Protein Sequencing Process

The protein sequencing process describes the steps that need to be taken in order to determine the amino acid sequence of a protein.

However, in order to begin, you must take some preliminary steps to ensure that your protein is ready to be sequenced:

  1. You must have a purified protein

After you confirmed or purified your protein, you can then move onto the actual sequencing process.

Step 1: Separate protein subunits

What this means: A protein can consist of 2 or more polypeptide chains that are linked together by disulfide bonds (see graphic below). For sequencing to properly work, you must break all protein subunits into individual strands.

N-terminal analysis of your protein provides information on the different types of subunits.

Since each polypeptide chain has an N-terminus (also known as the “end group” of a protein) it is possible to use a fluorescent compound such as 5-dimethylamino-1-naphthalenesulfonyl chloride (aka “dansyl chloride”) to cleave to the primary amine (N-terminal of a protein is the amine group)

Quick Recap: N-terminal vs C-terminal of amino acids

So What Does Dansyl Chloride Do?

What dansyl chloride does is it cleaves or attaches itself onto the n-terminus of an amino acid, thus fluorescing the amino acid.

Acid hydrolysis is then used to separate the dansylated amino acid from free amino acids as shown below:

Source: Fundamental of Biochemistry (Voet, Voet, and Pratt)

Chromatography is used to compare the fluoresced amino acid to a known standard to determine the amino acid.

Step 2: Produce Smaller Peptide Fragments

Remember, protein sequences can be very long!

In fact there are 20n sequence combinations possible (n = # of amino acid residues)

If the polypeptides that are longer than 40 to 100 residues cannot be sequenced directly.

So to remedy this situation, you can produce smaller peptide fragments to work with via chemical or enzymatic hydrolysis

Chemical Cleavage:

CNBr (cyanogen bromide) cleaves onto the methionine (Met) residues of an amino acid.

Enzymatic Cleavage:

Enzyme cleavage can be tricky because various enzymes cleave differently. 

There are:

Endopeptidases: 

Endopeptidases are enzymes that cleave to internal peptide bonds and hydrolyze them.

Exopeptidases:

Exopeptidases are enzymes that cleave to terminal C or N residues of amino acids in order to fragment polypeptides.

Proteases: 

Proteases act as both exopeptidases and endopeptidases.

However, these enzymes have specific side chain requirements in order for cleavage to occur. This means that the identity of the amino acid before the one being cleaved matters.

Step 3: Sequence the protein fragment

Once you have your fragment. You are ready to sequence your protein. Two common methods are Edman Degradation and Tandem Mass Spec

Step 4: Repeat steps 2 and steps 3 using a different cleavage/hydrolysis method

A different cleavage method will give you different peptide fragments that will overlap your initial sequencing. At the end, you will put together your overlapping sequences to get your final sequence.

Source: Voet, Voet, and Pratt

Step 5: Assemble the overlapping fragments for the full protein sequence

As stated, different fragments derived from different cleavage mechanisms yield various sequences. At the end, the sequences are overlapped to obtain the full ordered sequence.

How to calculate formal charge

How to calculate formal charge

 

Not all atoms within a neutral molecule need be neutral. An atom can have the following charges: positive, negative, or neutral, depending on the electron distribution. This is often useful for understanding or predicting reactivity. Identifying formal charges helps you keep track of the electrons.

The formal charge is the charge on the atom in the molecule. The term “formal” means that this charge is not necessarily on the presented atom because in some cases, it is also prevalent on other atoms present in the molecule. It is actually spread out through the other atoms and is not only on the one atom. Identifying a formal charge involves:

  1. Determining the appropriate number of valence electrons for an atom – This can be accomplished by inspecting the periodic table. The group number indicates the appropriate number of valence electrons for each atom.
  2. Determining whether the atom exhibits the appropriate number of electrons – In the Lewis structure, determine whether some of the atoms show an unexpected number of electrons.

The formal charge on an atom can be calculated using the following mathematical equation.

 

 

Lewis structures also show how atoms in the molecule are bonded. They can be drawn as lines (bonds) or dots (electrons). One line corresponds to two electrons. The nonbonding electrons, on the other hand, are the unshared electrons and these are shown as dots. One dot is equal to one nonbonding electron. The valence electrons are the electrons in the outermost shell of the atom.

 

 

Examples:

 

CH4, methane

A number of bonding electrons: 2 for H, 8 for C

A number of non-bonding electrons: 0 for both H and C

[Formal charge]H = 1 – (1/2) × 2 – 0 = ⇒ This applies to each hydrogen. These hydrogens are all zero.

 [Formal charge]c = 4 – (1/2) × 8 – 0 = 0

⇒ This molecule is neutral.

 

 

CH3+, methyl cation

 A number of bonding electrons: 2 for H, 6 for C

A number of non-bonding electrons: 0 for both H and C

[Formal charge]H = 1 – (1/2) × 2 – 0 = ⇒ This applies to each hydrogen. These hydrogens are all zero.

[Formal charge]c = 4 – (1/2) × 6 – 0 = 4 – 3 – 0 = +1 ⇒ This is a cation.

 

 

CH3‾, methyl cation

 A number of bonding electrons: 2 for H, 6 for C

A number of non-bonding electrons: 0 for H, 2 for C

[Formal charge]H = 1 – (1/2) × 2 – 0 = ⇒ This applies to each hydrogen. These hydrogens are all zero.

[Formal charge]c = 4 – (1/2) × 6 – 2 = 4 – 3 – 2 = -1 ⇒ This is a anion.