What is the fastest way to crystallise lysozyme (for student course)?

What is the fastest way to crystallise lysozyme (for student course)?

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High school sudents are going to visit my university and I plan to demonstrate crystallisation of lysozyme. I ordered pure lysozyme from VWR. I can easily crystallise this within 15 min in batch (4% w/v NaCl, 35 mg/mL Lysozyme dissolved in 0.2M NaAc). However, as many different student groups will go through this experiment sequentially, it is preferable that it takes even less time (5 min waiting time maximum). I can of course try to optimise this on my own, but I am wondering if anyone has experience with the absolute fastest way lysozyme forms crystals in a batch setup (to save time and chemicals).

In summary: What are the most optimal buffer/pH/lysozyme concentrations that will give crystals (in a batch experiment) within a very short timeframe (<5-6 min)?

AP Biology question

This is from my daughter's AP Biology homework, at the end of the section on free energy, activation energy, enzymes, cofactors, etc. The section on the immune system is not until the end of the year. I think the question is too vague, makes too many assumptions, and requires a very complex answer. I think the teacher wrote it herself, thinking there is a relatively simple answer. What do you think? About the question? About the teacher? I can't imagine this question made it into a decently edited textbook.

The common cold is a viral infection that affects the upper respiratory tract. Symptoms include a cough, runny nose, and fever. a) Identify what happens to the body when it has a fever. b) Explain why having a fever is beneficial in fighting off the virus. c) Explain why having a long term fever (3+ days) can be dangerous to the human body.

I don’t think the question is too vague? There’s not really anything particular about immunology that corresponds with having a fever. From the section on enzymes, activation energy, and general principles, you should be able to answer why having an elevated body temperature would affect a [viral] infection, and why having one for a prolonged period of time would be detrimental to the host (human). I agree that it is a little vague, as I think the answer to the first part is just something along the lines of “raised temperature above homeostasis.”

Edit: It is expected in an AP biology course to make the connection between “standard” and non-standard conditions for metabolism to occur. pH range, temperature, etc. all contribute to an enzymes efficacy. Why do some enzymes work better in the saliva vs stomach, lysosomes or cytoplasm? Similarly, if you disrupt these (ie raise the body temperature by 4F), what might happen to these processes (and to the virus, by extension)?

What’s ‘diffraction’?

Diffraction involves passing photons through a crystal, which scatters them to produce a recognisable pattern. High-energy photons like X-rays provide the best resolution, hence ‘X-ray diffraction’.

A crystal is an ordered collection of a defined set of molecules, in a repeat pattern occurring over such a large volume that you can see it with the naked eye. Table salt crystals provide a familiar example, with sodium and chlorine atoms neatly packaged up. But while neat packages are rare in biology, crystals can be coaxed out of far more complex molecules, like proteins.

In a perfect crystal, every instance of an atom has an identical relationship to its neighbours. Because of this arrangement, photons are scattered in a highly regular way when they are passed through the crystal. This scattering is called ‘diffraction’. The resulting pattern (captured on film in the old days and now detected with a digital sensor) is determined by:

  • the relative positions of the atoms
  • the number of electrons they have and
  • how well ordered they are.

Lawrence Bragg was the first person to realise the potential of diffraction and worked out the fundamentals of the process. A new law in physics, Bragg’s Law, was named after him.

