CHEM 440
Biochemistry I
J. D. Cronk   Syllabus [ Previous | Next ] Pick a lecture:
8.header

Lecture 8. Protein methods

Wednesday 21 September 2011

Ch.5 topics: Polypeptide diversity; protein purification and analysis; protein sequencing; protein evolution. Class discussion: Review for Exam 1; uric acid/urate as a product of purine metabolism and its role in the disease gout. Importance of protein primary structure. Protein solubility.

Reading: Voet, Voet, and Pratt; Ch.5 (pp.91-120) Link to Ch.5 PowerPoint

8. Summary
 

Lecture 8 Summary

The conformational space of a polypeptide chain can be represented as an ordered set of points on a Ramachandran plot. Locally repeating patterns of main-chain dihedral angles give rise to what we call secondary structure - principally involving α-helix and β-strand conformations - correspond to clusters of points on a Ramachandran plot.

  Ramachandran plot graphic  
An example of a protein topology diagram   Proceeding from secondary structure "elements", we can describe how these elements are linked in a particular protein, and the order of β strands in a β sheet structure. This is called protein topology, and can be represented by means of a topology diagram (see figure at left). The topology diagram helps us see how the three-dimensional structure of a long polypeptide chain - i.e. protein tertiary structure - can be built up from its secondary structure elements. Topological representation, although abstract, reveals patterns commonly repeated and reiterated throughout structural biology. The recognition of motifs or supersecondary structures as simple "compounds" of secondary structure that bridge the hierarchical gap between secondary and tertiary protein structure can be seen in this light. Given the many thousands of protein structures that have been obtained by the methods of X-ray crystallography and multidimensional NMR, a number of general principles of protein tertiary structure seem well-established.  
Above: Example of a topology diagram for a polypeptide chain. The parts of the chain that adopt β-strand conformation are shown as yellow arrows, α-helices are shown as red cylinders. These secondary structure elements are connected by the blue loops
Another level of the protein structure hierarchy, quaternary structure, describes how polypeptide chains with well-defined tertiary structures associate together in specific, and most often symmetric, forms. Proteins that adopt quaternary structural forms can have functional advantages over proteins with the same basic activity but no quaternary associations (i.e. those existing as unassociated molecules, or monomers).

Motifs and supersecondary structure

Small motifs, like the β-α-β unit introduced earlier, can combine to form larger elements of structure. Motifs, supersecondary structure, and domains are terms used to describe structural levels that bridge from simple, repetitive secondary structures to more complex and unique tertiary structures.

The figure at right (click on figure to show large version) shows two ways in which β-α-β units can combine to form a four-stranded parallel β-sheet. In the figure, the β-α-β motifs are labeled 1 and 2, and the connecting helices are shown as green cylinders. Part (a) shows a sequential parallel sheet (the order of the strands is 1-2-3-4, from left to right, and all helices lie on the same face of the sheet. In part (b), the order of strands is 4-3-1-2, and the helices from motifs 1 and 2 lie on opposing faces of the sheet. In addition, in this case a crevice or cleft forms between motifs (indicated by the green triangle) at the sheet's C-terminal edge, which is often the location of binding sites for small-molecule ligands or substrates.   Topology diagrams for two ways to connect beta-alpha-beta motifs to form 4-stranded parallel beta-sheets
The bottom portion of the figure shows a representation of each supersecondary structure as one might imagine it to appear upon a 90° rotation so that the C-terminal edge of the sheet is pointing toward the observer. This shows more clearly the side of the sheet that where the helices are found.

α/β structures

The parallel β-sheets that result from the stringing together of βα units take on two typical tertiary structure patterns. If the sheet curves around to form a closed cylinder, a structure called an alpha/beta (α/β) barrel forms. This is sometimes called a TIM barrel, after the enzyme triose phosphate isomerase (TIM) in which this tertiary structure was first observed. In the barrel, eight beta strands form the core of the protein, while α-helices all lie on the outside.
 

Protein structure determination: X-ray crystallography and multidimensional NMR. X-ray crystallography provides a high-resolution view of the structures of molecules. As applied to proteins, it typically reveals the locations of all non-hydrogen atoms that are well-localized in a tertiary structure to a resolution range of 3 - 1 Å.

Structural hierarchy: Subdomains. Domains and tertiary structure. "Invariant" features of protein tertiary structure. Quaternary (4°) structure - "multimers" of polypeptide chains.

Structural bioinformatics.

"Invariant" features of protein tertiary structure

We can summarize much of what has been learned about protein structure in the form of some generalizations that describe features common to most if not all proteins.

  • Proteins have well-packed, non-polar interiors, whereas polar and charged groups are accessible to the outside. Where polar groups occur in the interior (e.g. main chain carbonyls and amides), they tend to be paired.
  • The interior of proteins are close-packed, i.e. there are no large cavities.
  • The protein chains do not generally form "knots" (although see Ref. 4 below).
  • In comparing proteins, it is found that sequence (1° structure) similarity usually implies structural similarity. Note that there are numerous instances in which two proteins with little or no detectable sequence similarity nonetheless adopt very similar structures.
  • Overall, there is very little strain in protein structures. In cases where local strain exists, the energetic cost is covered by numerous favorable interactions within the rest of the structure.

Quaternary (4°) structure

The top of the protein structure hierarchy is the description of how polypeptide chains with defined tertiary structures associate (noncovalently) to form stable complexes called multimers. The classic example illustrating the difference between tertiary and quaternary structure is the comparison between myoglobin (Mb) and hemoglobin (Hb).

Learning objectives

  • Define the terms primary, secondary, tertiary, and quaternary structure.
  • Learn how to use a structure viewing program (such as PyMOL or Swiss PDB Viewer).
  • Describe motifs and supersecondary structure
  • Represent motifs with topology diagrams.
  • Describe and explain general features of protein tertiary structure.
  • For each of the following techniques, describe the principles upon which it is based, the type of information obtained through its use, and any significant limitations:
    • X-ray crystallography
    • multidimensional NMR

Page updated 08-04-2010

References:

  1. Branden, Carl & Tooze, John Introduction to Protein Structure (1st ed. 1991, Garland Publishing)
  2. Lesk, AM. Introduction to Protein Architecture (2001, Oxford University Press).
  3. Creighton, TE. Proteins: Structure and Molecular Properties (2nd ed, 1993. Freeman)
  4. Taylor,WR, Lin, K. "Protein knots: A tangled problem" (2003) Nature 421: 25.
footer
[ E-mail: cronk@gonzaga.edu ]