Protein Primary Structure
The primary structure of peptides and proteins refers to the linear number
and order of the amino acids present. The convention for the designation of the
order of amino acids is that the N-terminal end (i.e. the end bearing the
residue with the free a-amino group) is to the left
(and the number 1 amino acid) and the C-terminal end (i.e. the end with the
residue containing a free a-carboxyl group) is to the
right.
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Protein Secondary Structure
The ordered array of amino acids in a protein confer regular conformational
forms upon that protein. These conformations constitute the secondary structures
of a protein. In general proteins fold into two broad classes of structure
termed, globular proteins or fibrous proteins. Globular proteins are compactly folded
and coiled, whereas, fibrous proteins are more filamentous or elongated. It is
the partial double-bond character of the peptide bond that defines the
conformations a polypeptide chain may assume. Within a single protein different
regions of the polypeptide chain may assume different conformations determined
by the primary sequence of the amino acids.
The Alpha-Helix
The a-helix is a common secondary structure
encountered in proteins of the globular class. The formation of the a-helix is spontaneous and is stabilized by H-bonding between
amide nitrogens and carbonyl carbons of peptide bonds spaced four residues
apart.
This orientation of H-bonding produces a helical coiling of the peptide
backbone such that the R-groups lie on the exterior of the helix and
perpendicular to its axis.
Not all amino acids favor the formation of the a-helix due to steric constraints of the R-groups. Amino
acids such as A, D, E, I, L and M favor the formation of a-helices, whereas, G and P favor disruption of the helix.
This is particularly true for P since it is a pyrrolidine based imino acid (HN=)
whose structure significantly restricts movement about the peptide bond in which
it is present, thereby, interfering with extension of the helix. The disruption
of the helix is important as it introduces additional folding of the polypeptide
backbone to allow the formation of globular proteins.
b -Sheets
Whereas an a-helix is composed of a single linear
array of helically disposed amino acids, b-sheets are
composed of 2 or more different regions of stretches of at least 5-10 amino
acids. The folding and alignment of stretches of the polypeptide backbone aside
one another to form b-sheets is stabilized by H-bonding
between amide nitrogens and carbonyl carbons. However, the H-bonding residues
are present in adjacently opposed stretches of the polypetide backbone as
opposed to a linearly contiguous region of the backbone in the a-helix.
b-Sheets are said to be pleated. This is due to
positioning of the a-carbons of the peptide bond which
alternates above and below the plane of the sheet.
b-Sheets are either parallel or antiparallel. In
parallel sheets adjacent peptide chains proceed in the same direction (i.e. the
direction of N-terminal to C-terminal ends is the same), whereas, in
antiparallel sheets adjacent chains are aligned in opposite directions.
Super-secondary Structure
Some proteins contain an ordered organization of secondary structures that
form distinct functional domains or structural motifs. Examples include the
helix-turn-helix domain of bacterial proteins that regulate transcription and
the leucine zipper, helix-loop-helix and zinc finger domains of eukaryotic transcriptional
regulators. These domains are termed super-secondary
structures.
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Tertiary Structure
Tertiary structure refers to the complete three-dimensional structure of the
polypeptide units of a given protein. Included in this description is the
spatial relationship of different secondary structures to one another within a
polypeptide chain and how these secondary structures themselves fold into the
three-dimensional form of the protein. Secondary structures of proteins often
constitute distinct domains. Therefore, tertiary
structure also describes the relationship of different domains to one another
within a protein. The interactions of different domains is governed by several
forces: These include hydrogen bonding, hydrophobic
interactions, electrostatic interactions and van
der Waals forces.
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Forces Controlling Protein Structure
Hydrogen Bonding:
Polypeptides contain numerous proton donors and acceptors both in their
backbone and in the R-groups of the amino acids. The environment in which
proteins are found also contains the ample H-bond donors and acceptors of the
water molecule. H-bonding, therefore, occurs not only within and between
polypeptide chains but with the surrounding aqueous medium.
