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Objectives for protein section II
• Explain conformational changes in proteins.
• Explain how GTP binding & hydrolysis allows ras
& other G-proteins to function as molecular on/off
switches.
• Discuss the relationship of ras normal function, ras
mutants, & ras’ role in cancer.
• Predict how amino acid substitutions in a protein
will affect its structure and function.
• Define protein families and discuss them in terms
of gene duplication, evolution, and functional
constraints.
• Compare and contrast protein domains and protein
motifs.
Ras protein & the relationship of form
& function
•
•
•
•
•
Protein = single polypeptide
189 amino acids
21 kD (kiloDalton)
Relays signals in cytoplasm
“Small” G-protein = Guanine nucleotide
binding protein
• Involved in cancer as a protooncogene
Ras is a molecular on/off switch
GDP
GTP
Exchange
SOS
GDP
GTP
Off
On
Hydrolysis
raf
on
Pi
mitosis
Ras undergoes a conformational
change when bound to GTP
Conformational change = a predictable change
in protein structure that is associated with
biological activity.
Conformational change could be due to the
binding of another protein, a certain
nucleotide, the addition of a phosphate, etc…
Ribbon Cartoon of Ras with GTP
Guanine
Base
Ribose sugar
3 phosphates
switch II
switch I
g -phosphate
can be
hydrolyzed
From C. Branden & J. Tooze. 1999. Introduction to Protein Structure, 2nd Ed.
Ras conformational changes upon
GTP binding (switch I, switch II)
GDP
GTP
sII
sI
With GTP, more of switch I becomes b sheet.
This b strand forms a b sheet with 2 strands in raf.
The preceding function of ras is required
for the proper function of all NORMAL
mammalian cells. Ras only participates in
the development of cancer when the gene
for ras is mutated (an oncogene) and the
resulting ras protein functions incorrectly
(an oncoprotein).
Mutated ras is involved in cancer
• 25-30% of all human cancers contain
mutated ras
• mutant gene causing cancer = oncogene
• resulting mutant protein causing cancer =
oncoprotein
• Oncogenic mutations in ras result in
SINGLE AMINO ACID CHANGES
• ras leads to cancer when it is overactive
Mutations in GTP binding region disrupt
hydrolysis and create oncoproteins
Essential glycines
(gly-12) required to
position GTP
Glutamine-61 required for
GTP hydrolysis.
Links to resources and tutorials on ras and
protein structures
• Chime tutorial on ras structure and oncogenic
mutations
– http://webhost.bridgew.edu/fgorga/ras/default.htm
• A cartoon movie of ras conformational changes
– http://bioinfo.mbb.yale.edu/MolMovDB/cgibin/morph.cgi?ID=55051-2222
• Basic tutorial on protein structure with G protein
(good for alpha helix & beta sheet)
– http://info.bio.cmu.edu/Courses/BiochemMols/ProtG/
ProtGMain.htm
Ras is part of a protein family
• Protein family = similar, but not identical
proteins within an organism.
• Proteins within a family have similar AA
sequences, and therefore, similar (but generally
not identical) structure and function.
• Protein families are created by duplication of an
ancestral gene.
• The genes then diverge over time, potentially
developing distinct, specific functions.
Mammals have three different Ras
genes and proteins
• H-Ras – most abundant in skin.
• K-Ras – most abundant in gut and thymus.
• N-Ras – most abundant in testis and thymus.
Ras is also part of G-protein
superfamily
• Other G-proteins
–
–
–
–
–
Rho
Rac
Rap1
Ran
Arf
Unrooted tree based on AA sequence
Rap1A
H-ras
Rac1
K-ras
RhoA
N-ras, M
N-ras, H
Ran
Arf1
In Class Problems
1. Explain why rap1A is located closer to the ras
proteins than the other proteins are.
Rap1A binds to raf and is involved in the same
signaling pathways, so you would expect its structure
to be most similar to ras.
2. Explain the grouping of rac1 and rhoA together
on a branch.
Rac1 and rhoA are both involved in signaling via cadherins
and the actin cytoskeleton. They share the most functions,
so they are the most similar to each other.
3. Explain the grouping of ran and arf1 together on a
branch. Ran and arf1 share somewhat similar
functions in that they are both involved in transport within
the cell, so they are similar. However, their functions are
very distinct, & so are their sequences.
10
30
20
40
Green = 100%
identical
50
60
70
80
100
90
Yellow =
many identical
Blue = similar
110
120
130
140
150
#s = AA # based
on human N-ras
160
170
180
In Class Problem
• Refer to the sequence alignment in the preceding
figure. Which nine amino acids are identical
between all the proteins shown? Write their
number and single letter code. Predict the area
where these nine amino acids would be located in
the tertiary structure of ras.
gly-10, gly-15, lys-16, thr-35, asp-57, gly-60, lys-117,
asp-119, ala-146
The one function that all of these proteins share is
the binding of GDP/GTP; therefore, you would
predict that the most commonly shared AAs would be
those involved in binding the nucleotide.
The 9 completely conserved AAs (green) in our
G protein examples are located precisely
around the nucleotide (blue) and most bond
with it.
C
N
Rotated ~180o
Switch II
Switch I
Movie available on Blackboard site under Course Documents