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Old 04-01-2009, 03:49 PM   #1
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Default Introduction to Protein Structure

Introduction to Proteins: A Structural Perspective.
By Kyle Nordquist (Center for Structural Biology, Department of Biochemistry, Vanderbilt University, Nashville, TN)
April, 2009

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.”


Figure 1



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.

Enzymatic activity

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).

Figure 3


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.




Figure 4
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Old 04-01-2009, 03:50 PM   #2
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Precipitation & Temperature stability – Hazed and Infoamed.

When we bring a pot of collected wort to a boil, we start to see lots of foam collect at the top of the brew kettle. Due to the properties of some proteins, high temperatures cause that tertiary structure to unfold. The main cause of proteins coming out of solution, or precipitation, (i.e. the foam or “break” that we observe), is exposure of previously hydrophobic surfaces of the protein to a water-based (aqueous) medium (the wort). When this occurs, the protein is no longer soluble, and has no choice but to come out of solution. This happens when proteins undergo shock as well, i.e., when a particular protein is still soluble at boiling temps, but is shocked to cool temps (such as the cold break)– again causing the proteins to unfold and fall out of the wort.
Just like how every person is different – every protein is different as well. Some proteins are extremely soluble at a wide range of temperatures and pHs, while others aren’t. Some proteins are best soluble at room temperature, others at refrigeration temps, and others at thermophilic temps – into the hundreds of degrees. Some proteins need high ionic strength buffers (relegated by different salt concentrations) and some are happy in pure water. Some proteins need low pH buffers, others need neutral pH, and still others need basic (high) pH conditions.
The temperature based properties of how a protein will either stay in solution or not is the most likely factor of various hazes in the beer. Most of the proteins come out of solution during the actual brewing process – but some are left behind in the finished, served product and are direct contributors to some aspects of mouthfeel, head retention, and how the beer clears (the latter also affected by residual yeast in suspension of course, but that’s not part of this discussion). It is not just a matter of more proteins = more haze, but it is more insoluble proteins = more haze. This is why it’s referred to as chill haze. These particular proteins are insoluble at colder temperatures and come out of solution. However, when the warm back up, they go back into solution, giving a clearer beer.
The same is somewhat true for head retention. When a beer is released from being under pressure, we know that the dissolved carbon dioxide begins to come out of solution. Bubbles are formed from this gas and travel through the beer upward, since the gas is less dense than the liquid, and therefore lighter in mass. On its way to the top, proteins in the beer will latch onto the CO2 bubbles and travel to the top with them. If a protein is too small, the bubbles will miss them (why a protein rest can be bad¥). If a protein is too large, the bubbles cannot carry them (why a protein rest can be good¥). So, only proteins in the middle range are trapped, and get transported. Once at the top, a matrix between gas and proteins is formed, creating the head or foam we like to see on the top of the beer. And we all know that this leads to increased sensory perception of the finished product in multiple ways (olfactory, mouthfeel, etc).
¥ Again, because it’s covered in many texts, I won’t get into protein rests here – just save to say that at particular temperatures proteins can also be chewed up by specific enzymes called proteases. If too many are chewed up (why it can be detrimental), the proteins are too small and won’t aid in head retention. If they are too big, however, a protein rest can be beneficial to help break them down into the median mass range. This all, of course, depends on the original modification of the malt, which is out of my scope.

So that’s what I have to say about proteins. I hope this has offered a new perspective in some ways – actually knowing what a protein looks like. If your curiosity has been peaked, and/or you just want to know what other proteins may look like, take a look around the Protein Data Bank. Here is the universal depository for all protein structures that have been determined.
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Old 04-01-2009, 04:29 PM   #3
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Great stuff PseudoChef. Can you go into the relationship between proteins and polyphenols? My extremely limited understanding is that polyphenols and proteins undergo reactions such that proteins help remove polyphenols and vice-versa. So ideally you want 'just the right amount of each'.

Somewhat related: I also read something that confused me a little. This article (regarding polyphenols and proteins) said that Polyclar and Silica Gel both had the correct (negative) charge to remove chill haze...but then said that the Polyclar attaches to the polyphenol end of the polyphenol-protein chain where the Silica Gel attaches to the protein side. But if they are the same charge...why would one bond to the polyphenol-end and the other bond to the protein-end? Hope I'm not going off on a tangent too much here.

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Old 04-01-2009, 04:40 PM   #4
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Good information. Thanks

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Old 04-01-2009, 04:48 PM   #5
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Originally Posted by SpanishCastleAle View Post
Great stuff PseudoChef. Can you go into the relationship between proteins and polyphenols? My extremely limited understanding is that polyphenols and proteins undergo reactions such that proteins help remove polyphenols and vice-versa. So ideally you want 'just the right amount of each'.

