Biochemistry I Fall Term, 2000

Lecture 6: Protein Primary & Secondary Structure

Assigned reading in Campbell: Chapter 4.1-4.3.

The Protein Architecture Tutorial introduces the basic principles of protein 3-D structure using Chime images of Protein G.
Conformational Elements of Protein Structure provides side-by-side Chime images of peptides in several secondary structure conformations.
Pauling, et al. (1951): The classic paper that anounced the structure of the a-Helix.
(Not assigned reading, however, this paper is an elegant description of protein structure "at the beginning".)

4.1 The Structures of Proteins Determine Their Function

Overview of the next two lectures:
Protein structures, even of the smallest proteins, can appear at first glance to be a daunting jumble of variously-colored balls and sticks. A typical (and reasonable) response from first-time viewers is, "What am I looking at?" or "What's important here?". In fact, practicing structural biologists ask themselves the same questions on viewing a new structure for the first time. The only difference between us and them, is that they have more experience in dissecting and reducing the complexity that is inherent in a structure that contains many thousands of atoms.
An efficient route to the goal of understanding structure is to first, organize our learning, and then our thinking into a hierarchy. For proteins, the structural hierarchy is:

1. Primary structure (1°): the amino acid sequence.
2. Secondary structure (2°): helices, sheets and turns.
3. Tertiary structure (3°): side chain packing in the 3-D structure.
4. Quaternary structure (4°): association of subunits.

Viewing protein structures at the various hierarchial levels listed above is an essential part of understanding the overall and the detailed aspects of protein structure and function. The textbook illustrations capture many of these features, but in the past few years, the availabilty of stand-alone programs (RasMol) and web-based visualization (Chime) has made it possible to view and to manipulate structural models in almost any way we want to see them.

The native conformation of a given protein is its functionally active conformation. The catalytic, binding, and structural roles played by proteins are all dependent on the the correct folding of the protein into a unique 3-D structure. In many cases, this native structure can be reversibly unfolded and then renatured to the native state. In other cases the denaturation is irreversible e.g. a boiled egg (extreme heat) or cottage cheese (extreme pH).

4.2 Primary Structure of Proteins

The strategies described in Campbell for sequencing proteins, i.e. determining their primary structure, illustrate these important principles:

1. Amino acid composition using an amino acid analyzer instrument following complete hydrolysis of the protein (6 M HCl, 110°, 24 hrs).
2. N-terminal and C-terminal analysis of the protein or more commonly, shorter peptide fragments (produced by chemical or enzymatic digestion).
3. Fragmentation of the protein into smaller peptides having overlapping sequences. Note these three examples:
   1. Cyanogen bromide (CNBr) cleaves the peptide bond after Met residues.
   2. Chymotrypsin hydrolyzes the peptide bonds that follow large hydrophobic residues, e.g. Phe, Tyr, Trp.
   3. Trypsin hydrolyzes the peptide bonds that follow positively charged residues, e.g. Lys and Arg.
4. Sequencing of the fragments with an automated instrument uses the Edman degradation method. The chemical mechanism of cleavage described in Campbell (and that described for CNBr cleavage) will not be covered in this course.

4.3 Secondary Structure of Proteins

A. Helical Structures

1. a-Helix
1. Pauling's discovery
2. Dimensions, geometry, & H-bonds
   3.6 residues/turn
   pitch = 5.4 Å/turn
   rise/residue = 1.5 Å
2. Other helices e.g. the 3₁₀ helix.

1. b-Hairpins
2. b-Sheets
1. parallel
2. antiparallel

Examples of three reverse turns are shown in Fig. 4.10 of Campbell. These short segments, frequently including Gly and/or Pro residues, have specific conformations in proteins and serve to connect the regular, repeating elements of secondary structure in a predictable way.

D. Fibrous Proteins

1. Keratin: coiled-coil of two a-Helices.
2. Silk: b-sheet.
3. Collagen: Three-stranded coiled-coil (See text).
4. Wool: a-helix with disulfide cross-links.

Each peptide bond is rigid and planar in the trans conformation. As noted in the Peptides lecture, however, the two single bonds adjacent to the a-carbon can rotate to three preferred torsion angles, each. These angles, are about the same for each of the peptide units in an a-helix: Y = -47°, and F = -57°.
Similarly, in b-structures, the angles assume values of: Y = 120°, and F = 125°. These are the ideal values for the dihedral angles of helices and sheets; a real protein shows minor (and a few major) deviations from the ideal values. An example of a "Ramachandran Plot" for Protein G (56 residues) is shown below:

If we have all of the Y and F angles in a protein, do we then have enough information to describe the 3-D structure? No, because the detailed packing of the amino acid side chains is not revealed from this information. However, the Y and F angles do determine the entire secondary structure of a protein.

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