Background Information

Background Information

Virtually every chemical reaction and activity that occurs in a living cell requires proteins. A multitude of different types of proteins perform a wide range of functions that include roles in cell support and shape, cell motility, cell communication, protection against foreign materials, cell reception, cell adhesion, catalytic functions as enzymes, and the transport of molecules. This great diversity of protein functions is a direct result of the structural organization and structural properties of proteins.

The three-dimensional conformation of a protein, also known as protein structure, is determined by the arrangement of amino acids that are held together by peptide bonds to form a polypeptide (20 or more amino acids linked together by peptide bonds). The specific sequence or order of amino acids in a polypeptide is known as the primary structure of a protein. In many proteins, chemical bonding between amino acids produces proteins with higher-order arrangements known as secondary and tertiary structure. In addition, for certain proteins­particularly enzymes, structural proteins, and transport proteins­a complete and functional protein consists of multiple polypeptide chains (subunits) that must wrap around each other in an arrangement known as quaternary structure. The overall conformation or structure of a given protein provides that protein with the unique structural characteristics that are necessary for the proper functions of that protein. Hence, disruption of protein structure, a process known as protein denaturation, drastically alters the functions of a protein.

One of the most extensively studied examples of the relationship between protein structure and function involves hemoglobin, the oxygen-transporting protein in human red blood cells. Adult human red blood cells contain relatively few organelles compared with other body cells. In the most basic sense, human red blood cells are essentially membrane sacs filled with hemoglobin. On average, a single red blood cell contains approximately 250 million hemoglobin molecules! The abundance of hemoglobin in red blood cells and the unique structure of the hemoglobin molecule itself accomplish the primary function of red blood cells: to transport oxygen from the lungs to body cells, tissues, and organs.

In addition to transporting oxygen, the conformation of hemoglobin contributes to the biconcave disk shape of human red blood cells. The shape of these cells provides them with the necessary flexibility to flow through thin-diameter blood vessels, such as capillaries, with a minimal amount of friction.

A single hemoglobin molecule consists of four subunits of a polypeptide known as globin. Globin polypeptides are synthesized from a large family of genes that are highly conserved among many species of vertebrate and invertebrate organisms. This family includes relatives such as myoglobin, an oxygen-storage protein present in most vertebrates. Several different globin polypeptides are used to transport oxygen in red blood cells, including some that are used only during fetal development. Adult human red blood cells contain hemoglobin molecules, which involve two alpha globin (a-globin) subunits and two beta globin (b-globin) subunits that wrap around each other. In the center of each globin subunit is a single iron-containing organic ring known as the heme group. One oxygen molecule can bind to the iron atom in each heme group; therefore, each hemoglobin molecule can bind to and transport a maximum of four molecules of oxygen.

The oxygen-carrying capability of hemoglobin depends on the electron configuration of the iron atom. Within the heme group, the iron atom exists as a transition metal in a divalent state called ferrous iron (Fe+2). The charged nature of an iron ion is essential for oxygen to bind to the heme group. In addition, oxygen binding to the heme group requires a change in the oxidation state of iron that allows the iron to bind oxygen without oxidizing the oxygen or the iron atom itself. This is accomplished by complexing the iron to four nitrogen atoms in the porphyrin ring and one amino acid in the globin subunit, and surrounding the ring with a cluster of hydrophobic amino acids in each globin subunit, thereby creating a hydrophobic pocket around the heme group. This configuration holds the iron atom at a displaced position above the plane of the heme group, which is the ideal conformation for binding and holding oxygen.

In addition, hemoglobin is an efficient carrier of oxygen because the globin chains exhibit an interaction known as cooperativity. In cooperativity, binding of a single oxygen molecule to one heme group results in an increased binding affinity for oxygen to the three other heme groups. Cooperativity occurs because the binding of one oxygen molecule to one heme group creates a shift in the conformation of each globin chain that produces a change in the overall quaternary structure of the entire hemoglobin molecule. This transition in conformation creates a molecule that favors the binding of additional oxygen molecules. The conformation change that occurs during cooperativity is due to the bonds or contacts of specific amino acids that connect the alpha and beta chains to each other and allow these chains to interact with each other.

In 1949, Linus Pauling provided biologists with a significant insight into the molecular basis for sickle-cell disease (sickle-cell anemia) by using gel electrophoresis to demonstrate that hemoglobin molecules isolated from normal patients and from those with sickle-cell disease differed in their rate of migration. This observation led Pauling to suggest that a difference in amino acid sequence accounted for the migration differences of these proteins. Using peptide sequencing techniques, Vernon Ingram subsequently demonstrated that the differences between normal hemoglobin and sickle-cell hemoglobin are due to an amino acid difference in the primary structure of the two proteins. This amino acid difference occurs because of a point mutation in one of the globin genes.

The most common mutation in a globin gene of an individual with sickle-cell disease involves a substitution in the codon that codes for the amino acid glutamic acid at position 6 in the b-globin polypeptide. Recall that single-nucleotide changes in the DNA sequence of a gene are known as point mutations. Point mutations in a gene sequence can result in the synthesis (transcription) of messenger RNA (mRNA) molecules with an altered base sequence. Depending on the location of a mutation within a codon, a mutation may or may not affect the protein coded for by a particular mRNA. The altered codon in sickle-cell disease results in the substitution of valine for glutamic acid at position 6. This disruption in the primary structure of the globin polypeptide occurs in a location on the globin subunit that is necessary for the proper folding of hemoglobin into its three-dimensional conformation that is essential for it to function as an oxygen-transport protein. As a direct result of this change in hemoglobin structure, sickle-cell hemoglobin binds oxygen with a much lower affinity than normal hemoglobin. In addition, red blood cells in the sickle-cell patient lose their characteristic biconcave disk shape and assume an irregular, elongated sickled configuration that greatly diminishes their movement through blood vessels. Sickled red blood cells frequently clump together and block blood flow through the capillaries. Because of changes both in hemoglobin and red blood cell structure, the sickle-cell patient suffers from decreased oxygen delivery to body organs and a variety of other painful conditions related to poor circulation of red blood cells and inadequate oxygen content within the body. The study of hemoglobin biochemistry and sickle-cell disease has provided biologists with an invaluable understanding of the molecular basis for disease.

In this laboratory you will have the opportunity to study the importance of amino acid sequence to the structure of the normal hemoglobin protein and normal human red blood cells. You will also investigate the connections between the nucleotide sequence, the physical properties of the hemoglobin polypeptide, the structure of red blood cells, and the physiological effects of a hemoglobin mutation.

References

  1. Ingram, V.M. Gene mutations in human hemoglobin: The chemical difference between normal and sickle cell hemoglobin. Nature 180 (1957).

  2. Dickerson, R.E., and Geis, I. Hemoglobin: Structure, Function, Evolution, and Pathology. Menlo Park, CA: Benjamin/Cummings, 1983.

  3. Pauling, L., Itano, H.A., Singer, S.J., and Wells, I.C. Sickle cell anemia, a molecular disease. Science 110 (1949).

  4. Klug, W.S., and Cummings, M.R. (1996): Essentials of Genetics, 2nd ed. Upper Saddle River, NJ: Prentice Hall Inc, 1996.