PART II
Analysis and Characterization of
Proteins
Topics covered:
• Protein Structure and Assays
• Differential Solubility
• Chromatography
• Membranes and Detergents
• Electrophoresis
• Overview on Protein Purification
59
, CHAPTER 7--INTRODUCTION TO PROTEINS
Proteins typically make up more than half the dry weight of cells. They contribute to the
structure of a cell and are responsible for cellular functions such as catalysis and molecular
recognition.
L-α-AMINO ACIDS
PROTEIN STRUCTURE
Nonpolar Polar
Proteins are polymers of L-α-amino acids. The α refers
to a carbon with a primary amine, a carboxylic acid, a hydrogen Alanine Arginine
and a variable side-chain group, usually designated as 'R'. Glycine Asparagine
Carbon atoms with four different groups are asymmetric and Isoleucine Aspartic a.
can exhibit two different arrangements in space due to the Leucine Cysteine
tetrahedral nature of the bonds. The L refers to one of these two Methionine Glutamic a.
possible configurations the four different groups on the α- Phenylalanine Glutamine
Proline Histidine
carbon can exhibit. Amino acids of the D-configuration are not Tryptophan Lysine
found in proteins and do not participate in biological reactions. Valine Serine
Threonine
Twenty different amino acids, distinguished by their side-chain Tyrosine
groups, are found in proteins (Box). The side-chain groups vary
in terms of their chemical properties such as polarity, charge and size. These various side-chain
groups will influence the chemical properties of proteins as well as determine the overall
structure of the protein (see Appendix). For example, the polar amino acids tend to be on the
outside of the protein where they interact with water and the nonpolar amino acids are on the
inside forming a hydrophobic core.
The covalent linkage between two amino acids
is known as a peptide bond. A peptide bond is formed
when the amino group of one amino acid condenses
with the carboxyl group of another amino acid to form
an amide (Figure). This arrangement gives the
polypeptide chain a polarity in that one end will have a
free amino group, called the N-terminus, and the other
end will have a free carboxyl group, called the C-
terminus.
Peptide bonds tend to be planar which gives
the polypeptide backbone some rigidity. However,
rotation can occur around both of the α-carbon bonds
resulting in a polypeptide backbone with different potential conformations in regards to the
relative positions of the R-groups. (Conceptually this can be viewed as the R-groups projection
into or out from the page in the figure.) Although many conformations are theoretically
possible, interactions between the R-groups will limit the number of potential conformations
and proteins tend to only fold into a single functional conformation. In other words, the confor-
mation or shape of the protein is due to the interactions of the side-chain groups with one
another and with the polypeptide backbone. The interactions can be between amino acids that
61
,are close together in a polypeptide or between amino acids that are far apart or even on
different polypeptides. These different types of interactions are often discussed in terms of
primary, secondary, tertiary and quaternary protein structure (Table).
Levels of Protein Structure
Refers to the amino acid sequence and the location of disulfide
Primary
bonds between cysteine residues (i.e., covalent bonds).
Refers to interactions between amino acids that are close
Secondary
together (eg., α-helix, β-sheet, β-turn, random coil).
Refers to interactions between amino acids that are far apart
Tertiary
(eg., motifs, domains).
Refers to interactions between two or more polypeptide chains
Quaternary
(i.e., protein subunits).
The primary amino acid sequence and positions of disulfide bonds strongly influence
the overall structure of protein. In regarads to the primary amino acid sequence, certain side-
chains will permit, or promote, hydrogen-bonding between neighboring amino acids of the
polypeptide backbone resulting in secondary structures such as β-sheets or α-helices. Alterna-
tively, certain R-groups may interfere with each other and prevent certain conformations.
In the α-helix conformation the peptide backbone takes on a 'sprial staircase' shape
which is stabilized by H-bonds between carbonyl and amide groups of every fourth amino acid
residue. This restricts the rotation of the bonds in the peptide backbone resulting in a rigid
structure. β-sheets are also rigid structures in which the polypeptide chain is nearly fully
extended with the R-groups alternating between pointing up and down. β-sheets interact either
in parallel (both with same orientation in regards to N- and C-termini) or anti-parallel fashion.
Certain amino acids promote the formation of either α-helices or β-sheets due to the nature of
the side-chain groups. Some side-chain groups may prevent the formation of secondary
structures and result in a more flexible polypeptide backbone, which is often called random coil
conformation.
The other aspect of primary protein
structure is the position of disulfide bonds. The
amino acid cysteine has a free thiol group that
can be oxidized to form a covalent bond with
another cysteine (Figure). These disulfide bonds
can form between cysteine residues that are relatively close or far apart within a single polypep-
tide chain, or even between separate polypeptide subunits with a protein. In this regard,
disulfide bonds can contribute to secondary, tertiary and quaternary aspects of protein structure.