Hydrophobic Forces:
Proteins are composed of amino acids that contain either hydrophilic or
hydrophobic R-groups. It is the nature of the interaction of the different
R-groups with the aqueous environment that plays the major role in shaping
protein structure. The spontaneous folded state of globular proteins is a
reflection of a balance between the opposing energetics of H-bonding between
hydrophilic R-groups and the aqueous environment and the repulsion from the
aqueous environment by the hydrophobic R-groups. The hydrophobicity of certain
amino acid R-groups tends to drive them away from the exterior of proteins and
into the interior. This driving force restricts the available conformations into
which a protein may fold.
Electrostatic Forces:
Electrostatic forces are mainly of three types; charge-charge, charge-dipole
and dipole-dipole. Typical charge-charge
interactions that favor protein folding are those between oppositely charged
R-groups such as K or R and D or E. A substantial component of the energy
involved in protein folding is charge-dipole interactions. This refers to the
interaction of ionized R-groups of amino acids with the dipole of the water
molecule. The slight dipole moment that exist in the polar R-groups of amino
acid also influences their interaction with water. It is, therefore,
understandable that the majority of the amino acids found on the exterior
surfaces of globular proteins contain charged or polar R-groups.
van der Waals Forces:
There are both attractive and repulsive van der Waals forces that control
protein folding. Attractive van der Waals forces involve the interactions among
induced dipoles that arise from fluctuations in the charge densities that occur
between adjacent uncharged non-bonded atoms. Repulsive van der Waals forces
involve the interactions that occur when uncharged non-bonded atoms come very
close together but do not induce dipoles. The repulsion is the result of the
electron-electron repulsion that occurs as two clouds of electrons begin to
overlap.
Although van der Waals forces are extremely weak, relative to other forces
governing conformation, it is the huge number of such interactions that occur in
large protein molecules that make them significant to the folding of proteins.
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Quaternary Structure
Many proteins contain 2 or more different polypeptide chains that are held
in association by the same non-covalent forces that stabilize the tertiary
structures of proteins. Proteins with multiple polypetide chains are termed
oligomeric proteins. The structure formed by
monomer-monomer interaction in an oligomeric protein is known as quaternary structure.
Oligomeric proteins can be composed of multiple identical polypeptide chains
or multiple distinct polypeptide chains. Proteins with identical subunits are
termed homooligomers. Proteins containing several
distinct polypeptide chains are termed heterooligomers.
Hemoglobin,
the oxygen carrying protein of the blood, contains two a and two b subunits arranged with a
quaternary structure in the form, a2b2. Hemoglobin is, therefore, a hetero-oligomeric
protein.
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Complex Protein Structures
Proteins also are found to be covalently conjugated with carbohydrates.
These modifications occur following the synthesis (translation)
of proteins and are, therefore, termed post-translational modifications. These
forms of modification impart specialized functions upon the resultant proteins.
Proteins covalently associated with carbohydrates are termed glycoproteins.
Glycoproteins are of two classes, N-linked and O-linked, referring to the site
of covalent attachment of the sugar moieties. N-linked sugars are attached to
the amide nitrogen of the R-group of asparagine; O-linked sugars are attached to
the hydroxyl groups of either serine or threonine and occasionally to the
hydroxyl group of the modified amino acid, hydroxylysine.
There are extremely important glycoproteins found on the surface of
erythrocytes. It is the variability in the composition of the carbohydrate
portions of many glycoproteins and glycolipids of erythrocytes that determines
blood group specificities. There are at least 100 blood group determinants, most
of which are due to carbohydrate differences. The most common blood groups, A,
B, and O, are specified by the activity of specific gene products whose
activities are to incorporate distinct sugar groups onto RBC membrane
glycoshpingolipids as well as secreted glycoproteins.
Structural complexes involving protein associated with lipid via noncovalent
interactions are termed lipoproteins. The distinct roles of lipoproteins are
described on the linked page. Their major function in the body is to aid in the
storage transport of lipid and cholesterol.
