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A zinc finger is a small protein motif (a small protein fragment consisting of several secondary structural elements, i.e., alpha helices and beta sheets) that functions by binding to a specific sequence of DNA and regulates the expression of an adjacent gene (Rhodes and Klug, 1993). It consists of only 25 amino acid residues. Therefore, it is a good first model to bend before tackling some of the larger proteins. It is also useful in that it consists of the two most common secondary structures found in proteins - an alpha helix and a two-stranded beta-sheet. Multiple zinc fingers are found in a wide variety of different DNA-binding proteins (see the zif268 protein in this collection). All zinc fingers (of this class) contain two histidine and two cysteine residues that are uniquely positioned in the motif to bind one atom of zinc. The coordinate binding of zinc by the side chains of these four amino acids stabilizes this structural motif - with great "structural economy". More information about the zinc finger motif can be found in papers by Struhl (1989) or by Rhodes and Klug (1993).
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Maltoporin is a protein that is embedded in the outer bacterial membrane where it functions to transport nutrients (maltose) into the cell. Its structure is remarkable. It is made up of 18 strands of anti-parallel beta-sheet. These 18 strands wrap around to form a "beta-barrel". As a result, a hole or pore is formed through which the maltose can pass into the cell. Many of the "loops" that connect adjacent strands of beta-sheet dive down into the pore where they partially plug the hole - and impart specificity to the porin by only allowing maltose to enter. This protein is an excellent example of the structure/function relationship. Hydrophobic amino acid side chains protrude from the outside surface of the barrel where they interact with the hydrophobic lipid membrane. Hydrophilic amino acid side chains are positioned on the inside surface of the barrel where they allow the polar maltose to enter the pore. What better way to accomplish the transport of the polar maltose molecule across the hydrophobic lipid bilayer?
Quicktime Movie of rotating Maltoporin model [270k]
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Flavodoxins are small proteins (180 residues) that function as electron carriers in a variety of oxidation-reduction reactions (Mayhem and Tolling, 1992). This ability to accept electrons from one source and pass them along to a second is mediated through a single bound flavin mono-nucleotide (FMN). Therefore, just as beta-globin is uniquely designed to bind a heme group, flavodoxin adopts a three-dimensional shape that allows it to bind FMN and interact with a variety of electron donors and acceptors. The structure of flavodoxin is very informative. It is a compact globular protein composed of 5 strands of beta-pleated sheet surrounded on the outside by 4 stretches of alpha-helix. Interestingly, if the flavodoxin model is "denatured" - that is, if all the turns between the alpha helices and beta sheets were removed and the protein viewed as a continuous, linear sequence of amino acids, - the alpha helices are found to be interspersed in the continuous amino acid sequence. Their sequential order is: helix 1 >> sheet 1 >> helix 2 >> sheet 2 >> helix 3 >> sheet 3 >> sheet 4 >> helix 4 >> sheet 5. How these discrete units of secondary structure that are interspersed in the linear sequence of the protein fold into a three-dimensional shape with all 5 beta-sheets on the inside and the alpha-helices on the outside, remains the focus of scientists who are working to understand the forces that drive protein folding. The FMN group is bound at one end of the protein by amino acid side chains contributed from turns between strands of beta-sheet and alpha helices.
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p53 is the most commonly mutated protein found in human cancers (Cho et.al., 1994). It is another sequence-specific DNA binding protein. It functions as a tumor suppressor protein. Therefore, cells lacking a functional p53 protein are at risk of losing their normal growth control and developing into a cancer. The protein is large (289 residues) and model should be attempted only after some familiarity with the bender is developed by bending smaller proteins. However, the model is instructive in several ways. First, in contrast to beta-globin which is made up entirely of alpha-helices, p53 is predominantly composed of beta-sheets. There is only one short stretch of alpha helix in the model. Second, the protein is of intrinsic interest to students due to its important role in the development of human cancer. From a structure/function point of view, it is interesting to note that mutations that have been found to inactivate this protein in cancer patients are clustered around selected residues of the single alpha helix. p53 is known to contact DNA through this alpha-helix. Therefore, the current model suggests that a mutation in DNA that changes a single amino acid in the alpha-helix of p53 destroys its ability to bind DNA and suppress the formation of tumors.
