Introduction and Goals
A typical eukaryotic cell is mostly water (70% by weight), whereas the remainder of the bulk of the cell is composed of carbon-based compounds. These carbon-based compounds fall into four major families of macromolecules, which make up the form and carry out the many diverse functions of a cell. In this and the next tutorial, we will focus on four macromolecules: proteins, nucleic acids, polysaccharides and lipids. Proteins, nucleic acids and polysaccharides are long chains of repeating subunits. Proteins are composed of chains of amino acids, nucleic acids are composed of chains of nucleotides, and polysaccharides are composed of chains of simple sugars. Strictly speaking, lipids, which are composed of hydrocarbons, are not macromolecules because they are not composed of repeating subunits; however, their large molecular weight and importance in cellular processes warrants their inclusion in this class of molecules. We will examine the composition and structure of these macromolecules, investigating the different types of chemical bonds used in their formation. Additionally, we will explore the distinct properties of these macromolecules; this will become relevant when we discuss how these macromolecules function in the context of cellular structure and/or function. In this first tutorial we will concentrate on one major family of macromolecules, proteins. The other three will be discussed in the next tutorial. By the end of this tutorial you should know:
- The basic structure of amino acids, and the distinct properties of their side chains
- How amino acids are linked to form a polypeptide
- The four levels of hierarchy of protein conformation
- The various types of bonds that regulate protein structure, and how different amino acids contribute to these bonds
- The importance of protein conformation to protein activity
Diverse functions in cells
Of the four types of macromolecules to be discussed, proteinsare the most abundant and diverse. Proteins participate in almost all cellular functions. Proteins facilitate practically every chemical reaction that occurs in a cell, as well as facilitate the transport of many small molecules in and out of the cell. Proteins relay and receive messages between cells, and trigger changes in a cell in response to these signals. Finally, proteins can act as motors and provide the force to move a cell, as well as move components within a cell.
A polymer of amino acids
All proteins are polymers (large molecules made up of repeating units). The repeating units, termed monomers, are the amino acids. There are twenty different amino acids, and they share a similar general structure. The basic structure is a central carbon (referred to as an alpha carbon), an amino group (NH3?), a carboxyl group (COO?) and an R-group (also termed a side chain). The R-group is variable for each amino acid, and it defines the different properties of each amino acid. There are two stereoisomers(chemical compounds with the same molecular formulas but differing in arrangement such that they are mirror images of each other) of amino acids, L and D, although only L-amino acids make up proteins.
The twenty amino acids are generally classified as nonpolar (hydrophobic, meaning they do not readily interact with water), polar (uncharged and hydrophilic, meaning they readily interact with water), or charged (either positively or negatively). The chemical composition of the R-group determines their classification. The various properties of the R-groups greatly influence how the amino acids interact with each other and their environment.Amino Acids are Linked via a Peptide Bond
A polymer of amino acids is termed a polypeptide. Polypeptides are formed by the sequential addition of amino acids. This occurs via a specific covalent bond (a bond formed between two or more atoms by sharing electrons) termed a peptide bond, which links the carboxyl group of the first amino acid to the amino group of the second amino acid. This is illustrated in the animation below. Peptide bond formation is a condensation reaction, which results in a release of water. The polypeptide grows by the addition of another amino acid to the free carboxyl group of the last amino acid added. This generates an inherent directionality to the polypeptide. The first amino acid is distinguished by a free amino group and the last amino acid added is distinguished by a free carboxyl group, thereby defining the beginning of the polypeptide as the amino terminus (N-terminus) and the end of the polypeptide as the carboxyl terminus (C-terminus). It is conventional to write the amino
acid sequence from the N-terminus to the C-terminus. The amino acids in the polypeptide chain are sometimes referred to as residues.
Although the amino acid sequence of a polypeptide is written as a simple linear arrangement, this does not reflect the actual structure of the polypeptide. Most proteins will spontaneously fold into specific, stable, three-dimensional shapes (conformations). The conformation of a protein is dictated by the amino acid sequence via chemical interactions between the peptide bonds, the side chains and the environment. The conformation of a protein is critical to its activity; therefore, if the amino acid sequence is correct but the conformation is altered, most proteins will not be active. Furthermore, understanding the conformation of a protein is necessary for determining how it functions and interacts with other molecules. This information is invaluable when designing therapeutic drugs that affect the activity of a specific protein.