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Clinical Significances
Visit the Inborn
Errors page for a more complete listing of diseases related to abnormal
proteins. Several brief examples are presented below.
The substitution of a hydrophobic amino acid (V) for an acidic amino acid
(E) in the b-chain of hemoglobin results in sickle cell
anemia (HbS). This change of a single amino acid alters the structure of
hemoglobin molecules in such a way that the deoxygenated proteins polymerize and
precipitate within the erythrocyte, leading to their characteristic sickle
shape.
Collagens are the most abundant proteins in the body. Alterations in
collagen structure arising from abnormal genes or abnormal processing of
collagen proteins results in numerous diseases, including Larsen syndrome,
scurvy, osteogenesis imperfecta and Ehlers-Danlos syndrome.
Ehlers-Danlos syndrome (see OMIM
links) is actually the name associated with at least ten distinct disorders
that are biochemically and clinically distinct yet all manifest structural
weakness in connective tissue as a result of defective collagen structure.
Osteogenesis imperfecta (see OMIM
links) also encompasses more than one disorder. At least four biochemically
and clinically distinguishable maladies have been identified as osteogenesis
imperfecta, all of which are characterized by multiple fractures and resultant
bone deformities. Marfan's
syndrome manifests itself as a disorder of the connective tissue and was
originally believed to be the result of abnormal collagens. However, recent
evidence has shown that Marfan's syndrome results from mutations in the
extracellular protein, fibrillin, which is an
integral constituent of the non-collagenous microfibrils of the extracellular
matrix.
Several forms of familial
hypercholesterolemia (see also OMIM
links) are the result of genetic defects in the gene encoding the receptor
for low-density lipoprotein (LDL). These defects result in the synthesis of
abnormal LDL receptors that are incapable of binding to LDLs, or that bind LDLs
but the receptor/LDL complexes are not properly internalized and degraded. The
outcome is an elevation in serum cholesterol levels and increased propensity
toward the development of atherosclerosis.
A number of proteins can contribute to cellular transformation and
carcinogenesis when their basic structure is disrupted by mutations in their
genes. These genes are termed proto-oncogenes.
For some of these proteins, all that is required to convert them to the oncogenic form is a single amino acid substitution. The
cellular gene, c-Ras, is observed to sustain
single amino acid substitutions at positions 12 or 61 with high frequency in
colon carcinomas. Mutations in c-Ras are most frequently observed genetic
alterations in colon cancer.
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Amino-Terminal Sequence Determination
Prior to sequencing peptides it is necessary to eliminate disulfide bonds
within peptides and between peptides. Several different chemical reactions can
be used in order to permit separation of peptide strands and prevent protein
conformations that are dependent upon disulfide bonds. The most common
treatments are to use either 2-mercaptoethanol or
dithiothreitol. Both of these chemicals reduce
disulfide bonds. To prevent reformation of the disulfide bonds the peptides are
treated with iodoacetic acid in order to alkylate
the free sulfhydryls.
There are three major chemical techniques for sequencing peptides and
proteins from the N-terminus. These are the Sanger, Dansyl
chloride and Edman techniques.
Sanger's Reagent: This sequencing technique utilizes the compound,
2,4-dinitrofluorobenzene (DNF) which reacts with the N-terminal residue under
alkaline conditions. The derivatized amino acid can be hydrolyzed and will be
labeled with a dinitrobenzene group that imparts a yellow color to the amino
acid. Separation of the modified amino acids (DNP-derivative) by electrophoresis
and comparison with the migration of DNP-derivative standards allows for the
identification of the N-terminal amino acid.
Dansyl chloride: Like DNF, dansyl chloride reacts with the N-terminal
residue under alkaline conditions. Analysis of the modified amino acids is
carried out similarly to the Sanger method except that the dansylated amino
acids are detected by fluorescence. This imparts a higher sensitivity into this
technique over that of the Sanger method.