Proteins containing disulfide bonds will be sensitive to reducing agents (such as β-mercapto-
ethanol) which can break the disulfide bond.
The various secondary structures can interact with other secondary structures within the
same polypeptide to form motifs or domains (i.e., tertiary structure). A motif is a common
combination of secondary structures and a domain is a portion of a protein that folds
independently. The tertiary structure will represent the overall three dimensional shape of a
62
, polypeptide. A typical protein structure is a compact entity composed of the various secondary
structural elements with protruding loops of
flexible (ie, random coil) sequence. This is often
depicted in a ribbon diagram (Figure) in which β-
sheets are drawn as flat arrows with the arrowhead
representing the N-terminal side and α-helices are
drawn as flat spirals. The flexible loops are repre-
sented by the strings connecting the secondary
structural elements.
Many proteins are composed of multiple subunits,
or distinct polypeptide chain that interact with one
another. This is referred to as quaternary structure.
PROTEIN STABILITY
Proteins are often fragile molecules
that need to be protected during purifi-
cation and characterization. Protein dena-
turation refers the loss of protein structure
due to unfolding. Maintaining biological
activity is often important and protein
denaturation should be avoided in those
situations. Elevated temperatures, extremes in pH, and changes in chemical or physical
environment can all lead to protein denaturation (Table). In general, things that destabilize H-
bonding and other forces that contribute to secondary and tertiary protein structure will promote
protein denaturation. Different proteins exhibit different degrees of sensitivity to denaturing
agents and some proteins can be re-folded to their correct conformations following
denaturation.
Factors Affecting Protein Stability
Factor Possible Remedies
temperature Avoid high temperatures. Keep solutions on ice.
Determine effects of freezing. Include glycerol in buffers. Store in
freeze-thaw
aliquots.
physical Do not shake, vortex or stir vigorously. (Protein solutions should
denaturation not foam.)
solution effects Mimic cellular environment: neutral pH, ionic composition, etc.
dilution effects Maintain protein concentrations > 1 mg/ml as much as possible.
oxidation Include 0.1-1 mM DTT (or β-ME) in buffers.
heavy metals Include 1-10 mM EDTA in buffers.
microbial growth Use sterile solutions, include anti-microbials, and/or freeze.
proteases Include protease inhibitors. Keep on ice.
The optimal conditions for maintaining the stability of each individual protein need to
63
Analysis and Characterization of
Proteins
Topics covered:
• Protein Structure and Assays
• Differential Solubility
• Chromatography
• Membranes and Detergents
• Electrophoresis
• Overview on Protein Purification
59
, CHAPTER 7--INTRODUCTION TO PROTEINS
Proteins typically make up more than half the dry weight of cells. They contribute to the
structure of a cell and are responsible for cellular functions such as catalysis and molecular
recognition.
L-α-AMINO ACIDS
PROTEIN STRUCTURE
Nonpolar Polar
Proteins are polymers of L-α-amino acids. The α refers
to a carbon with a primary amine, a carboxylic acid, a hydrogen Alanine Arginine
and a variable side-chain group, usually designated as 'R'. Glycine Asparagine
Carbon atoms with four different groups are asymmetric and Isoleucine Aspartic a.
can exhibit two different arrangements in space due to the Leucine Cysteine
tetrahedral nature of the bonds. The L refers to one of these two Methionine Glutamic a.
possible configurations the four different groups on the α- Phenylalanine Glutamine
Proline Histidine
carbon can exhibit. Amino acids of the D-configuration are not Tryptophan Lysine
found in proteins and do not participate in biological reactions. Valine Serine
Threonine
Twenty different amino acids, distinguished by their side-chain Tyrosine
groups, are found in proteins (Box). The side-chain groups vary
in terms of their chemical properties such as polarity, charge and size. These various side-chain
groups will influence the chemical properties of proteins as well as determine the overall
structure of the protein (see Appendix). For example, the polar amino acids tend to be on the
outside of the protein where they interact with water and the nonpolar amino acids are on the
inside forming a hydrophobic core.
The covalent linkage between two amino acids
is known as a peptide bond. A peptide bond is formed
when the amino group of one amino acid condenses
with the carboxyl group of another amino acid to form
an amide (Figure). This arrangement gives the
polypeptide chain a polarity in that one end will have a
free amino group, called the N-terminus, and the other
end will have a free carboxyl group, called the C-
terminus.