Hierarchical levels of organization
There are four hierarchical levels of organization of protein structure: primary structure, secondary structure, tertiary structure and quaternary structure. Primary structure refers to the sequence of amino acids. Secondary and tertiary structures refer to local and global interactions, respectively. Quaternary structure refers to the assembly of multiple polypeptide subunits in a multimeric (comprised of two or more polypeptides) protein. Proteins can be illustrated in a variety of ways, including space-filling (see Figure 3 for an example) or ball-and-stick (see Figure 3for an example) models, which depict every atom of the polypeptide. For a large polypeptide, this can be very complex and it is often difficult to focus on structural features. Therefore, a trace of the polypeptide backboneis often used to illustrate larger polypeptides. The backbone illustrates the path of the protein by tracing the connection between alpha carbons of consecutive amino acids. This allows one to visualize the path of the polypeptide (its conformation).
As stated above, the primary structure of a protein is simply the amino acid sequence of a polypeptide. All subsequent levels of protein structure are dependent on primary structure. The bonds responsible for the primary structure are the peptide bonds between consecutive amino acids. Primary structure is generally indicated by the polypeptide sequence, starting from the amino terminus to the carboxyl terminus.
The secondary structure of a protein is the local structure assumed by a portion of polypeptide via regular hydrogen bonds(bonds resulting from intermolecular attractions between molecules containing hydrogen and an electronegative element) between adjacent amino acids. There are two common secondary structures, the alpha helix (? helix) and the beta sheet (? sheet). These structures are formed by interactions between the peptide bonds rather than the side chains of the amino acids; specifically, hydrogen bonds between the imino group (NH) of one peptide bond and the carbonyl group (CO) of a nearby peptide bond.
The alpha helix
If one were to trace the polypeptide backbone of an alpha helix, it would resemble a spiral staircase; each amino acid represents one step and the side chains protrude along the outside (Figure 4). In this structure, there are hydrogen bonds between the imino group (NH) of one peptide bond and the carbonyl group (CO) of an amino acid directly above it in the helix. Returning to our spiral staircase analogy, the imino group of one "step" would hydrogen bond with the carbonyl group of the "step" directly above, which, in fact, is three steps away.
The Beta Sheet
A beta sheet is formed by hydrogen bonding between one stretch of amino acids and another stretch of amino acids in another portion of the same polypeptide (Figure 5). Imagine that the polypeptide backbone is a strip of paper folded back and forth, with a crease at every alpha carbon along the length of the backbone. Using this analogy, the side chains are above or below the crease in each pleat. Each peptide bond of one ? sheet (sometimes referred to as a ?-pleated sheet) is juxtaposed to another peptide bond in another nearby beta sheet via hydrogen bonding between the imino and carbonyl groups. The two stretches of polypeptide are held together by hydrogen bonding, which maintains the distinct pleated structure. The nature of the hydrogen bonds is the same as for an alpha helix - that is, the imino group of one peptide bond bonded to the carbonyl group of another peptide bond. The two peptide bonds involved can be between amino acids far apart in the primary sequence but brought together in the overall folding of the polypeptide. Beta sheets can be formed between regions of the polypeptide in the same orientation (parallel) or in opposite orientations (antiparallel).
Proteins are composed of different numbers and arrangements of alpha helices and beta sheets (Figure 6). For example, hemoglobin, the protein that carries oxygen in your bloodstream, is composed of alpha helices exclusively. Immunoglobulins, which make up the circulating antibodies in your bloodstream, are composed of polypeptides that are predominantly beta sheets. Hexokinase, the first enzyme in glycolysis, is composed of alpha helices and beta sheets. The ability to form an alpha helix or beta sheet is determined by the amino acid sequence. Some amino acids have a greater propensity for adopting one conformation over another, based on their side chains. For instance, prolines are rarely found in alpha helices. However, it should be noted that although the side chains can influence the likelihood of forming an alpha helix or beta sheet, they do not participate directly in the hydrogen bonding that determines these structures.
