BCH 4053 Biochemistry I
Fall 2001
Dr. Michael Blaber


Lecture 9

Chemical groups in proteins, Purification of proteins, Amino acid sequencing


Chemical Groups in Proteins

 

The following is a list of commonly observed prosthetic groups in conjugated proteins

 

Purification of Protein Mixtures

Selective purification of a particular protein from a mixture of different proteins takes advantage of the unique physical and chemical properties of the protein of interest.

What common properties distinguish different proteins?

An important key point about fractionation steps is that you need to be able to know which fraction contains your protein of interest

Before you can purify something, you must have an assay

More detailed information on protein purification and chromatography can be found at:

Protein purification: Assays and Initial Steps in a Purification

Protein purification: Ion exchange, Dialysis and Concentration

Protein purification: Gel Filtration, Hydrophobic Interaction Chromatography, Affinity Chromatography, Plumbing

Protein purification: Running the experiment

 

Determination of the amino acid sequence of a protein

 

Proteins are chemically well-defined. All the molecules of a given sample of pure protein have an identical primary sequence.

The analytical chemistry for determination of the sequence of amino acids in a polypeptide has been worked out, and to a large degree, automated.

It should be noted that proteins can also be sequenced by sequencing the DNA that codes for them (and subsequently translating the DNA sequence into protein sequence). Many proteins are actually sequenced in this way. But direct protein sequencing is still important in many cases.

 

Peptide Mapping

How can sequence information for the entire polypeptide be obtained?

How do we order the different sequences that we obtain?

Name

Source

Specificity

Chymotrypsin

Bovine Pancreas

Cleavage after Tyr, Phe and Trp; some cleavage after Leu, Met and Ala

Bromelain

Pineapple

Cleavage after Lys, Ala and Tyr

Trypsin

Bovine Pancreas

Cleavage after Arg, less after Lys

V8 protease

Staphylococcus aureus

Cleavage after Glu, less after Asp

 

Overlapping sequence information can allow you to align the peptides in the correct order and determine the sequence of the original large polypeptide (i.e. protein).

 

A single polypeptide will give an unambiguous amino terminal sequence:

Cycle

1

2

3

4

Amino acid

Alanine

Phenylalanine

Asparagine

Lysine

However, a disulfide-linked pair of polypeptides will give an ambiguous sequence:

Cycle

1

2

3

4

Amino acid(s)

Alanine, Asparagine

Proline, Phenylalanine

Aspartic acid, Asparagine

Lysine, Methionine

 

One of the first steps in protein sequencing is to therefore reduce any disulfide bonds and to separate individual polypeptide chains.


Other considerations prior to amino or carboxyl terminal sequencing


For more information see the following:

Protein Sequencing, Peptide Mapping, Synthetic Genes

 

Sequence Determination by Mass Spectrometry

Mass spectrometry is a method that separates and quantitates molecules based upon their mass to charge ratio (m/z). It is so accurate that it can assign a mass to a molecule to within 1 Da of accuracy. Therefore, the composition of atoms within the molecule can be accurately identified.

 

The Nature of Amino Acid Sequences

When scientists first began sequencing proteins there were many unanswered questions regarding proteins and the amino acids.

 

With regard to amino acids in proteins, it was found that while each amino acid can be found in proteins, some (e.g. alanine) are present in larger amounts, and some are relatively infrequent (e.g. Tryptophan):

Amino Acid

Frequency of Occurrence in Proteins (%)

Ala

9.0

Arg

4.7

Asn

4.4

Asp

5.5

Cys

2.8

Gln

3.9

Glu

6.2

Gly

7.5

His

2.1

Ile

4.6

Leu

7.5

Lys

7.0

Met

1.7

Phe

3.5

Pro

4.6

Ser

7.1

Thr

6.0

Trp

1.1

Tyr

3.5

Val

6.9

 

One surprises for scientists who studied homologous proteins between different species, involved the comparison of human with other great apes, in particular, the chimpanzee.

Protein sequence analysis provided a way to determine the "similarity" of species on a molecular level.

Another side of sequence similarity is the following: Proteins with similar functionalities often have similar tertiary structures, and therefore, similar amino acid sequences

 

Yet another surprise was related to the utility with which nature can produce a variety of functional proteins using a relatively small "toolbox" of tertiary structures.


© 2001 Dr. Michael Blaber