Cell and Molecular Biology

1. Protein Structure
and Function

2. Proteins
• Make up about 15% of the cell
• Have many functions in the cell
– Enzymes
– Structural
– Transport
– Motor
– Storage
– Signaling
– Receptors
– Gene regulation
– Special functions

3. Shape = Amino Acid Sequence
• Proteins are made of amino acids (20 kinds)
linked by peptide bonds
• Polypeptide backbone is the repeating
sequence of the C and N atoms linked by
peptide bond
• The side chain or R group gives the amino
acid it’s unique property and is not part of the
backbone or the peptide bond

4. Polypeptide
Backbone

5. Amino Acids
Hydrophilic Hydrophobic

6. Protein Folding
• The peptide bond allows for rotation around it
and therefore the protein can fold and orient
the R groups in favorable positions
• Weak non-covalent interactions will hold the
protein in its functional shape – these are
weak and will take many to hold the shape

7. Non-covalent Bonds in Proteins

8. • The side chains will help determine the
conformation in an aqueous solution
Globular Proteins

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

10. Protein Folding
• Proteins shape is determined by the sequence of
the amino acids
• The final shape is called the conformation and
has the lowest free energy possible
• Denaturation is the process of unfolding the
protein
– Can be down with heat, pH or chemical compounds
– In the chemical compound, can remove and have the
protein renature or refold
• Molecular chaperones are small proteins that
help guide the folding and can help keep the new
protein from associating with the wrong partner

11. Refolding
• The conformation of a protein is determined solely by
its amino acid sequence. (B) The structure of urea. Urea is
very soluble in water and unfolds proteins at high
concentrations, where there is about one
urea molecule for every six water molecules.

12. Protein Folding
• 2 regular folding patterns
have been identified –
formed between the bonds
of the peptide backbone
• -helix – protein turns like a
spiral – fibrous proteins
(hair, nails, horns)
• -sheet – protein folds back
on itself as in a ribbon –
globular protein

13.  Sheets • Core of many proteins is
the  sheet
• Form rigid structures with
the H-bond
• Can be of 2 types
– Anti-parallel – run in an
opposite direction of its
neighbor (A)
– Parallel – run in the same
direction with longer
looping sections between
them (B)

14.  Helix • Formed by a H-bond
between every 4th peptide
bond – C=O to N-H
• Usually in proteins that
span a membrane
• The  helix can either coil
to the right or the left
• Can also coil around each
other – coiled-coil shape –
a framework for structural
proteins such as nails and
skin

15. Levels of Organization
• Primary structure
– Amino acid sequence of the protein
• Secondary structure
– formed by H bonds in the peptide chain
backbone
• -helix and -sheets
• Tertiary structure
– non-covalent interactions between the R groups
within the protein
• Quarternary structure
– Interaction between 2 polypeptide chains

16. Protein Structure

17. Domains
• A domain is a substructure produced by
any part of a polypeptide chain that can
fold independently into a compact, stable
structure.
• usually contains between 40 and 350
amino acids, and it is the modular unit from
which many larger proteins are
constructed.
• The different domains of a protein are
often associated with different functions.

18. Domains
(A) Cytochrome b562, a single-domain protein involved in electron transport
in mitochondria. This protein is composed almost entirely of α helices. (B) The
NAD-binding domain of the enzyme lactic dehydrogenase, which is
composed of a mixture of α helices and β sheets. (C) The variable domain
of an immunoglobulin (antibody) light chain, composed of a sandwich of two
β sheets.

19. Useful Proteins
• There are thousands and thousands of different
combinations of amino acids that can make up
proteins and that would increase if each one had
multiple shapes
• Proteins usually have only one useful
conformation because otherwise it would not be
efficient use of the energy available to the system
• Natural selection has eliminated proteins that do
not perform a specific function in the cell

20. Protein
Families
• Have similarities in amino acid sequence and 3-D
structure
• Have similar functions such as breakdown
proteins but do it differently

21. Proteins – Multiple Peptides
• Non-covalent bonds can form interactions
between individual polypeptide chains
– Binding site – where proteins interact with one
another
– Subunit – each polypeptide chain of large protein
– Dimer – protein made of 2 subunits
• Can be same subunit or different subunits

22. Single Subunit Proteins
The enzyme neuraminidase exists as a
ring of four identical polypeptide chains.
The small diagram shows how the
repeated use of the same binding
interaction forms the structure.
Two identical protein
subunits binding together
to form a symmetric protein
dimer.
The Cro repressor protein
from bacteriophage lambda
binds to DNA to turn off viral
genes.