Part 2: HIV and Structural Biology

00:00:07.28 Hello, my name is David Haas,
00:00:09.29 and in my previous presentation,
00:00:12.26 I explained to you how cryo-cooling was invented
00:00:16.23 and has contributed substantially
00:00:18.25 to the field of structural biology.
00:00:23.09 And now, I'd like to give you an example
00:00:26.00 of an actual benefit that was derived from that,
00:00:28.22 which is the HIV epidemic
00:00:31.05 and some of the drugs
00:00:33.29 that were developed through structural biology.
00:00:38.17 Once again, we need to thank Max Perutz,
00:00:40.24 who began the field of structural biology
00:00:44.25 and had the great vision
00:00:46.27 that three-dimensional structures
00:00:48.17 not only permit the identification
00:00:51.05 of how the molecules act in living organisms
00:00:55.22 but in fact how the structures can be determined,
00:00:59.06 as well as how drugs can now be designed
00:01:01.27 using the process called structure-based drug design.
00:01:05.28 So, now I'd like to tell you a story
00:01:08.17 of how I have experienced
00:01:12.04 "science for the benefit of humanity."
00:01:15.17 While I was at the Cold Spring Harbor Laboratory,
00:01:18.02 I was told
00:01:21.06 that my cryo-cooling experiments and publications
00:01:24.04 probably advanced structural biology
00:01:26.07 by 10 or 20 years.
00:01:28.24 And at the time, you know,
00:01:30.15 10 to 20 years of scientific discovery
00:01:32.27 is really usually not a big deal,
00:01:34.16 and it didn't have any significance to me.
00:01:37.05 until while I was preparing for a presentation this last April,
00:01:42.10 at the Protein Data Bank in Rutgers,
00:01:45.23 they asked me to talk about exactly what I'm telling you:
00:01:49.21 the invention of cryo-crystallography.
00:01:51.25 And while I was doing that,
00:01:53.15 I discovered the AIDS epidemic
00:01:56.23 and the Lazarus effect.
00:01:58.21 And for those of you who don't know
00:02:01.00 what the Lazarus effect is,
00:02:02.24 in medicine, the Lazarus effect
00:02:05.06 is when many people are dying
00:02:07.08 and then some event occurs
00:02:09.24 where these vast numbers of people stop dying.
00:02:12.21 And that's exactly what happened
00:02:14.28 with the AIDS epidemic in 1996.
00:02:18.28 This diagram here is from the United States
00:02:23.00 statistics from the CDC.
00:02:24.28 But let me just tell you first about the worldwide AIDS epidemic,
00:02:27.20 which began in 1980.
00:02:31.06 There have been 76 million people who have been
00:02:36.04 infected with HIV virus since 1980,
00:02:38.23 of whom 39 million have died.
00:02:41.01 And today, worldwide,
00:02:43.02 there were 37 million people still infected.
00:02:47.15 There is no cure for AIDS,
00:02:49.13 as you know,
00:02:51.17 but there is a therapeutic treatment,
00:02:54.13 and that's what I'm gonna explain.
00:02:56.09 So, now if I can talk about the United States,
00:02:58.06 this is the death curve and the infection curve.
00:03:01.13 The red one's the infection occur
00:03:04.18 for AIDS in the United States.
00:03:07.15 So far, since 1980,
00:03:09.15 700,000 Americans have died from AIDS.
00:03:12.28 And as you see,
00:03:15.15 the curve begins in 1980
00:03:18.05 and continually increases,
00:03:20.04 with hundreds of thousands of individuals
00:03:22.23 becoming infected with AI.
00:03:24.10 with HIV virus each year.
00:03:26.16 Since the average lifetime,
00:03:28.10 if you had caught HIV in.
00:03:31.14 during the '80s,
00:03:33.26 was typically 2-4 years,
00:03:35.28 your statistics would move from the red curve to the blue curve,
00:03:39.11 which is the death rate,
00:03:41.14 in about 2 years.
00:03:43.12 And you see that the death rate is continuous
00:03:45.11 from 1980 to 1996.
00:03:47.14 It's a straight line, basically,
00:03:49.16 which means that none of the drugs
00:03:51.24 -- and there were many drugs that were tried --
00:03:53.25 produced any effect on the death rate.
00:03:59.27 In 1984,
00:04:02.10 the HIV virus was identified as being
