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Introduction toStudying Proteins
Chapter 5
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Learning Outcomes
✤Describe the structure of proteins, including the significance of amino acid R-groups and their impact on the three-dimensional structure of proteins.
✤Explain the steps of transcription and translation in protein synthesis.
✤Discuss the role of naturally occurring proteins and recombinant proteins in biotechnology.
✤Differentiate proteins that function as part of structure, as antibodies, and as enzymes.
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Learning Outcomes
✤Describe the structure of antibodies and explain the relationship between antibodies and antigens.
✤Discriminate among the classes of enzymes and discuss the effect of reaction conditions on enzyme activity.
✤Summarize polyacrylamide gel electrophoresis and identify its usefulness for studying proteins.
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5.1 The Structure and Function of Proteins
Virtually all biotechnology products have something to do with proteins.
Protein function is determined by the three-dimensional structure(shape) of the protein.
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5.1 The Structure and Function of Proteins
Virtually all biotechnology products have something to do with proteins.
Protein function is determined by the three-dimensional structure(shape) of the protein.
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Protein Molecule Structure
Proteins are polymers composed of amino acids
carboxylic acidgroup
aminogroup
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Twenty different kinds of amino
acids are found in proteins
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Protein Molecule StructureMost proteins contain tens of hundreds of
amino acids chained together by peptide bonds.
Molar mass and the specific amino acid sequence can be determined by mass
spectrometry
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Protein Molecule StructureMost proteins contain tens of hundreds of
amino acids chained together by peptide bonds.
Molar mass and the specific amino acid sequence can be determined by mass
spectrometry
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Protein Molecule Structure
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Protein Molecule Structure
Primary protein structure is the linear amino acid sequence including disulfide bridges between cystines.
covalent bonds
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Protein Molecule Structure
Secondary protein structure is the folding and twisting that varies depending on the amino acid side chains.
hydrogen bonds
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Secondary conformations include the alpha helix and the beta pleated sheet
Protein Molecule Structure
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Secondary conformations include the alpha helix and the beta pleated sheet
Protein Molecule Structure
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Protein Molecule Structure
Tertiary protein structure is the total 3-D shape.hydrogen bonds, ionic bonds, and
hydrophobic interactions
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Protein Molecule Structure
Tertiary protein structure can be predicted from amino acid sequences or determined by x-ray crystallography.
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Protein Molecule Structure
Quaternary protein structure results from combining more than one polypeptide.
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Function of Structural Proteins
STRUCTURE = FUNCTION
Viral recognition proteins
Glycoprotein 120 on the surface of HIV, must
exactly match its human cell membrane receptors to recognize, attach, and
infect a T-helper cell.
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Because proteins are often
modified after transcription and
they have complex 3-D structures,
studying protein is much more
challenging than studying DNA.
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GFP protein
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GFP protein is used extensively in biotech as a
marker. When attached to a
molecule of interest it glows!
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GFP protein
239 aa28,870 Daltons
Dalton = 1 H atomDalton = 1 amuKDa = 1000amu
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BIO-RAD’s GFP has 3 mutations which allow for
higher solubility in water.
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Proteomics = study of proteins
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Proteome = entire
collection of an organism’s
proteins
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Separation and identification of proteins using SDS-PAGE
Sodium DodecylSulfate = detergent used to denature protein and equalize amino acid
charges
PolyAcrylamide Gel Electrophoresis separates proteins by size and sometimes
shape.
http://www.jove.com/video/758/electrophoretic-separation-of-proteins
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Samples are mixed in Laemmli sample buffer containing
Tris buffer- keeps pH 6.8 for electrophoresis
Glycerol- weighs sample down for loading
Bromophenol blue- colors sample
*SDS*dithiothreitol (DTT)
Separation and identification of proteins using SDS-PAGE
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SDSProtein charges vary depending on the specific amino acid sequence. SDS equally coats all proteins in negative charge so that migration is only based on protein size.
Separation and identification of proteins using SDS-PAGE
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*DTT-
breaks -S-S- bridges creating a linear polypeptide so migration is based only on size.
can be left out to provide info on disulfide bond locations
Separation and identification of proteins using SDS-PAGE
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Heat is often used to ensure
complete denaturing
Separation and identification of proteins using SDS-PAGE
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Separation and identification of proteins using SDS-PAGE
protein standards of known mass are included
for comparison
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Vocabulary
• X-ray crystallography – a technique used to determine the three-dimensional structure of a protein
• Polar – the chemical characteristic of containing both a positive and negative charge on opposite sides of a molecule
• Primary structure – the order and type of amino acids found in a polypeptide chain
• Secondary structure – the structure of a protein (alpha helix and beta sheets) that results from hydrogen bonding
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Vocabulary
• Tertiary structure – the structure of a protein that results from several interactions, the presence of charged or uncharged “R” groups, and hydrogen bonding
•Quaternary structure – the structure of a protein resulting from the association of two or more polypeptide chains
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5.1 Review Questions
1. How many different kinds of amino acids are found in proteins? What distinguishes one
amino acid from another?
2. What causes polypeptide chains to fold into functional proteins?
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Questions and Comments?