy of transition state and
therefore accelerates the reactiontherefore accelerates the reaction

44. Reaction RatesReaction Rates
 KKMM – [S] that allows rxn to proceed at ½ it– [S] that allows rxn to proceed at ½ it
maximum ratemaximum rate

45. Prosthetic GroupsProsthetic Groups
 Occasionally the sequence of the protein isOccasionally the sequence of the protein is
not enough for the function of the proteinnot enough for the function of the protein
 Some proteins require a non-protein moleculeSome proteins require a non-protein molecule
to enhance the performance of the proteinto enhance the performance of the protein
 Hemoglobin requires heme (iron containingHemoglobin requires heme (iron containing
compound) to carry the Ocompound) to carry the O22
 When aWhen a prosthetic groupprosthetic group is required by anis required by an
enzyme it is called aenzyme it is called a co-enzymeco-enzyme
 Usually a metal or vitaminUsually a metal or vitamin
 These groups may be covalently or non-These groups may be covalently or non-
covalently linked to the proteincovalently linked to the protein

47. Regulation of EnzymesRegulation of Enzymes
 Regulation of enzymaticRegulation of enzymatic
pathways prevent thepathways prevent the
deletion of substratedeletion of substrate
 Regulation happens atRegulation happens at
the level of the enzymethe level of the enzyme
in a pathwayin a pathway
 Feedback inhibition isFeedback inhibition is
when the end productwhen the end product
regulates the enzymeregulates the enzyme
early in the pathwayearly in the pathway

48. Feedback RegulationFeedback Regulation
 Negative feedbackNegative feedback ––
pathway is inhibited bypathway is inhibited by
accumulation of finalaccumulation of final
productproduct
 Positive feedbackPositive feedback – a– a
regulatory moleculeregulatory molecule
stimulates the activity ofstimulates the activity of
the enzyme, usuallythe enzyme, usually
between 2 pathwaysbetween 2 pathways
 ↑↑ ADP levels cause theADP levels cause the
activation of theactivation of the
glycolysis pathway toglycolysis pathway to
make more ATPmake more ATP

49. AllosteryAllostery
 Conformational coupling of 2 widelyConformational coupling of 2 widely
separated binding sites must beseparated binding sites must be
responsible for regulation – active siteresponsible for regulation – active site
recognizes substrate and 2recognizes substrate and 2ndnd
sitesite
recognizes the regulatory moleculerecognizes the regulatory molecule
 Protein regulated this way undergoesProtein regulated this way undergoes
allosteric transition or a conformationalallosteric transition or a conformational
changechange
 Protein regulated in this manner is anProtein regulated in this manner is an
allosteric proteinallosteric protein

50. Allosteric RegulationAllosteric Regulation
 Method of regulation is also used in otherMethod of regulation is also used in other
proteins besides enzymesproteins besides enzymes
 Receptors, structural and motor proteinsReceptors, structural and motor proteins

51. Allosteric RegulationAllosteric Regulation
 Enzyme is only partially active with sugar only butEnzyme is only partially active with sugar only but
much more active with sugar and ADP presentmuch more active with sugar and ADP present

52. PhosphorylationPhosphorylation
 Some proteins are regulated by theSome proteins are regulated by the
addition of a POaddition of a PO44 group that allows forgroup that allows for
the attraction of + charged side chainsthe attraction of + charged side chains
causing a conformation changecausing a conformation change
 Reversible protein phosphorylationsReversible protein phosphorylations
regulate many eukaryotic cell functionsregulate many eukaryotic cell functions
turning things on and offturning things on and off
 ProteinProtein kinaseskinases add the POadd the PO44 and proteinand protein
phosphatasephosphatase remove themremove them

53. Phosphorylation/DephosphorylationPhosphorylation/Dephosphorylation
 Kinases capable ofKinases capable of
putting the POputting the PO44 on 3on 3
different amino aciddifferent amino acid
residuesresidues
 Have a –OH group onHave a –OH group on
R groupR group
 SerineSerine
 ThreonineThreonine
 TyrosineTyrosine
 Phosphatases thatPhosphatases that
remove the POremove the PO44 maymay
be specific for 1 or 2be specific for 1 or 2
reactions or many bereactions or many be
non-specificnon-specific

54. GTP-Binding ProteinsGTP-Binding Proteins
(GTPases)(GTPases)
 GTP does not release its POGTP does not release its PO44
group but rather the guaninegroup but rather the guanine
part binds tightly to thepart binds tightly to the
protein and the protein isprotein and the protein is
activeactive
 Hydrolysis of the GTP toHydrolysis of the GTP to
GDP (by the protein itself)GDP (by the protein itself)
and now the protein isand now the protein is
inactiveinactive
 Also a family of proteinsAlso a family of proteins
usually involved in cellusually involved in cell
signaling switching proteinssignaling switching proteins
on and offon and off

55. MolecularMolecular
SwitchesSwitches

56. Motor ProteinsMotor Proteins
 Proteins can move in theProteins can move in the
cell, say up and down acell, say up and down a
DNA strand but with veryDNA strand but with very
little uniformitylittle uniformity
 Adding ligands to changeAdding ligands to change
the conformation is notthe conformation is not
enough to regulate thisenough to regulate this
processprocess
 The hydrolysis of ATP canThe hydrolysis of ATP can
direct the the movement asdirect the the movement as
well as make it unidirectionalwell as make it unidirectional
 The motor proteins thatThe motor proteins that
move things along the actinmove things along the actin
filaments or myosinfilaments or myosin

57. Protein MachinesProtein Machines
 Complexes of 10 orComplexes of 10 or
more proteins that workmore proteins that work
together such as DNAtogether such as DNA
replication, RNA orreplication, RNA or
protein synthesis, trans-protein synthesis, trans-
membrane signalingmembrane signaling
etc.etc.
 Usually driven by ATPUsually driven by ATP
or GTP hydrolysisor GTP hydrolysis
 See video clip on CD inSee video clip on CD in
bookbook