Edman degradation: The utility of the Edman degradation technique is
that it allows for additional amino acid sequence to be obtained from the
N-terminus inward. Using this method it is possible to obtain the entire
sequence of peptides. This method utilizes phenylisothiocyanate to react with
the N-terminal residue under alkaline conditions. The resultant
phenylthiocarbamyl derivatized amino acid is hydrolyzed in anhydrous acid. The
hydrolysis reaction results in a rearrangement of the released N-terminal
residue to a phenylthiohydantoin derivative. As in the Sanger and Dansyl
chloride methods, the N-terminal residue is tagged with an identifiable marker,
however, the added advantage of the Edman process is that the remainder of the
peptide is intact. The entire sequence of reactions can be repeated over and
over to obtain the sequences of the peptide. This process has subsequently been
automated to allow rapid and efficient sequencing of even extremely small
quantities of peptide.
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Protease Digestion
Due to the limitations of the Edman degradation technique, peptides longer
than around 50 residues can not be sequenced completely. The ability to obtain
peptides of this length, from proteins of greater length, is facilitated by the
use of enzymes, endopeptidases, that cleave at specific sites
within the primary sequence of proteins. The resultant smaller peptides can be
chromatographically separated and subjected to Edman degradation sequencing
reactions.
Specificities of Several Endoproteases
| Enzyme |
Source |
Specificity |
Additional
Points |
| Trypsin |
Bovine pancreas |
peptide bond C-terminal to R, K, but not if
next to P |
highly specific for positively charged
residues |
| Chymotrypsin |
Bovine pancreas |
peptide bond C-terminal to F, Y, W but not if
next to P |
prefers bulky hydrophobic residues, cleaves
slowly at N, H, M, L |
| Elastase |
Bovine pancreas |
peptide bond C-terminal to A, G, S, V, but not
if next to P |
|
| Thermolysin |
Bacillus
thermoproteolyticus |
peptide bond N-terminal to I, M, F, W, Y, V,
but not if next to P |
prefers small neutral residues, can cleave at
A, D, H, T |
| Pepsin |
Bovine gastric mucosa |
peptide bond N-terminal to L, F, W, Y, but
when next to P |
exhibits little specificity, requires low
pH |
| Endopeptidase V8 |
Staphylococcus aureus |
peptide bond C-terminal to E |
|
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Carboxy-Terminal Sequence Determination
No reliable chemical techniques exist for sequencing the C-terminal amino
acid of peptides. However, there are enzymes, exopeptidases, that
have been identified that cleave peptides at the C-terminal residue which can
then be analyzed chromatographically and compared to standard amino acids. This
class of exopeptidases are called, carboxypeptidases.
Specificities of Several Exopeptidases
| Enzyme |
Source |
Specificity |
| Carboxypeptidase A |
Bovine pancreas |
Will not cleave when C-terminal residue = R, K
or P or if P resides next to terminal residue |
| Carboxypeptidase B |
Bovine pancreas |
Cleaves when C-terminal residue = R or K; not
when P resides next to terminal reside |
| Carboxypeptidase C |
Citrus leaves |
All free C-terminal residues, pH optimum =
3.5 |
| Carboxypeptidase Y |
Yeast |
All free C-terminal residues, slowly at G
residues |
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Chemical Digestion of Proteins
The most commonly utilized chemical reagent that cleaves peptide bonds by
recognition of specific amino acid residues is cyanogen
bromide (CNBr). This reagent causes specific cleavage at the
C-terminal side of M residues. The number of peptide fragments that result from
CNBr cleavage is equivalent to one more than the number of M residues in a
protein.
The most reliable chemical technique for C-terminal residue identification
is hydrazinolysis. A peptide is treated with
hydrazine, NH2-NH2, at high temperature
(90oC) for an extended length of time (20-100hr). This treatment
cleaves all of the peptide bonds yielding amino-acyl hydrazides of all the amino
acids excluding the C-terminal residue which can be identified
chromatographically compared to amino acid standards. Due to the high percentage
of hydrazine induced side reactions this technique is only used on
carboxypeptidase resistant peptides.
