1 INTRODUCTION TO PROTEIN STRUCTURE AND MODELING I. PROTEINS ARE LARGE MOLECULES MADE OF AMINO ACIDS Proteins fold up in a hierarchical manner....that is, in stages. Stretches of amino acids within the entire amino acid sequence of a protein (the primary structure) will spontaneously adopt one of several ordered structures (the secondary structure) which then folds into an overall energetically favorable tertiary structure. This tertiary structure is uniquely suited to carry out a specific function. Basic chemical rules govern the formation of a specific tertiary structure, depending on the primary sequence of amino acids. This sequence of amino acids is determined by the genetic information in DNA.
II. AMINO ACIDS: THE BUILDING BLOCKS Amino acids are molecules built around a central carbon atom (called an “alpha carbon”. To this alpha carbon, four different chemical groups are covalently bonded: an amino group (NH2), a carboxyl group O || C-OH
, a hydrogen, and an “R” group. “R”
stands for any of 20 different chemical groups. Thus, the alpha carbon, together with the hydrogen, amino group, and carboxyl group, is the same for all amino acid; they form what is called the amino acid “backbone”. The “R” group, on the other hand, is the only chemical group that differs among the amino acids; it gives each amino acid its “chemical personality”. Examples of different “R”groups (also called “side chains”) are shown on the left.
MolyMod® kits can be used to model amino acids. Notice that the amino acid model does not lie flat, like the diagram above. This is because the four electrons shared in carbon covalent bonds have tetrahedral geometry.
2 III. POLYPEPTIDES: A LINEAR CHAIN OF AMINO ACIDS Amino acids are joined together covalently to form polypeptides peptides in the ribosome, according to instructions provided by the genetic information in DNA. This linking is accomplished by “condensation”: an OH group is removed from the carboxyl end of one amino acid and an H is removed from the amino group of another amino acid. This forms water and a “dipeptide”. The bond joining one amino acid to another is called a “peptide bond”. Using the MolyMod® kit, you can construct a dipeptide, as shown on the left. At the bottom is the water that is split out. The problem with this model, however, is that there is complete rotation about the peptide bond joining the two amino acids. In reality, there is not free rotation due to the peculiar nature of this bond. The only free rotation is about each of the alpha carbon atoms. The angles of rotation about the alpha carbon atom are: PHI (Φ; the NH2 Cα bond) and PSI (Ψ; the CαCOOH bond).
Φ
peptide bond
Ψ
Cα
IV. POLYPEPTIDES CAN ADOPT A PREFERRED SPATIAL STRUCTURE
In an aqueous environment, stretches of 10 – 20 amino acids in a polynucleotide will SPONTANEOUSLY adjust the phi/psi angle to form a preferred “secondary structure”. Depending on the specific amino acid sequence, the two most common secondary structures are the ALPHA HELIX and BETA SHEET. Both are helical patterns: one looks like a winding staircase; the other lik zig-zag sheet. In both cases, the secondary structure is stabilized by several hydrogen bonds; while these are much weaker than covalent bonds, several of them in one region can provide significant stability. The Alpha Helix and Beta Sheet Construction Kits allow you to model these two secondary structures. Each kit contains amino backbone units that assemble magnetically to form one helix or the other. Each set of amino acids has a permanently fixed phi/psi angle consistent with either an alpha helix or beta sheet. To each backbone unit, one of 20 different side chains can be attached magnetically.
Note that because each amino acid is asymmetric (different chemical groups attached to the alpha carbon), a polypeptide is asymmetric as well. Thus, there are two different ends, which are called the N terminus (the end that terminates with the amino group of an ultimate amino acid) and a C terminus (the end that terminates with the carboxyl group of an ultimate amino acid).
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If we represent a beta sheet by an arrow, with the tail being the N terminal end and the head being the C terminal end (N C), and we have two such sequences separated by some other secondary structure: These sequences could hydrogen bond to form beta sheets in two ways:
anti-parallel beta sheet
parallel beta sheet
V. POLYPEPTIDE TERTIARY STRUCTURE CAN BE MODELED IN DIFFERENT WAYS This representation of a tetrapeptide shows all of the atoms in each amino acid. There are occasions in which this representation is useful; but there are other times....especially when you are trying to visualize a protein with hundreds of amino acids....when a more simplified representation is better.
4 In this diagram, we’ve first stripped off all the atoms in the amino acids that comprise the backbone of the tetrapeptide, and left just those of the side chains. Then, we connected each of the alpha carbons by a “pipe”. Further simplification involves removing all atoms, and just showing an idealized trace of what the backbone looks like in space. This is called an “alpha carbon backbone” representation.
Shown below are two representations of the beta-globin molecule (one of the components of hemoglobin). A beta globin has 141 amino acids that fold into eight alpha helices to form its tertiary structure. The “ball and stick” representation on the left shows all the atoms; the alpha carbon backbone representation on the right shows just the trace of the alpha carbons in space, allowing easier visualization of the eight alpha helices.
VI. PROTEINS FOLD UP SPONTANEOUSLY INTO A TERTIARY STRUCTURE With 15 tacks and a 4 foot Toober, you can explore the chemical forces that drive protein folding.
The Toober represents a polypeptide backbone; the colored tacks represent different side chains.
Distribute the tacks randomly but evenly along the Toober. Then fold the Toober according to the following chemical “rules”:
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You should have no trouble folding your toober so that all of the yellow, hydrophobic tacks are clustered together in the central core of the folded structure. However, it may be difficult to maintain this structure while simultaneously: • Pairing blue and red tacks (positive and negative charges that neutralize each • AND pairing orange tacks that represent disulfide bonds, • AND keeping all the polar white tacks on the surface of the protein Once everyone has completed the folding, hold them up. Although the same toober and tacks were used, each assorted the tacks differently (i.e. different amino acid sequence); thus, there are different folded shapes caused by the underlying chemical forces on the different sequences.
VII. THE THREE DIMENSIONAL STRUCTURE HAS BEEN DETERMINED FOR THOUSANDS OF PROTEINS AND IS IN THE PUBLIC DOMAIN To determine the three dimensional structure of a protein, the spatial coordinates of each atom in the protein must be determined. The two most common methods to do this are x-ray diffraction and nuclear magnetic resonance. The data is then filed in the Protein Data Bank, a non-profit on-line data bank, and can be accessed without charge by the public. For x-ray diffraction, the protein must first be crystallized. Data extracted from its diffraction pattern is used to calculate a three dimensional electron density map of the protein. At this point, the known amino acid sequence of the protein is then modeled into the experimentally determined electron density. The result is the 3D structure of the protein, with each atom assigned a x,y,z coordinate that uniquely positions it in 3D space.
6 This modeled structure is stored as data giving the x,y,z coordinates of each atom. These data can then be used by computers to generate a visual picture of the model, and a physical model of the protein.
