- Apr 18, 2007
- Reaction score
- West Chicago 'Burbs
Introduction to Proteins: A Structural Perspective.
By Kyle Nordquist (Center for Structural Biology, Department of Biochemistry, Vanderbilt University, Nashville, TN)
At this point, we have all undoubtedly come across the term ‘protein’ during our brewing research. Most likely, we brush it off to our generalized understanding that they are some sort of molecule, it coagulates (or precipitates) during hot and cold break, and that, some brewers do a protein rest during the mash. Some of you may be aware that the enzymes we are interested in for starch conversion are actually proteins themselves (enzyme is a fancy word to denote a particular protein has catalytic activity). Let’s go back to that first statement, though: some sort of molecule. What does this molecule look like? What does any molecule look like, for that matter? You’re probably flashing back to high-school chemistry now – erector set-like contraptions of bonds and carbons and oxygens and ohgodshootmenow. But it doesn’t have to be like that (at least, I hope it doesn’t). To really get at why these molecules precipitate, why they can cause haze and foam, and why they undergo rests during the mash, I think it will help if you have a better understanding of what proteins actually consist of. And like anything when you’re trying to learn, pictures always help. Lucky for us, technological advancement is booming these days, and under the right conditions, seeing what proteins look like (what we call determining their molecular structures at the atomic level) has never been easier. Structure determination helps us understand how proteins bind to different proteins facilitating molecular reactions and propogating cellular signals. Proteins, as they relate to biology – are at the heart of everything (after all, there’s around 20,000 different proteins in your body right now). They’re pretty important in beer, too.
What makes a protein?
Well, that’s easy: Amino acids. In biology, there are exactly 20 different amino acids that we deal with. Just as a cell is the building block of life, amino acids are the building blocks of protein. They are small molecules composed of hydrogen, carbon, nitrogen, oxygen, and sometimes sulfur atoms. As you would imagine, each amino acid has distinct chemical properties that, in various combinations, dictate the overall chemical properties of the protein they encompass. To make this easy, let’s simply say that amino acids have two parts: their backbone and their sidechain. Amino acids are named as such because their backbone consists of an amino group (NH3) and a carboxylic acid group (COOH). Now, if a couple amino acids string together as shown in Figure 1, it is simply referred to as a peptide – a fancy word for small protein. When the peptide elongates past, say, 10-15 amino acids (or residues) or so, we start getting into protein territory. Proteins span a gigantic gamut in size – 3-4 kilodaltons* to Megadaltons. Amino acids bind together by linkages between carboxy end (terminus) of one molecule to the amino terminus of the next. They keep stringing together in this fashion as the translational need dictatesǂ.
*a Dalton refers to a unit of mass used in science: 1 Da is equal to the molecular weight of 1 hydrogen atom.
ǂIn biology, proteins are the translated product of RNA, which is the transcripted product of DNA, the stuff that makes up our genes. For a more elaborate description, you can Google “The Central Dogma of Molecular Biology.”
Protein structure and folding – Secondary, Tertiary, and Quaternary
Now, the majority of proteins aren’t just straight chains of amino acids. Think of a rogue string that you just pulled off the seam of your shirt. Now, it’s probably not perfectly straight. There are likely some kinks, bends, and maybe even a loop or two. This is basically what is called “secondary structure” of the protein (with primary structure defined as the specific sequence of amino acids). There are basically 3 types of secondary structure in reference to a protein: alpha helices, beta sheets/strands, and random coil. Helices are long coiled segments, beta sheets are straight sections of the protein, and random coil is basically just random loops and otherwise regions void of well-defined structure.
Tertiary structure is the overall structure of one specific protein. With alpha helices and beta sheets, etc., these different foundations (or motifs) will still be attracted to opposite charges, hydrophobic (not water soluble) patches, and other elements (hydrogen bonding, salt bridges, van der Waals interactions, etc), which will cause the overall protein fold. See Figure 2 – this is our protein of interest for the moment, alpha amylase derived from barley. It has a good dispersion of alpha helices, beta sheets, and random coil – but overall, it is quite globular (spherical) due to it folding into and onto itself in various places.
Figure 2, PDB ID: 1RPK, Robert, X. et al. (All molecular structure figures made with the Pymol Molecular Graphics Suite.)
Sometimes, having one copy of a particular protein is not enough for a functional molecule. Many proteins actually self-assemble, having multiple copies of the same protein binding together to help facilitate its particular activity. This is called oligomerization. The overall assembly of the protein when it contains multiple copies of the same unit refers to its quaternary structure.
So the big deal about the proteins that we are concerned about is their enzymatic activity. I previously referenced this a little bit, and gave a little sneak peak in the structure I pictured above, but the enzymes we depend on to convert those starches to sugars are proteins themselves. We primarily hear about alpha and beta amylase. These proteins are responsible for recognizing starch molecules and breaking them apart (cleaving). I won’t get into what specifically the amylases do, as this is covered already in various brewing literature. What I’d like to do is show you why they do what they do – with reference to the structure. When we have a catalytic protein such as an enzyme, it will most likely have an active site – a region in the structure that serves to attract another molecule of interest and facilitates some type of molecular mechanism with regards to this molecule.
Looking now at Figure 3, we see a surface representation of the protein – something a little more accurate to how it actually appears in the cell. It is a solid species, containing various mountains, valleys, and other deformations throughout. Based on the type of amino acids comprising this particular structure, different regions of this protein will embody different characteristics:
- Electrostatics: basically this is the specific charge at that particular surface of the protein. Some surfaces may be highly negative – serving to attract positively charged species, and vice versa. Lots of regions will also just be neutral – a type of tether, possibly used to separate the negative regions from the positive regions.
- Hydrophobicity: When regions of the protein are comprised mostly of amino acids that are not water soluble, they fold in on themselves creating “cores” or “pockets” that actually help stabilize the overall fold of the protein since they pack tightly together.
- Surface exposed side chains: This characteristic may not embody a specific role in any protein, but often times in a structure, the side chains of various amino acids will poke out into solution. This offers avenues of modification, where not only other molecules can interact, but where binding and other types of modification can occur (for instance, lysine side chains can be what’s called ‘ubiquitinated’ – addition of another molecule to it – and thus the protein can be signaled for other cellular roles).
Now see that orange molecule in that center valley in the structure (Figure 4)? Packs nicely in there, doesn’t it? That orange molecule is a sugar packed in the active (or catalytic) site of the amylase. This is where the cleavage takes place. Based on the chemical properties of the residues that form that active site, it serves to attract the starch or sugar molecule, and also based on the properties, the amylase is able to destabilize the bonds between the sugar molecules, breaking them down (cleaving them) in simpler and simpler forms – what we brewers see as starch conversion into sugar.