Objectives for protein section II

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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.

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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

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Ras is a molecular on/off switch
Exchange
GDP
GTP
GDP
raf
Off
On
Hydrolysis
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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

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Ribbon Cartoon of Ras with GTP
switch I
switch II
From C. Branden J. Tooze. 1999. Introduction to
Protein Structure, 2nd Ed.
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Ras conformational changes upon GTP binding
(switch I, switch II)
GDP
GTP
sI
sII
With GTP, more of switch I becomes b sheet.
This b strand forms a b sheet with 2 strands in
raf.
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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).
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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

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Mutations in GTP binding region disrupt
hydrolysis and create oncoproteins
Essential glycines (gly-12) required to position
GTP
Glutamine-61 required for GTP hydrolysis.
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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/cgi-bin/morph
  .cgi?ID55051-2222
• Basic tutorial on protein structure with G
  protein (good for alpha helix beta sheet)
• http//info.bio.cmu.edu/Courses/BiochemMols/ProtG/
  ProtGMain.htm

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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.

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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.

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Ras is also part of G-protein superfamily

• Other G-proteins
• Rho
• Rac
• Rap1
• Ran
• Arf

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Unrooted tree based on AA sequence
Rap1A
H-ras
Rac1
K-ras
RhoA
N-ras, M
N-ras, H
Ran
Arf1
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In Class Problems

• 1. Explain why rap1A is located closer to the ras
  proteins than the other proteins are.
• 2. Explain the grouping of rac1 and rhoA
  together on a branch.
• 3. Explain the grouping of ran and arf1 together
  on a branch.

Rap1A binds to raf and is involved in the same
signaling pathways, so you would expect its
structure to be most similar to ras.
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.
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.
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Green 100 identical
Yellow many identical
Blue similar
s AA based on human N-ras
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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.
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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
Switch II
Rotated 180o
Switch I
Movie available on Blackboard site under Course
Documents