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Size Exclusion Chromatography
This chromatographic technique is based upon the use of a porous gel in the
form of insoluble beads placed into a column. As a solution of proteins is
passed through the column, small proteins can penetrate into the pores of the
beads and, therefore, are retarded in their rate of travel through the column.
The larger proteins a protein is the less likely it will enter the pores.
Different beads with different pore sizes can be used depending upon the desired
protein size separation profile.
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Ion Exchange Chromatography
Each individual protein exhibits a distinct overall net charge at a given
pH. Some proteins will be negatively charged and some will be positively charged
at the same pH. This property of proteins is the basis for ion exchange
chromatography. Fine cellulose resins are used that are either negatively
(cation exchanger) or positively (anion exchanger) charged. Proteins of opposite charge to
the resin are retained as a solution of proteins is passed through the column.
The bound proteins are then eluted by passing a solution of ions bearing a
charge opposite to that of the column. By utilizing a gradient of increasing
ionic strength, proteins with increasing affinity for the resin are
progressively eluted.
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Affinity Chromatography
Proteins have high affinities for their substrates or co-factors or
prosthetic groups or receptors or antibodies raised against them. This affinity
can be exploited in the purification of proteins. A column of beads bearing the
high affinity compound can be prepared and a solution of protein passed through
the column. The bound proteins are then eluted by passing a solution of unbound
soluble high affinity compound through the column.
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High Performance Liquid Chromatography (HPLC)
In column chromatography the smaller and more tightly packed a resin is the
greater the separation capability of the column. In gravity flow columns the
limitation column packing is the time it takes to pass the solution of proteins
through the column. HPLC utilizes tightly packed fine diameter resins to impart
increased resolution and overcomes the flow limitations by pumping the solution
of proteins through the column under high pressure. Like standard column
chromatography, HPLC columns can be used for size exclusion or charge
separation. An additional separation technique commonly used with HPLC is to
utilize hydrophobic resins to retard the movement of nonpolar proteins. The
proteins are then eluted from the column with a gradient of increasing
concentration of an organic solvent. This latter form of HPLC is termed reversed-phase HPLC.
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Electrophoresis of Proteins
Proteins also can be characterized according to size and charge by
separation in an electric current (electrophoresis) within solid sieving gels
made from polymerized and cross-linked acrylamide. The most commonly used
technique is termed SDS polyacrylamide gel electrophoresis
(SDS-PAGE). The gel is a thin slab of acrylamide polymerized between
two glass plates. This technique utilizes a negatively charged detergent (sodium
dodecyl sulfate) to denature and solubilize proteins. SDS denatured proteins
have a uniform negative charge such that all proteins will migrate through the
gel in the electric field based solely upon size. The larger the protein the
more slowly it will move through the matrix of the polyacrylamide. Following
electrophoresis the migration distance of unknown proteins relative to known
standard proteins is assessed by various staining or radiographic detection
techniques.
The use of polyacrylamide gel electrophoresis also can be used to determine
the isoelectric charge of proteins (pI). This
technique is termed isoelectric focusing.
Isoelectric focusing utilizes a thin tube of polyacrylamide made in the presence
of a mixture of small positively and negatively charged molecules termed
ampholytes. The ampholytes have a range of pIs that establish a pH gradient
along the gel when current is applied. Proteins will, therefore, cease migration
in the gel when they reach the point where the ampholytes have established a pH
equal to the proteins pI.
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Centrifugation of Proteins
Proteins will sediment through a solution in a centrifugal field dependent
upon their mass. Analytical centrifugation measure the rate that proteins
sediment. The most common solution utilized is a linear gradient of sucrose
(generally from 5-20%). Proteins are layered atop the gradient in an
ultracentrifuge tube then subjected to centrifugal fields in excess of 100,000 x
g. The sizes of unknown proteins can then be determined by comparing their
migration distance in the gradient with those of known standard proteins.
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This article has been modified by Dr. M. Javed Abbas.
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20:37 21/12/2002
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