Protein Structure/Function Relationships

Transcription

1 Protein Structure/Function Relationships W. M. Grogan, Ph.D. OBJECTIVES 1. Describe and cite examples of fibrous and globular proteins. 2. Describe typical tertiary structural motifs found in proteins. 3. Relate the primary amino acid sequence of a protein to its tertiary structure. 4. Describe the concept of domains and relate it to protein function. RESOURCES Lehninger (5 th Ed.), Chapter 4 (continued) OVERVIEW AND PERSPECTIVES Proteins fold into specific structures to generate the shapes, spatial relationships, environments, reactive groups and physical properties necessary to carry out their functions. We have already seen how the primary amino acid sequence of a protein can promote the formation of a certain class of secondary structure. We will now learn how secondary structural elements can assemble themselves into a variety of tertiary structural motifs and multisubunit quaternary structures. It should be noted that the folding of a protein is, for the most part, a synergistic process in which each successive stage in the process is enabled by and, in turn, stabilizes earlier stages. I. Fibrous proteins consist of extensive supramolecular complexes of monomers formed entirely from a single type of secondary structure (helix or sheet). They are insoluble in water due to a high content of amino acid residues with hydrophobic R groups, which are largely buried in the interior of the complexes. These proteins contribute strength and/or flexibility in structural roles. A. Keratin (see Figure 1)

2 Lehninger Figure α helix, cross linked by disulfide bonds to stabilize quaternary structure. 2. The biochemical basis for the permanent wave (see Figure 2). Lehninger Box 4 2

3 B. Collagen, the matrix for connective tissue (see Figure 3) Lehninger Figure The basic subunit is a tightly wound left handed helix (not α) which forms a 3 stranded superhelix with a right handed twist. 2. Repeating subunit Gly X Y, where X is often Pro and Y is often HyPro. Gly is required at the junction where the three strands make very close contact. [What would be the consequences of a genetic defect resulting in substitution of Ala for a Gly residue in collagen?] 3. Collagen fibrils are reinforced by covalent cross links between Lys and HyLys. Vitamin C (ascorbic acid) is required for hydroxylation of both HyPro and HyLys. [How might this observation relate to the symptoms of scurvy?] C. Silk: An exceptionally strong and flexible fiber based on a fibrous protein consisting entirely of β sheet structure.

4 II. Globular Proteins are by definition much more compact structures than fibrous proteins, approximating spheres in their overall shapes (see Figure 4). This class of proteins is much more diverse than fibrous proteins at all structural levels and includes enzymes, transporters, regulatory proteins, immunoglobulins (note the name), receptors, signal transducers, apolipoproteins and membrane structural proteins, as well as proteins with other functions. This structural and functional diversity is achieved using various combinations of a limited number of secondary structural motifs, primarily helical and sheet segments separated by turns and loops that permit folding of the protein into a compact structure (see, for example, Figure 5). Lehninger Figure 4 14

5 Lehninger Figure 4 15 A. What can be learned from examining the crystal structures of enzymes? 1. What classes of secondary structure are apparent in the structure of myglobin? Is this typical of all globular proteins? 2. How tightly packed are the amino acid residues in this structure? 3. Based on what we learned about protein folding earlier, what is holding this structure together? Is the distribution of amino acid side chains depicted in Figure 5 consistent with this conclusion? 4. Where in this structure would you expect to find the following amino acid residues? Pro, Glu, Arg, Leu, Thr, Asn?

6 5. Each of the α helices present in this structure has a series of highly polar peptide bonds as well as some residues with polar side chains. How are these structures incorporated into the hydrophobic core of this protein? 6. The heme group incorporates an iron atom that must be maintained in the reduced (Fe 2+ ) state in order to reversibly bind oxygen. Where is the oxygen binding heme group located in this protein? Why is this location important for the function of myoglobin? Can this function of the tertiary structure be generalized to other proteins? 7. The model shown in Figure 5 is constructed from a set of atomic coordinates obtained from the crystallized protein by x ray diffraction and submitted to the protein data bank. Does this model depict THE structure of myoglobin? Suggest some conditions that might cause this structure to change. [See Lehninger, Box 4 4 for an explanation of techniques for determining the threedimensional structure of proteins.] B. Figure 6 depicts the x ray crystal structures of two much more complex proteins with molecular weights of 62,000 and 74,000, respectively. 1. What types of secondary structure can you detect in these models? 2. These proteins have only 30% identity in amino acid composition. However, much of their secondary and tertiary structures are virtually superimposable. Moreover, the residues that participate in catalysis are spatially located in exactly the same positions relative to the rest of the protein structure. Both of these proteins are

7 enzymes that catalyze hydrolysis of lipophilic esters. What does this imply about the role of three dimensional structure in the function of enzymes? C. Common folding motifs: Many proteins are able to fold to their native conformations during their synthesis or even after being denatured. Others may require the assistance of proteins called chaperones for proper folding or may lose the information needed for correct folding due to posttranslational modifications such as proteolytic cleavage or formation of disulfide linkages. 1. Like the proverbial politics (All politics is local.), protein folding begins with local interactions to form secondary structure (see Figure 7). Lehninger Figure 4 19, Adjacent secondary structural elements then interact to form simple tertiary structural motifs, which eventually interact with each other to form more complex motifs (see Figure 8). [Why would this order of folding be favored over interactions between elements that are more distant in the amino acid sequence?]

8 Lehninger Figure 4 21 D. Large globular proteins often form distinct globular regions called domains. These domains tend to retain their characteristic three dimensional structures, even when separated from the rest of the protein. A domain may be associated with a particular function or property of the protein, for examples, binding of a substrate or generation of an immune response. The enzymes depicted in Figure 6 have a deep hydrophobic substrate binding pocket formed between amino terminal and carboxyl terminal domains. Both domains contribute residues to the catalytic active site that lies between them. E. Quaternary structure: The association of two or more polypeptide subunits (protomers), usually by non covalent interactions, forms a multimer. The subunits may or may not be identical and the individual subunits may play similar or identical roles. The multimeric structure may play a role in regulation, in that the complex includes both regulatory and catalytic subunits or binding to one subunit affects the activity of another. We have already examined some fibrous proteins that are multi subunit structures. Others include the ribosome which catalyzes protein synthesis, viral particles and the fatty acid synthase complex from E. coli, which catalyzes a series of reactions in the synthesis of palmitic acid. Hemoglobin is an example of a relatively simple tetrameric protein composed of α and β subunits (see Figure 9).

9 Lehninger Figure 4 22 F. Prions, infectious agents with no nucleic acids (see Lehninger, Box 4 6). 1. These proteins (Mr = 28,000) apparently produce disease by somehow inducing misfolding of proteins that are already present in the brain. 2. They cause fatal, incurable diseases referred to as spongiform encephalopathies, including mad cow disease, Creutzfeldt Jakob disease and kuru in humans and scrapie in sheep.