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Cell and Molecular Biology
Aug 22, 2009
Protein Structure and Function
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Assignment reading: chapter 3 and journal paper
'Glowing' jellyfish grabs Nobel
Jellyfish will glow under blue and ultraviolet light because of a
protein in their tissues. Scientists refer to it as green fluorescent
protein, or GFP.
Fluorescence protein is
part of the gene---cells
constantly emits green
fluorescence
Fluorescence protein has been
bounded to protein inside the cells--fade within weeks in the absence of
antifading mounting media
Brainbow
Glowing mouse
http://news.bbc.co.uk/2/hi/science/nature/7658945.stm
The monomer unit of protein: amino acid
Panel 3-1 Cell and Molecular Biology, 4th edition
Formation of peptide bond
Proteins form by condensation of
amino acids in a reaction that
releases water, is coupled to ATP
hydrolysis and is catalyzed by
enzymes within the ribosome.
Amino acids are commonly joined
together by an amide linkage
(peptide bond)
Figure 3-1. Cell and Molecular Biology, 4th edition
Figure 3-4 Cell and Molecular Biology, 4th
edition
Figure 3-2. Cell and Molecular
Biology, 4th edition
A protein consists of a polypeptide backbone with attached side chains. Protein
differs in its sequence and number of amino acids. The two ends of a
polypeptide are the amino terminus (N-terminus) and the carboxyl terminus (Cterminus). The amino acid sequence of a protein is always presented in the Nto-C direction, reading from left to right.
There are 20 amino acids
It is useful to remember their most important structural features.
At a minimum, memorize their names and one letter codes.
Know which one is acidic, basic, hydrophobic, polar, big, small,
reactive, inert
There are a few modifications done to amino acids as or immediately
after the protein is made:
e.g. phosphorylation, acetylation, acylation, glycosylation
Essential amino acids
Figure 3-3. Cell and Molecular Biology, 4th edition
The side chains
Panel 3-1 Cell and Molecular Biology, 4th edition
Panel 3-1 Cell and Molecular Biology, 4th edition
Peptide sequence
Figure 3-2. Cell and Molecular Biology, 4th edition
Non-covalent bonds in protein folding
Figure 3-5. Cell and Molecular Biology, 4th edition
Three types of noncovalent bonds that help proteins fold. Although a
single one of these bonds is quite weak, many of them often form
together to create a strong bonding arrangement. The final folded
structure, or conformation, adopted by any polypeptide chains is the
one with the lowest free energy.
Formation of hydrogen bonds
Figure 3-7. Cell and Molecular Biology, 4th edition
Large numbers of hydrogen
bonds form between adjacent
regions of the folded
polypeptide chain and help
stabilize its three-dimensional
shape. The protein depicted is a
portion of the enzyme
lysozyme, and the hydrogen
bonds between the three
possible pairs of partners have
been differently colored, as
indicated.
Proteins have hydrophobic cores
Figure 3-6. Cell and Molecular Biology, 4th edition
The polar amino acid side chains tend to gather on the outside of the
protein, where they can interact with water; the nonpolar amino acid
side chains are buried on the inside to form a tightly packed
hydrophobic core of atoms that are hidden from water. In this
schematic drawing, the protein contains only about 30 amino acids.
Changes in protein conformation
Figure 3-8. Cell and Molecular Biology,
4th edition
A protein can be unfolded, or denatured, by treatment with certain
solvents to disrupt the non-covalent bonds or heat (heat denaturation)
and cold (< 20C for certain antibodies)
Some proteins, often small ones, reach their proper folded state spontaneously.
Once unfolded, kT allows them to find their equilibrium structure when
returned to physiological conditions. Other proteins are metastable: they are
helped to fold to structures they would practically never find at random. Protein
folding in a living cell is often assisted by special proteins call molecular
chaperones.
Only a very small fraction of random sequences of amino acids make
polymers with a unique or stable structure. Nature has selected those
sequences with specific folded shapes. The shapes and therefore
functions can be very fragile to even tiny changes in atomic structure
(mutation). A single protein can have separate sections each with its own
folded domain, and linked by spacers.
Figure 19-53. Cell and Molecular Biology, 4th edition
http://www.ks.uiuc.edu/Research/fibronectin/
Disulfide bonds stabilize protein structure
Figure 3-29. Cell and Molecular
Biology, 4th edition
Figure 3-42. Cell and Molecular
Biology, 4th edition
Disulfide bond covalently link polypeptide chains together, providing
a major stabilizing effect on a protein.