Peptide bonds tend to be planar which gives
the polypeptide backbone some rigidity. However,
rotation can occur around both of the α-carbon bonds
resulting in a polypeptide backbone with different potential conformations in regards to the
relative positions of the R-groups. (Conceptually this can be viewed as the R-groups projection
into or out from the page in the figure.) Although many conformations are theoretically
possible, interactions between the R-groups will limit the number of potential conformations
and proteins tend to only fold into a single functional conformation. In other words, the confor-
mation or shape of the protein is due to the interactions of the side-chain groups with one
another and with the polypeptide backbone. The interactions can be between amino acids that
61
,are close together in a polypeptide or between amino acids that are far apart or even on
different polypeptides. These different types of interactions are often discussed in terms of
primary, secondary, tertiary and quaternary protein structure (Table).
Levels of Protein Structure
Refers to the amino acid sequence and the location of disulfide
Primary
bonds between cysteine residues (i.e., covalent bonds).
Refers to interactions between amino acids that are close
Secondary
together (eg., α-helix, β-sheet, β-turn, random coil).
Refers to interactions between amino acids that are far apart
Tertiary
(eg., motifs, domains).
Refers to interactions between two or more polypeptide chains
Quaternary
(i.e., protein subunits).
The primary amino acid sequence and positions of disulfide bonds strongly influence
the overall structure of protein. In regarads to the primary amino acid sequence, certain side-
chains will permit, or promote, hydrogen-bonding between neighboring amino acids of the
polypeptide backbone resulting in secondary structures such as β-sheets or α-helices. Alterna-
tively, certain R-groups may interfere with each other and prevent certain conformations.
In the α-helix conformation the peptide backbone takes on a 'sprial staircase' shape
which is stabilized by H-bonds between carbonyl and amide groups of every fourth amino acid
residue. This restricts the rotation of the bonds in the peptide backbone resulting in a rigid
structure. β-sheets are also rigid structures in which the polypeptide chain is nearly fully
extended with the R-groups alternating between pointing up and down. β-sheets interact either
in parallel (both with same orientation in regards to N- and C-termini) or anti-parallel fashion.
Certain amino acids promote the formation of either α-helices or β-sheets due to the nature of
the side-chain groups. Some side-chain groups may prevent the formation of secondary
structures and result in a more flexible polypeptide backbone, which is often called random coil
conformation.
The other aspect of primary protein
structure is the position of disulfide bonds. The
amino acid cysteine has a free thiol group that
can be oxidized to form a covalent bond with
another cysteine (Figure). These disulfide bonds
can form between cysteine residues that are relatively close or far apart within a single polypep-
tide chain, or even between separate polypeptide subunits with a protein. In this regard,
disulfide bonds can contribute to secondary, tertiary and quaternary aspects of protein structure.
Proteins containing disulfide bonds will be sensitive to reducing agents (such as β-mercapto-
ethanol) which can break the disulfide bond.
The various secondary structures can interact with other secondary structures within the
same polypeptide to form motifs or domains (i.e., tertiary structure). A motif is a common
combination of secondary structures and a domain is a portion of a protein that folds
independently. The tertiary structure will represent the overall three dimensional shape of a
62
, polypeptide. A typical protein structure is a compact entity composed of the various secondary
structural elements with protruding loops of
flexible (ie, random coil) sequence. This is often
depicted in a ribbon diagram (Figure) in which β-
sheets are drawn as flat arrows with the arrowhead
representing the N-terminal side and α-helices are
drawn as flat spirals. The flexible loops are repre-
sented by the strings connecting the secondary
structural elements.
Many proteins are composed of multiple subunits,
or distinct polypeptide chain that interact with one
another. This is referred to as quaternary structure.
PROTEIN STABILITY
Proteins are often fragile molecules
that need to be protected during purifi-
cation and characterization. Protein dena-
turation refers the loss of protein structure
due to unfolding. Maintaining biological
activity is often important and protein
denaturation should be avoided in those
situations. Elevated temperatures, extremes in pH, and changes in chemical or physical
environment can all lead to protein denaturation (Table). In general, things that destabilize H-
bonding and other forces that contribute to secondary and tertiary protein structure will promote
protein denaturation. Different proteins exhibit different degrees of sensitivity to denaturing
agents and some proteins can be re-folded to their correct conformations following
denaturation.
Factors Affecting Protein Stability
Factor Possible Remedies
temperature Avoid high temperatures. Keep solutions on ice.
Determine effects of freezing. Include glycerol in buffers. Store in
freeze-thaw
aliquots.
physical Do not shake, vortex or stir vigorously. (Protein solutions should
denaturation not foam.)
solution effects Mimic cellular environment: neutral pH, ionic composition, etc.
dilution effects Maintain protein concentrations > 1 mg/ml as much as possible.
oxidation Include 0.1-1 mM DTT (or β-ME) in buffers.
heavy metals Include 1-10 mM EDTA in buffers.
microbial growth Use sterile solutions, include anti-microbials, and/or freeze.
proteases Include protease inhibitors. Keep on ice.
The optimal conditions for maintaining the stability of each individual protein need to
63
