Lesson 7
Protein Structure
Last week we learned that DNA is transcribed to mRNA which is then translated into a protein amino acid sequence. This week we will learn about the different types of protein structures.
Primary, secondary, tertiary, and quaternary structure
The amino acid sequence of a protein makes up the primary structure of a protein. When the amino acids of the polypeptide chain fold and form non-covalent interactions with one another they form secondary structure elements. There are two main types of secondary structure elements formed called beta-sheets and alpha-helices. When the entire amino acid chain folds and secondary structure elements interact with one another, the tertiary structure is formed. Finally, when multiple polypeptide chains interact with one another it is called its quaternary structure. Read this chapter from Molecular Biology of the Cell textbook for more detailed information about the energetics of protein folding, protein domains, etc. It is amazing how many different protein structures (some represented below) there are with a wide variety of functions (we will discuss protein function next week).
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Known protein structures
Protein structures are determined by X-ray crystallography, nuclear magnetic resonance (NMR), and cryo-electron microscopy (EM) (we will learn more about these techniques in Module 3). Known protein structures (where each atom is defined in space with three dimensional coordinates with respect to each other and at distances defined by the bonds formed between the atoms) are deposited in the Protein Data Bank (PDB). Each protein structure has a unique 4 letter/number code. For example, the first LspA structure is 5dir (Vogeley et al., 2016).
To visualize protein structures, you must use special computer programs. The most common is PyMOL.
Q1: Download the LspA pdb structure (5dir). Download PyMOL for educational use. Open the LspA structure in PyMOL. Show the protein structure as a cartoon. Change to your favorite color. What secondary structural elements are found in the LspA structure? PyMOL Wiki is a great site for learning different commands, but I will introduce some in the introductory meeting.
Determining protein size and structure
As each amino acid has a specific molecular weight, the primary structure of a protein therefore dictates overall molecular weight of the protein. It is important to know the molecular weight of a protein when you are purifying it (separating it from other cellular components, as we will also discuss in Module 3) so you know you have the correct protein. We use computer programs such as the ExPASy ProtParam tool to determine the protein's molecular weight, along with other important information such as the number of each amino acid, pI (the pH at which the protein has a neutral charge), and extinction coefficient which we will learn about later.
Q2: Using the LspA sequence below, determine the molecular weight of the protein.
MGSSHHHHHHSSGLVPRGSHMPDVDRFGRLPWLWITVLVFVLDQVSKAFFQAELSMYQQIVVIPDLFSWTLAYNTGAAFSF
LADSSGWQRWLFALIAIVVSASLVVWLKRLKKGETWLAIALALVLGGALGNLYDRMVLGHVVDFILVHWQNRWYFPAFNL
ADSAITVGAVMLALDMFRSKKSGEAAHG
Mass spectrometry can be used to determine the molecular weight of a protein in a sample. Read this brief description of mass spectrometry.
Q3: Which peak in the mass spectrometry plot below represents LspA? (Note: This is a spectrum that I took. Membrane proteins are sometimes difficult to assess via mass spectrometry as you can see by how noisy it is). The x-axis (labeled m/z) is the mass to charge ratio. We can assume that the protein has no charge so the x-axis just represents the mass of the protein.
One way to assess the secondary structure of a protein is by using circular dichroism (CD). The differential absorption of left and right circularly polarized light shows distinct spectra for proteins containing alpha-helices vs. containing beta-sheets vs. a random coil (unstructured regions of the protein) as can be seen in this figure. Beta-sheets have one minima while alpha-helices have two minima. If a protein contains both types of secondary structure, the spectra is additive with respect to the proportion of each.
Q4: Below is the CD spectrum of LspA. Again, CD is difficult to acquire with membrane proteins due to the detergent used to mimic the membrane. From this spectra what secondary structure elements does LspA contain?
Extra: FoldIt is a fun game which helps you understand the forces behind protein folding and allows you to try to fold a protein yourself. FoldIt for educators has additional tutorials about protein structure and folding.
[1] "File:Protein structure examples.png" by Axel Griewel is licensed under CC BY-SA 3.0