23. Different Subunit Proteins
• Hemoglobin
–2  globin
subunits
–2  globin
subunits

24. Protein Assemblies
• Proteins can form very
large assemblies
• Can form long chains if
the protein has 2 binding
sites – link together as a
helix or a ring
• Actin fibers in muscles
and cytoskeleton – is
made from thousands of
actin molecules as a
helical fiber

25. Types of Proteins
• Globular Proteins – most of what we have
dealt with so far
– Compact shape like a ball with irregular
surfaces
– Enzymes are globular
• Fibrous Proteins – usually span a long
distance in the cell
– 3-D structure is usually long and rod shaped

26. Important Fibrous Proteins
• Intermediate filaments of the cytoskeleton
– Structural scaffold inside the cell
• Keratin in hair, horns and nails
• Extracellular matrix
– Bind cells together to make tissues
– Secreted from cells and assemble in long fibers
• Collagen – fiber with a glycine every third amino acid
in the protein
• Elastin – unstructured fibers that gives tissue an
elastic characteristic

27. Collagen and Elastin

28. Extracellular Proteins Are Often Stabilized
by Covalent Cross-Linkages
• Cross linkages can be
between 2 parts of a
protein or between 2
subunits
• Disulfide bonds (S-S)
form between
adjacent -SH groups
on the amino acid
cysteine

29. Proteins at Work
• The conformation of a protein gives it a unique
function
• To work proteins must interact with other molecules,
usually 1 or a few molecules from the thousands to 1
protein
• Ligand – the molecule that a protein can bind
• Binding site – part of the protein that interacts with
the ligand
– Consists of a cavity formed by a specific arrangement of
amino acids

30. Ligand Binding

31. Formation of Binding Site
• The binding site forms when amino acids from within
the protein come together in the folding
• The remaining sequences may play a role in regulating
the protein’s activity

32. Antibody Family
• A family of proteins that can be created to
bind to almost any molecule
• Antibodies (immunoglobulins) are made in
response to a foreign molecule ie. bacteria,
virus, pollen… called the antigen
• Bind together tightly and therefore
inactivates the antigen or marks it for
destruction

33. Antibodies
• Y-shaped molecules with 2 binding sites at the
upper ends of the Y
• The loops of polypeptides on the end of the
binding site are what imparts the recognition
of the antigen
• Changes in the sequence of the loops make
the antibody recognize different antigens -
specificity

34. Antibodies

35. Binding Strength
• Can be measured directly
• Antibodies and antigens are mixing around in a
solution, eventually they will bump into each
other in a way that the antigen sticks to the
antibody, eventually they will separate due to the
motion in the molecules
• This process continues until the equilibrium is
reached – number sticking is constant and
number leaving is constant
• This can be determined for any protein and its
ligand

36. Equilibrium
Constant
• Concentration of antigen, antibody and
antigen/antibody complex at equilibrium can be
measured – equilibrium constant (K)
• Larger the K the tighter the binding or the more non-
covalent bonds that hold the 2 together

37. Enzymes as Catalysts
• Enzymes are proteins that bind to their ligand as
the 1st step in a process
• An enzyme’s ligand is called a substrate
– May be 1 or more molecules
• Output of the reaction is called the product
• Enzymes can repeat these steps many times and
rapidly, called catalysts
• Many different kinds –