Different levels of protein structure
Non-covalent bonding stabilizes protein folding, which
can be categorized by 4 different levels of structure.
Primary structure: linear amino acid sequence
Secondary structure: regular orientation due to H-bond -helix,
-pleated sheet
Tertiary structure: full 3-D organization of a polypeptide chain
Quaternary structure: multi-subunit complex consisting of
multiple polypeptide chains
Large proteins (~ 50-2000 amino acid long) come in a wide
variety of shapes, generally consisting of several distinct
protein domains—structural units that fold more or less
independently of each other.
Secondary structure: alpha helix
Figure 3-9. Cell and Molecular Biology, 4th edition
The regular conformation of the polypeptide backbone observed in the α
helix and the β sheet. (A, B, and C) The α helix. The N–H of every
peptide bond is hydrogen-bonded to the C=O of a neighboring peptide
bond located four peptide bonds away in the same chain.
Alpha helices can form a stable coiled-coil structure
http://www.bio.miami.edu/~cmallery/255/255prot/
gk2x37.coil.gif
Figure 3-11. Cell and Molecular Biology, 4th edition
Two (or three ) α-helices can wrap around each other to form a stable
coiled-coil structure. By doing so, both helices have most of their
nonpolar (hydrophobic) side chains lined up on one side so that
they can twist around each other with these side chains facing inward.
This long coiled-coil structural framework for many elongated
proteins such as -keratin, myosin, and collagen.
Secondary structure: beta sheet
Figure 3-9. Cell and Molecular Biology,
4th edition
(D, E, and F) The β sheet. In this example, adjacent peptide chains
run in opposite (antiparallel) directions. The individual polypeptide
chains (strands) in a β sheet are held together by hydrogen-bonding
between peptide bonds in different strands, and the amino acid side
chains in each strand alternately project above and below the plane
of the sheet
Beta sheets can have parallel or
antiparallel strands
Figure 3-10. Cell and Molecular Biology, 4th edition
Protein domains, e.g. Src protein
Figure 3-12. Cell and Molecular Biology, 4th edition
A single polypeptide can have
more than one independently
folded domain. This feature is
exploited in nature and
biotechnology to make
chimeric proteins.
It’s not yet possible to predict
the folded shape from amino
acid sequences. Good
predictive rule for 2nd structure
but not for the higher level
structures.
Proteins can be classified into many families
The present-day proteins can be grouped into protein families. Each family
member has amino acid sequences and 3-D conformation resembles those
of the other family members while performing different functions.
Family of serine proteases
(digestive enzymes)
Figure 3-12. Cell and Molecular
Biology, 4th edition
The two conformations are strikingly similar as well as their amino acid
sequences (green) and the location of active sites (red). However, these
active sites have different enzymatic activities to cleave different
peptide bonds. The difference is due to the genetic modification (copies
of genes) during evolution.
Function within a structure: protein modules
Figure 3-20. Cell and Molecular Biology, 4th edition
Figure 3-19. Cell and Molecular Biology, 4th edition
Protein module (~ 40-200 amino
acids) is a subset of protein domain
that has a versatile structure as
found in a variety of different
contexts in different molecules.
Protein modules provide a convenient
framework for the generation of
extended structure. The in-line
arrangement with N- and C- terminal in
opposite ends can readily link in series
to form extended structure either with
themselves or with other molecules.
Large protein molecules contain more than
one polypeptide chain
Any region of a protein’s surface that can interact with another
molecule through sets of non-covalent bonds is called a
binding site.
The tight binding of two folded polypeptide chains at this site
creates a larger protein molecule which a precisely defined
geometry. Each polypeptide chain in such a protein is called a
protein subunit.
Two identical subunits bind
head-to-head, held together by
a combination of hydrophobic
forces (blue) and a set of
hydrogen bonds (yellow region).
Figure 3-21. Cell and Molecular Biology, 4th edition
A long chain of identical
protein molecules can be
constructed if each
molecule has a binding site
complementary to another
region of the surface of the
same molecule. Therefore,
protein molecules can
assemble to form filaments
that may span the entire
length of a cell.
Figure 3-25. Cell and Molecular Biology, 4th edition
Hierarchy in protein assembly
Final shape (3 most common conformations)
www.bio.miami.edu/~cmallery/255/255prot/255proteins.htm
Globular
Helix
Figure 3-24
Figure 3-26
Fiber
Figure 3-28
Protein: classified by functions
Enzymes  catalytic activity and function (-ase)
Structural  collagen of tendons and cartilage, keratin
of hair and nails
Transport proteins  bind and carry ligand
Motor proteins  can contract and change the shape of
cytoskeleton
Defensive  antibodies, thrombin
Regulatory  growth factors, hormones, transcription factors
Receptor  cell surface receptors
Protein Function: How shape determins function?