The tertiary structure of a protein is the overall folding of the polypeptide. This level of structure involves various kinds of global interactions and varies amongst different proteins. The interactions that determine tertiary structure occur between the R-groups of the amino acids. These interactions include covalent and non-covalent bonds. The most common types of chemical interactions that determine tertiary structure, and thereby maintain the conformation of a polypeptide, are disulfide bonds, ionic bonds, hydrogen bonds and hydrophobic interactions. These are illustrated in Figure 7.
In proteins, a disulfide bond (S-S) is a covalent bond formed between the sulfhydryl (SH) groups of two cysteine residues. It can occur between two cysteines separated by many other amino acids in the polypeptide chain or between cysteines in two separate polypeptides, as long as they are brought together when the protein is folded.
An ionic bond (electrostatic bond) is a non-covalent bond formed between oppositely charged ions; in proteins, between the side chains of charged amino acids. Positively charged amino acids tend to repel other positively charged amino acids and attract negatively charged amino acids. Negatively charged amino acids repel other negatively charged amino acids and attract positively charged amino acids.
In proteins, hydrogen bonds are non-covalent bonds that can occur between the side chains of polar amino acids, between the side chains of polar amino acids and the peptide bonds, and between the side chains of polar amino acids and water in the environment.
Hydrophobic interactions describe the tendency for amino acids to be arranged based on their interaction with water. For a soluble protein, amino acids with hydrophilic side chains tend be found on the surface of the protein, where they can interact with water, whereas hydrophobic amino acids tend to be buried within the center of the protein structure, shielded from water.
The non-covalent bonds and interactions described above are not very strong on their own, however, in an average polypeptide they are very numerous and their combined effects on protein conformation are great. Ultimately, the balance between all of these types of bonds and interactions determines and stabilizes the conformation of a polypeptide.
The quaternary structure of a protein is the assembly of multiple polypeptides into a functional protein. Some proteins are composed of a single polypeptide and therefore do not have quaternary structure; their conformation is complete when their tertiary structure is achieved. However, other proteins are composed of several polypeptide subunits. Quaternary structure refers to the folding and association of these subunits into a multimeric protein. For instance, hemoglobin is actually composed of four polypeptide subunits: two alpha chain polypeptides and two beta chain polypeptides. In order for hemoglobin to function, the subunits must be folded correctly and interacting properly. The bonds that determine and stabilize subunit assembly are the same as those that determine tertiary structure: disulfide bonds, ionic bonds, hydrogen bonds and hydrophobic interactions.
Conformation Determines Activity
Most proteins spontaneously fold into a specific conformation, driven by the bonds and interactions described for secondary, tertiary and quaternary levels of protein organization. The correct folding of a protein is essential for normal activity. Disrupting the conformation of a protein without changing the primary sequence or cleaving any peptide bonds can inactivate a protein's activity. This alteration of protein conformation is referred to as denaturation, and it results in the loss of activity. Three common methods of protein denaturation involve heat, extreme pH and reducing agents. Heat will disrupt the many hydrogen bonds that dictate folding. Since most proteins function at near neutral pHs, altering the pH of the protein's environment will also affect its many ionic bonds. Finally, reducing agents are chemicals that cleave a disulfide bond and restore the sulfhydryl group to the cysteines involved.
Proteins are polymers of amino acids. The conformation of a protein is critical for its activity. Protein conformation is determined by the sequence of amino acids and their interactions with each other and the environment. The structure of a protein has several levels of organization. The primary structure of a protein is the amino acid sequence of a single polypeptide. The amino acids are linked via peptide bonds. The secondary structure of a protein describes the local features of organization, determined by hydrogen bonding between adjacent peptide bonds. Two common secondary structures are the alpha helix and the beta sheet. The tertiary structure of a protein describes the overall shape of the polypeptide, and is regulated by more long-range interactions that occur between the amino acids throughout the polypeptide. These interactions involve the R-groups of the amino acids and include disulfide bonds, ionic bonds, hydrogen bonds and hydrophobic interactions. The quaternary structure of a protein describes the assembly of multiple polypeptide chains into a functional protein. The quaternary structure of a protein is generated by the same types of interactions that determine the tertiary structure. A protein's conformation and activity can be altered by denaturation, most commonly involving heat, extreme pHs or reducing agents.