38. ENZYME REACTION CATALYZED
Hydrolases general term for enzymes that catalyze a hydrolytic cleavage reaction.
Nucleases break down nucleic acids by hydrolyzing bonds between nucleotides.
Proteases break down proteins by hydrolyzing bonds between amino acids.
Synthases general name used for enzymes that synthesize molecules in anabolic
reactions by condensing two smaller molecules together.
Isomerases catalyze the rearrangement of bonds within a single molecule.
Polymerases catalyze polymerization reactions such as the synthesis
of DNA and RNA.
Kinases catalyze the addition of phosphate groups to molecules. Protein
kinases are an important group of kinases that attach phosphate
groups to proteins.
Phosphatases catalyze the hydrolytic removal of a phosphate group from a molecule.
Oxido-Reductases general name for enzymes that catalyze reactions in which
one molecule is oxidized while the other is reduced. Enzymes of this
type are often calledoxidases, reductases, and dehydrogenases.
ATPases hydrolyze ATP. Many proteins with a wide range of roles have an
energy-harnessing ATPase activity as part of their function, for
example, motor proteins such as myosin and membrane
transport proteins such as the sodium–potassium pump.
Table 3-1Some Common Types of Enzymes

39. Enzymes at Work
• Lysozyme is an important enzyme that protects us from
bacteria by making holes in the bacterial cell wall and
causing it to break
• Lysozyme adds H2O to the glycosidic bond in the cell wall
• Lysozyme holds the polysaccharide in a position that
allows the H2O to break the bond – this is the transition
state – state between substrate and product
• Active site is a special binding site in enzymes where the
chemical reaction takes place

40. Features of Enzyme Catalysis

41. Prosthetic Groups
• Occasionally the sequence of the protein is not
enough for the function of the protein
• Some proteins require a non-protein molecule to
enhance the performance of the protein
– Hemoglobin requires heme (iron containing
compound) to carry the O2
• When a prosthetic group is required by an
enzyme it is called a co-enzyme
– Usually a metal or vitamin
• These groups may be covalently or non-covalently
linked to the protein

42. Retinal and heme. (A) The structure of retinal, the light-sensitive molecule
attached to rhodopsin in the eye. (B) The structure of a heme group. The
carbon-containing heme ring is red and the iron atom at its center
isorange.

43. Regulation of Enzymes
• Regulation of enzymatic
pathways prevent the
deletion of substrate
• Regulation happens at the
level of the enzyme in a
pathway
• Feedback inhibition is
when the end product
regulates the enzyme early
in the pathway

44. Feedback Regulation
• Negative feedback –
pathway is inhibited by
accumulation of final
product
• Positive feedback – a
regulatory molecule
stimulates the activity of
the enzyme, usually
between 2 pathways
–  ADP levels cause the
activation of the glycolysis
pathway to make more ATP

45. Allostery
• Conformational coupling of 2 widely separated
binding sites must be responsible for regulation
– active site recognizes substrate and 2nd site
recognizes the regulatory molecule
• Protein regulated this way undergoes allosteric
transition or a conformational change
• Protein regulated in this manner is an allosteric
protein

46. Allosteric Regulation
• This method of regulation is also used in other
proteins besides enzymes
– Receptors, structural and motor proteins

47. Allosteric Regulation
• Enzyme is only partially active with sugar only but
much more active with sugar and ADP present

48. Phosphorylation
• Some proteins are regulated by the
addition of a PO4 group that allows for the
attraction of + charged side chains causing
a conformation change
• Reversible protein phosphorylations
regulate many eukaryotic cell functions
turning things on and off
• Protein kinases add the PO4 and protein
phosphatase remove them

49. Phosphorylation/Dephosphorylation
• Kinases capable of
putting the PO4 on 3
different amino acid
residues
– Have a –OH group on R
group
• Serine
• Threonine
• Tyrosine
• Phosphatases that
remove the PO4 may be
specific for 1 or 2
reactions or many be
non-specific

50. GTP-Binding Proteins (GTPases)
• GTP does not release its
PO4 group but rather the
guanine part binds tightly
to the protein and the
protein is active
• Hydrolysis of the GTP to
GDP (by the protein itself)
and now the protein is
inactive

51. Molecular Switches

52. Motor Proteins • Proteins can move in the cell,
say up and down a DNA strand
but with very little uniformity
– Adding ligands to change the
conformation is not enough to
regulate this process
• The hydrolysis of ATP can direct
the the movement as well as
make it unidirectional
– The motor proteins that move
things along the actin
filaments or myosin

53. Protein Machines
• Complexes of 10 or more
proteins that work
together such as DNA
replication, RNA or
protein synthesis, trans-
membrane signaling etc.
• Usually driven by ATP or
GTP hydrolysis
• See video clip on CD in
book