Biotechnology Chapter Five Lecture- Proteins (part a)

Introduction toStudying Proteins

Chapter 5

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.

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.

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.

5.1 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.

Protein Molecule Structure

Proteins are polymers composed of amino acids

carboxylic acidgroup

aminogroup

Twenty different kinds of amino

acids are found in proteins

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

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

Protein Molecule Structure

Protein Molecule Structure

Primary protein structure is the linear amino acid sequence including disulfide bridges between cystines.

covalent bonds

Protein Molecule Structure

Secondary protein structure is the folding and twisting that varies depending on the amino acid side chains.

hydrogen bonds

Secondary conformations include the alpha helix and the beta pleated sheet

Protein Molecule Structure

Secondary conformations include the alpha helix and the beta pleated sheet

Protein Molecule Structure

Protein Molecule Structure

Tertiary protein structure is the total 3-D shape.hydrogen bonds, ionic bonds, and

hydrophobic interactions

Protein Molecule Structure

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

Protein Molecule Structure

Quaternary protein structure results from combining more than one polypeptide.

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.

Because proteins are often

modified after transcription and

they have complex 3-D structures,

studying protein is much more

challenging than studying DNA.

GFP protein

GFP protein is used extensively in biotech as a

marker. When attached to a

molecule of interest it glows!

GFP protein

239 aa28,870 Daltons

Dalton = 1 H atomDalton = 1 amuKDa = 1000amu

BIO-RAD’s GFP has 3 mutations which allow for

higher solubility in water.

Proteomics = study of proteins

Proteome = entire

collection of an organism’s

proteins

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

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

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

*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

Heat is often used to ensure

complete denaturing

Separation and identification of proteins using SDS-PAGE

Separation and identification of proteins using SDS-PAGE

protein standards of known mass are included

for comparison

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

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

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?

Questions and Comments?