Transcript Introduction to Studying Proteins

Introduction to Studying
Proteins
Chapter 5
Objectives
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Describe the main roles that proteins play in
organisms.
Describe the process by which proteins are
assembled.
Describe how amino acids differ from one
another.
Describe the process by which an enzyme
functions and list factors that influence their
ability to work properly.
Describe the process of SDS-PAGE.
Why study proteins?
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Proteins in organisms can
– Serve as structural components in cells
 i.e. collagen
– Act as enzymes
 Carry out essential chemical reactions
 i.e. catalase
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Enzymes are of extreme interest due to
application in medicine and potential
commercial use.
Protein Structure
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Protein structure is critical
in determining what the
protein can do.
Proteins are a type of
macromolecule that are
assembled from subunits
called amino acids.
– Usually tens or hundreds of
amino acids chained
together.
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All organisms build their
proteins from a pool of 20
amino acids.
Protein Structure
All amino acids have
the same basic
structure.
 What separates one
amino acid from
another?
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– “R group”
 Charged (+/-)
 Polar
 Uncharged
tyrosine (tyr)
lysine (lys)
glutamate (glu)
glycine (gly)
valine (val)
phenylalanine (phe)
methionine (met)
proline (pro)
Protein Structure
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How is one
amino acid
connected to
another to form
a polypeptide
chain?
– Condensation
reaction
Protein Structure
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Ways protein structure
can be represented:
– Primary structure
 Linear sequence of amino
acids.
 gly-ala-val-pro
– Secondary structure
 Three dimensional
structure of either alpha
helix or beta sheet.
Protein Structure
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Ways protein structure
can be represented:
– Tertiary structure
 Final three dimensional
shape a polypeptide takes.
– Quaternary structure
 Proteins with more than one
polypeptide chain.
– i.e. collagen or hemoglobin
Protein Synthesis
Protein production is controlled by a
variety of organelles in a cell.
 Cells are very precise in determining when
and how much of a protein is needed.
 In medicine and industry biotechnologist
have found ways to manipulate cells into
making large numbers of specific proteins.
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Protein Synthesis
The instructions for how to make a protein
reside in the cells DNA, found in the nucleus.
 The machinery for making the protein,
ribosomes, are found outside the nucleus.
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Protein Synthesis
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Getting the
instructions from the
DNA to the ribosomes
involves transcription.
– Reminder that bases
used for producing
RNA are A, U, C, G.
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The mRNA produced
leaves the nucleus.
Protein Synthesis
The actual protein
product is produced
during translation.
 Ribosomes use mRNA
and tRNA molecules
to assemble the a.a’s
in the correct
sequence.
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Protein Synthesis
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The order of the amino
acids is determined by
the three base sequences
of the mRNA.
– Codons
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tRNA molecules that
carry the amino acids
have complementary
anti-codons
Protein Synthesis
Protein Synthesis
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Once a polypeptide chain forms it can be
further modified (posttranslational
modifications).
– Addition of important chemical groups.
– Phosphorylation
– Cleavage
Enzymes
Enzymes represent one important type or
group of proteins.
 Cells carry out millions of chemical
reactions every second.
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– Without enzymes they would not occur fast
enough to meet the needs of the cells.
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The amount and type of enzyme produced
are tightly controlled by the cell.
Enzymes
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How do enzymes work?
– When an enzyme speeds up a reaction, the participating reactants
are called the enzyme’s substrates.
– Enzymes help speed up reactions that otherwise would occur on
there own.
– The end result of the reactions (degradation or synthesis) is the
formation of product(s).
– Enzymes themselves are NOT used up in the reaction.
– Some enzymes require cofactors.
Enzymes
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Factors that influence
how enzymes work.
– Temperature
– pH
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Swings outside
specified ranges can
result in denaturation
of the enzyme.
Enzymes
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Enzymes are divided in six categories based on their
function.
– Hydrolases…..break chemical bond
– Lyases…… break chemical bond
– Transferases….transfer of a functional group from one molec to
another
– Isomerases….. catalyses interconversion of isomers
– Oxidoreductases…. catalyzes transfer of electrons from one
molecule to another
– Synthetases….making new molec
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Note that enzymes are normally denoted by having the
suffix “-ase” added to their name.
– I.e. sucrase
Studying Proteins
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Companies that produce protein products
or study proteins must be able to:
– Separate the protein of interest.
– Determine that amount of protein present.
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Characteristics of proteins that make it
possible to achieve either one of both
points above:
– Overall charge, size, shape, and solubility
Studying Proteins
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Spotlight: SDS-PAGE
– Gel electrophoresis allows for the separation
of proteins based on charge, size, and shape.
– Polyacrylamide gel electrophoresis is utilized
(PAGE).
 Allows for better resolution
 4-18% gels most commonly used
– Higher concentration for smaller proteins
 When protein size unknown gradient gels can be
used.
– Less concentrated at the top than the bottom.
Studying Proteins
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Spotlight: SDS-PAGE
– Use of sodium dodecyl
sulfate (SDS)
 Denatures proteins into
polypeptide strands
 Gives each polypeptide
strand an overall negative
charge
 Proteins studied are
strictly being separated by
size.
Studying Proteins
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Spotlight: SDS-PAGE
– Visualization of the
proteins in the gel.
 Coomassie Blue
– Milligram amounts of
protein.
 Silver stain
– Microgram amounts of
protein.
– Size of unknown bands
can be determined from
comparison to the protein
molecular weight
standard.
Homework 5
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Review 5.1
– Questions 1, 2, 3
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Review 5.2
– Questions 1, 2, 3
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Review 5.3
– Questions 1, 2
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Review 5.4
– Questions 1, 2
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Think like Biotech
– Questions: 2, 3, 5, 6, 7
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