The specific binding of protein molecules determines their activity
and function--- 3-D shape/conformation matters. Binding always
shows great specificity.
Figure 3-37. Cell and Molecular Biology, 4th edition
Many weak bonds are needed to enable a protein to bind tightly to a
second molecule, which is called a ligand for the protein. A ligand
must therefore fit precisely into a protein's binding site, like a hand
into a glove, so that a large number of noncovalent bonds can be
formed between the protein and the ligand.
Protein conformation determines its chemistry.
• arrangement of neighboring parts of the polypeptide chain to
exclude water molecule
• the clustering of neighboring polar amino acid side chains can
alter their reactivity
Figure 3-38. Cell and Molecular
Biology, 4th edition
Figure 3-39. Cell and Molecular
Biology, 4th edition
Enzymes
• Enzymes are very important class of proteins that determine all the
chemical transformations that make and break covalent bonds in cells
• Enzymes bind to one or more ligands, called substrates, and convert
substrates to products.
• Enzymes may speed up reaction without themselves being changed--catalysts.
Enzymes accelerate chemical reactions by decreasing the activation energy.
Figure 3-46. Cell and Molecular Biology, 4th edition
Allosteric enzymes: feedback mechanism
Many enzyme has at least two different binding sites on their
surfaces:
active site--- recognizes the substrate
regulatory site--- recognizes regulartory molecule
Interaction depends on a conformational change in the protein:
binding at one of the sites causes a shift from one folded shape to
a slightly different folded shape.
positive regulation
Figure 3-57. Cell and Molecular Biology, 4th edition
negative regulation
Figure 3-58. Cell and Molecular Biology, 4th edition
Many protein functions are driven by phosphorylation
Phosphorylation regulates thousands of protein functions in a typical
eukaryotic cells. Phosphorylation occus by the addition of a phosphate
group to amino acid side chains, usually the OH- terminal of serine,
threonine and tyrosine. This cause a major conformational change in
protein due to charge interaction or the attached phosphate group
becomes part of the structure.
Figure 3-63. Cell and Molecular Biology, 4th edition
Protein kinases: catalyze phosphorylation (addition of phosphate)
Proten phosphatases: catalyze dephosphorylation (removal of phosphate)
Individual protein kinases serve as
microchips.
Cyclin-dependent protein kinase
(Cdk) regulates the cell cycle.
Figure 3-66. Cell and Molecular Biology, 4th edition
Cdk becomes active when:
1. Cyclin is present
2. Pi added to specific threonine
side chain
3. Pi removed from tyrosine side
chain
When all 3 requirements are met,
Cdk is turned on.
GTP binding proteins as molecular switches
The activity of a GTP-binding protein (also called a GTPase) generally
requires the presence of a tightly bound GTP molecule (switch “on”).
Hydrolysis of this GTP molecule produces GDP and inorganic phosphate
(Pi), and it causes the protein to convert to a different, usually inactive,
conformation (switch “off”). As shown here, resetting the switch
requires the tightly bound GDP to dissociate, a slow step that is greatly
accelerated by specific signals; once the GDP has dissociated, a molecule
of GTP is quickly rebound.
Figure 3-70. Cell and Molecular Biology, 4th edition
Phosphorylation in cell signaling
Many signaling pathways important for the cell survival involve GTPbinding proteins (GTPases). The phosphate group is part of GTP that
binds very tightly to the protein it regulates. When the tightly bound
GTP is hydrolyzed to GDP, this domain undergoes a conformational
change that inactivate it.
GTP = molecular switch
Ras protein
important role in cell
signaling
Figure 3-72. Cell and Molecular Biology, 4th edition
The large conformational change in GTP-bound domain
EF-Tu
Figure 3-74. Cell and Molecular Biology,
Ras protein
4th
edition
Figure 3-71. Cell and Molecular
Biology, 4th edition
The addition and dissociation of phosphate group causes a shift of a few
tenths of a nanometer at the GTP binding site. This tiny movement causes a
large conformational change to propagate along the switch helix. The helix
serves as a latch that adheres to a specific site in another domain of the
molecule. After GTP hydrolysis, the switch helix detaches, allowing the two
domains to swing apart over a distance of ~ 4 nm.