南京大学-杨荣武生物化学-英文版-第一篇-ProteinIV

Denaturation
Disruption of the normal 3D structure of a protein, such that it loses biological activity. Usually caused by heat, Chemicals or changes in pH. Usually irreversible. A cooked egg cannot be “uncooked.”
5. What is the structure of protein X? Methods for analyzing proteins
– – – – – – – Amino acid analysis Proteolytic digestion Peptide fingerprinting Peptide sequencing Multiple sequence alignment Mass spectrometry X-ray crystallography (Currently the best way to study protein structure) – Nuclear Magnetic Resonance
High
If you combine SDS-PAGE and isoelectric focusing, you get 2-dimensional gel electrophoresis (2D gel)
3. Is protein X found in other cell?
Method: Western blotting
1. How big is protein X?
Method: electrophoresis Separates protein molecules based on their sizes
Electrophoresis
Procedure: ① Incubate protein X with SDS so that it becomes negatively charged ② Load onto gel ③ Apply voltage to the gel ④ Stain gel with a protein dye (e.g. Coomasie Blue) This particular kind of electrophoresis is also called SDS-PAGE (Polyacrylamide Gel Electrophoresis)
Internal hydrolysis
– Trypsin - cleaves on the C-side of K or R; – Chymotrypsin - C-side of F, Y, W
Experimenting with proteins…
Let's say you discover a new protein: protein X You might ask the following questions: 1. How big is the protein? 2. What is its isoeletric point? 3. Is it present in other cells / organisms? 4. What is its sequence? 5. What is its structure?
PITC
PITC
G I
Identification of the amino acid at position 1 using HPLC
PITC
G
I
I
I
V E Q C C T S PITC-induced cleavage of the amino-terminal peptide bond
I
Identification of the amino acid at position 2 using HPLC
Protein sequencing is difficult and time-consuming DNA sequencing is much faster The genome projects will not be possible if we have to sequence protein one by one!
Cells contain many different protein, how does one identify a particular protein-of-interest from a gel?
With the help of antibodies!
Antibodies are defense proteins made by immune cells to recognize foreign proteins in the body
Properties of Proteins and their Research Methods
Some Properties of Proteins
pI value and solubility Denature and Re-nature Hydrolysis Other properties
Step 1: Make crystals of your favorite protein (to make sure all individual molecules are precisely oriented)
Crystals of myoglobin
X-ray Crystallography is used to determine the structure of proteins
Amino acids can be separated by HPLC
Analysis of Proteins: Peptide sequencing
G I V E Q C C T S I Label with phenyl isothiocyanate (PITC) V E Q C C T S I PITC-induced cleavage of the amino-terminal peptide bond V E Q C C T S Label with PITC
Remember proteins have different charges at different pH?
Procedure: ① Make a gel with pH gradient ② Load protein ③ Find the pH where the protein stops moving
PITC
I
V E Q C C Twenku.baidu.comS
I
Separation of peptides: Peptide fingerprinting
Proteolytic digests of proteins can be separated into individual peptides using 2-dimensional techniques. In the above example, the peptide mixture is spotted onto filter paper and initially separated by charge (electrophoresis) in one direction. The filter paper is then subjected to paper chromatography in the perpendicular direction (separating the peptides by size). Each spot on the filter paper in panel C represents a unique peptide.
Hemoglobin S can be separated from hemoglobin A by peptide fingerprinting
In the panels at the left, the peptide fingerprints are shown for proteolytic digests of hemoglobins A and S. Notice that only one spot has shifted positions in the fingerprint. This spot corresponds to the peptide that contains the valine at position 6 in hemoglobin S instead of a glutamic acid. As a result of this change, the peptide migrates differently in the fingerprint. Peptide fingerprinting was used to first identify the amino acid that is altered in hemoglobin S.
Enzymatic Hydrolysis
N -terminal hydrolysis C-terminal hydrolysis
– Carboxypeptidase A cleaves all except for P, R, K; – Carboxypeptidase B cleaves only R, and K; – Carboxypeptidase Y cleaves all; – Carboxypeptidase C cleaves all;
Electrophoresis
Molecular Weight Markers
2. What is protein X's isoelectric point?
Method: isoelectric focusing Separates protein molecules based on their isoelectric point
Amino acid analysis gives the composition of proteins
Acid hydrolysis results in cleavage of the peptide bonds in a protein. Acid hydrolysis of the insulin A chain is shown. The relative numbers of each amino acid in the hydrolyzed protein can be quantified by HPLC.
Protein solution Ammonium sulfate solution
In the hanging drop method of protein crystallization, a concentrated protein solution is spotted on a coverslip and placed above an ammonium sulfate solution. The ammonium sulfate solution absorbs water from the surrounding air, dehydrating the protein solution. As a result, the concentration of the protein solution gradually increases, resulting in crystal formation.
How are antibodies against the protein-ofinterest obtained?
This is what a real Western blot looks like
4. What is the sequence of protein X?
Method: Protein sequencing