Somewhat related: I also read something that confused me a little. This article (regarding polyphenols and proteins) said that Polyclar and Silica Gel both had the correct (negative) charge to remove chill haze...but then said that the Polyclar attaches to the polyphenol end of the polyphenol-protein chain where the Silica Gel attaches to the protein side. But if they are the same charge...why would one bond to the polyphenol-end and the other bond to the protein-end? Hope I'm not going off on a tangent too much here.
I'm not up to verse with regards to polyphenols, or much of organic chemistry, for that matter - so I have limited understanding of that aspect as well.

As for why they have the same charge but bind different regions of the complex, my first instinct would be that the acceptor regions are different. There could be steric hindrence based on size difference between PolyClar and the Silica - so one may be too big to bind to one region, while the other is the correct size. This is also along the lines of perhaps there is a specific bond or side-chain interaction that must take place, and only one offers that scenario.
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Old 04-01-2009, 05:09 PM   #6
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Can you tell me more about cleavage?

Good work, PC. I read it all. You did a good job of making it fairly east to read and follow for us non scientists.

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Old 04-01-2009, 05:44 PM   #7
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¥ Again, because it’s covered in many texts, I won’t get into protein rests here – just save to say that at particular temperatures proteins can also be chewed up by specific enzymes called proteases. If too many are chewed up (why it can be detrimental), the proteins are too small and won’t aid in head retention. If they are too big, however, a protein rest can be beneficial to help break them down into the median mass range. This all, of course, depends on the original modification of the malt, which is out of my scope.
Great job, P-chef. I'd like to talk about this more here and this is probably the best thread in which to do it. Assuming proteolytic enzymes are most "active" in the 113°F-131°F (-ish) range, I'd like to know how one can look at a malt analysis and determine the best rest temperature(s) (and time) to maximize head retention while minimizing haze formation for any given malt.

Noonan briefly discusses protein rests and Soluble Nitrogen Ratio and that the higher the SNR, the higher the "protein rest" temp (or lack thereof). But I'd like to be a bit more precise about it. In fact, Noonan's info is even a bit outdated because most malt today has a higher SNR than what he accounts for.

Look at Duvel, for example. The high carbonation helps, but that is one killer head... like meringue. It's my understanding that Moortgat uses several different pilsener malts of a modification (and therefore SNR) that they specify to the maltster and then utilize specific protein-related rest temperature(s). (I believe they step-mash.)

Obviously, it would be difficult for homebrewers to get variously under-modified pilsener malts, but understanding how rest temps affect the protein-related parameters of a malt analysis would help us homebrewers to maximize head retention in our beers by using the malt analysis to determine rest temps. I'd like to know if we can determine rest temps more precisely by looking at SNR, total protein, etc.

We talk about proteins being too big, too small, and just right for head retention. So, using SNR as an example for a determining malt parameter, if I have a malt with an SNR of 42, what proportion/size proteins (small, large, just right) are already present in the malt? How will various rest temps affect it?... say a rest at 131°F vs. a rest at 141°F, for example.
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Old 04-01-2009, 09:53 PM   #8
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I, too, have always been enamored with the head formation and stability of Duvel as well as Houblon Chouffe (albeit the latter is loaded with hops, which have head formation/retention abilities of their own). It is very interesting to hear about the different malts that they may use. Have you checked out the Less Modified Pils available at MoreBeer?

I haven't deluged into any protein rest experiments. I conducted one on a dubbel I did about a year ago (133 for 30 minutes, which I understand to be bad at this point) and it absolutely killed my head retention. I love brewing Belgian beers, but have never been able to reproduce the head one sees of the commercial variety.

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Old 05-24-2009, 08:40 AM   #9
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*squeeees* pictures! and molecular structures! haha, thanks for the neat info!

I read somewhere recently that the larger proteins from beta protease are the head positive proteins, which should lead to the conclusion that a short, higher temp protein rest = better head retention? I'll see if I can find that article again now...

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Old 05-24-2009, 01:06 PM   #10
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I read somewhere recently that the larger proteins from beta protease are the head positive proteins, which should lead to the conclusion that a short, higher temp protein rest = better head retention? I'll see if I can find that article again now...
I'd be interested to see it. I think, ideally, one should know the malt's soluble and total protein content before choosing a protein rest temperature (and/or duration). But higher temperature or shorter time duration is definitely safer than lower temp./longer duration with most of the malt today. For British pale ale malts, I wouldn't do any protein-related rest.
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