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The green fluorescent protein (GFP) is responsible for the green fluorescence of the jellyfish, Aequorea victoria. Its structure is striking. It is remarkably similar to a lantern. Eleven strands of anti-parallel beta sheet form a barrel similar to that seen in maltoporin. The top and bottom of the barrel - or lantern - are closed off by short alpha-helices or loops connecting the beta strands. Where does the light come from? A single alpha-helix runs exactly through the center of the lantern, from bottom to top, and forms the "fluorophore" - the light-emitting structure. The formation of this fluorophore is also interesting and points out the close relationship between biology and chemistry. Three consecutive amino acids in this central alpha-helix - serine65, tyrosine66 and glycine67 - undergo a cyclization reaction to form a complex chemical entity with an extended pattern of alternating single- and double-bonds. This fluorophore absorbs light at a lower wavelength, and emits a bright green light via a fluorescence mechanism. GFP is a good protein with which to introduce the new field of biotechnology. This protein has become a popular marker for gene expression in mammalian cells. Therefore, the structure of the wild-type jellyfish protein has been altered - or engineered - to optimize its properties in mammalian cells. For example, its codons have been "humanized". Jellyfish use a different subset of the degenerate genetic code to translate GFP mRNA into protein. Biotechnologists have changed the sequence of the gene encoding GFP such that a different subset codons that are more common in human cells are used. In addition, mutations have been introduced into the gene that alter the absorption and emission spectra of the protein so that it is more useful in fluorescence-activated cell sorting techniques. More information regarding the GFP protein can be found in the paper by Chalfie et.al. (1994).
Quicktime Movie of rotating GFP model [184k]
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Max is a small protein that interacts both with itself or with another protein, Myc to form a functional DNA-biding protein (Blackwood and Eisenman, 1991). The Myc/Max heterodimer has been implicated in the development of numerous human cancers. A model of the Max/Max homodimer (two identical Max proteins that form a stable complex) is instructive for several reasons. First, it is possible to clearly represent the binding of this protein dimer to DNA. Therefore, this brings the story of the "central dogma" full-circle. That is, the information in DNA is expressed as a messenger RNA which is in turn translated into protein -- and then, a small subset of those proteins function by returning to the DNA and regulating the expression of certain genes whose protein products control the normal growth and development of the cell. Second, the structure of Max is unusual in that it is essentially two long alpha helices connected by a loop. The protein forms a homodimer via a "leucine zipper" (O'Shea et.al., 1989; Struhl, 1989). Because an alpha helix consists of ~3.5 residues per turn of the helix, every 7th residue is positioned on one face of the helix. In a leucine zipper, every 7th amino acid is a hydrophobic leucine residue. Two alpha-helices with this "heptad repeat of leucines" can therefore interact with one another through hydrophobic interactions of their leucines. This simple interaction domain can be easily demonstrated with the Max homodimer model.
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Zif268 is a zinc finger protein that binds to the nucleotide sequence GCG TGG GCG and regulates the expression of downstream genes. Zif268 contains three zinc fingers in tandem. Close examination of the zinc finger model will reveal that the amino-terminal end (the beta-sheet) and the carboxy-terminal end ( the alpha-helix) lie on opposite ends of the motif. This arrangement has significant consequences for the evolution of multiple-finger proteins. In Zif268, three fingers are joined together, C-terminus to N-terminus, such that the three alpha-helicies form a half-circle, with the alpha-helices on the inside. This inner surface of alpha-helices is precisely shaped to lie in the major groove of double-stranded DNA. Several amino acid side chains near the base of each alpha-helix form salt-bridges or hydrophobic interactions with the bases that comprise the Zif268 binding site. Each helix binds to three successive base-pairs -- therefore, the Zif268 binding site is nine base-pairs long. Structural biologists some day hope to design artificial zinc finger proteins that can be directed to any target DNA sequence where they will be equipped with additional protein domains that will either inhibit or enhance the expression of a target gene. More information regarding Zif268 and the structure-based design of novel zinc finger proteins can be found in papers by Pavletich and Pabo (1991) and by Greisman and Pabo (1997).
Quicktime Movie of rotating Zif model [270k]
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