Biotechnology Chapter Five Lecture- Protein Structure and SDS-PAGE

1. Introduction to
Studying Proteins
Chapter 5

2. 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.

3. 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.

4. 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.

5. 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.

6. Protein Molecule Structure
Proteins are polymers composed of
amino acids
amino
group
carboxylic acid
group

7. Twenty
different
kinds of
amino
acids are
found in
proteins

8. Protein Molecule Structure
Most 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

9. Protein Molecule Structure
Most 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

10. Protein Molecule Structure

11. Protein Molecule Structure
Primary protein structure is the linear amino acid
sequence including disulfide bridges between cystines.
covalent bonds

12. Protein Molecule Structure
Secondary protein structure is the folding and twisting
that varies depending on the amino acid side chains.
hydrogen bonds

13. Protein Molecule Structure
Secondary conformations include the
alpha helix and the beta pleated sheet

14. Protein Molecule Structure
Secondary conformations include the
alpha helix and the beta pleated sheet

15. Protein Molecule Structure
Tertiary protein structure is the total 3-D shape.
hydrogen bonds, ionic bonds, and
hydrophobic interactions

16. Protein Molecule Structure
Tertiary protein structure can be predicted from amino
acid sequences or determined by x-ray crystallography.

17. Protein Molecule Structure
Quaternary protein structure results from combining
more than one polypeptide.

18. 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.

19. Because proteins
are often
modified after
transcription and
they have
complex 3-D
structures,
studying protein
is much more
challenging than
studying DNA.

20. GFP
protein

21. GFP protein is used
extensively in
biotech as a
marker.
When attached to a
molecule of
interest it glows!

22. GFP protein
239 aa
28,870 Daltons
Dalton = 1 H atom
Dalton = 1 amu
KDa = 1000amu

23. BIO-RAD’s GFP
has 3 mutations
which allow for
higher solubility in
water.

24. Proteomics =
study of proteins

25. Proteome =
entire
collection of
an organism’s
proteins

26. 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/electrophoreticseparation-of-proteins

27. Separation and identification of proteins
using SDS-PAGE
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)

28. Separation and identification of proteins
using SDS-PAGE
SDS
Protein 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.

29. Separation and identification of proteins
using SDS-PAGE
*DTTbreaks -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

30. Separation and identification of proteins
using SDS-PAGE
Heat is often
used to
ensure
complete
denaturing

31. Separation and identification of proteins
using SDS-PAGE
protein
standards of
known mass
are included
for
comparison

32. 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

33. 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

34. 5.1 Review Questions
1.
2.
How many different kinds of amino acids are
found in proteins? What distinguishes one
amino acid from another?
What causes polypeptide chains to fold into
functional proteins?

35. Questions and
Comments?