Primary structure of proteins

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 α-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 α-carboxyl group) is to the right.

Secondary structure in proteins

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 α-Helix

The α-helix is a common secondary structure encountered in proteins of the globular class. The formation of the α-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 (α-helix due to steric constraints of the R-groups. Amino acids such as A, D, E, I, L and M favor the formation of α-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.

β-Sheets

Whereas an α-helix is composed of a single linear array of helically disposed amino acids, β-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 β-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 α-helix. β-sheets are said to be pleated. This is due to positioning of the α-carbons of the peptide bond which alternates above and below the plane of the sheet. β-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. β-sheets can be depicted in ball and stick format or as ribbons in certain protein formats.

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.

 

Tertiary structure of proteins

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.

 

Protease digestion

Due to the limitations of the so-called 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 not when next to P	exhibits little specificity, requires low pH
Endopeptidase V8	Staphylococcus aureus	peptide bond C-terminal to E

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 (90°C) 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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Protein separation and purification

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.

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.

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.

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.

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.

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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Protein functions in the cell

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Clinical significance

Just a few of the many examples of disorders that result from defects in protein structure and function:

1. The substitution of a hydrophobic amino acid (V) for an acidic amino acid (E) in the β-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.

2. 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 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.

3. Several forms of familial hypercholesterolemia 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.

4. 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 RAS is observed to sustain single amino acid substitutions at positions 12 or 61 with high frequency in colon carcinomas. Mutations in RAS are most frequently observed genetic alterations in colon cancer.