00:04:06.03 the causal agent of HIV.
00:04:08.01 And then, in 1986,
00:04:10.07 proteins were isolated,
00:04:12.07 one of which is this protein,
00:04:13.25 which is HIV protease,
00:04:15.23 one of the enzymes that makes the HIV virus.
00:04:19.28 The crystal structure was actually determined,
00:04:23.26 the three-dimensional structure, in 1989.
00:04:26.14 And we can thank Merck Laboratory in New Jersey
00:04:29.22 for not only solving the structure,
00:04:31.22 but they published the data to the whole world
00:04:34.20 in the Protein Data Bank in 1989.
00:04:38.28 This gave the opportunity
00:04:41.11 for hundreds of other laboratories
00:04:42.23 to work on the HIV protease.
00:04:47.08 It turned out that inhibitors
00:04:50.07 were actually made,
00:04:51.22 and they were made using structural biology,
00:04:54.13 using cryo-cooling.
00:04:56.11 And in 1996, the therapy was introduced.
00:05:02.18 It was this cocktail of three drugs,
00:05:05.04 of which the HIV protease inhibitor was the most important.
00:05:09.08 And instead of dying, you would.
00:05:12.05 you would live a normal life,
00:05:15.17 with in fact.
00:05:17.24 it was called a manageable disease.
00:05:19.15 And because of that,
00:05:21.28 the individuals did not transfer to the death curve,
00:05:25.21 and you see this precipitous fall
00:05:28.10 in the death curve in 1996.
00:05:30.20 The death rate fell from 96%
00:05:34.04 down to below 20%.
00:05:36.13 So, you can just imagine,
00:05:38.06 between 1980 and 1996,
00:05:41.02 if you had become infected with HIV
00:05:44.04 and you went to your physician,
00:05:45.26 and he would tell you had a 4% chance of surviving.
00:05:50.16 But in 1996,
00:05:53.09 you would go in to. go to your doctor,
00:05:56.03 and he would say, yes,
00:05:58.06 today there's a therapy.
00:05:59.25 You will not be cured of HIV,
00:06:02.09 but it will in fact become a manageable disease.
00:06:05.09 And this dramatic fall in the death rate in 1996
00:06:09.23 is called the Lazarus effect.
00:06:11.25 Tens if not hundreds of thousands of Americans
00:06:15.08 lived who would not live.
00:06:18.18 And basically, the HIV protease inhibitor drugs
00:06:22.21 which were developed
00:06:24.20 were developed years and years faster
00:06:27.00 than they would have been
00:06:29.09 if there had not been structural biology,
00:06:31.09 and as it turns out, cryo-cooling,
00:06:33.17 which participated in that.
00:06:35.29 So, it makes me feel very good
00:06:37.25 that, literally, my research, done 50 years ago,
00:06:41.00 not ending up in dusty pages in a journal
00:06:44.15 but actually saving thousands of lives.
00:06:46.29 That really is science for the benefit of humanity.
00:06:51.29 Experiments that you do,
00:06:54.17 and publications that you make,
00:06:56.03 as a young scientist,
00:06:57.16 will in fact. could contribute very much to the future.
00:06:59.21 So, I hope you've become a little bit inspired
00:07:02.24 with the work that I have done,
00:07:05.01 and the fact that it inspired me,
00:07:06.29 and maybe it will inspire you to continue.
00:07:09.26 Thank you.

  • Part 1: Cryo-cooling Protein Crystals: The First 52 Years

The home lab expriments

A second approach is to design experiments around what can be readily found at home. A huge amount of physics, chemistry and biology can be investigated using regular everyday items.

For example, students can measure the force of gravity with a simple pendulum, or find the latent heat of ice by observing the temperature change when added to a glass of water.

What is the latent heat of ice? Source: Travis Nep Smith/Flickr

This has enormous appeal as it not only saves costs but also may improve learning outcomes for the students by making experiments more relatable to the world around us.

The downside is that some key experiments might require specialist, expensive apparatus, such as a decent optical microscope, well beyond what could be expected to be performed at home.

Nucleic acids questions

Hey guys, I guess you can say this is quite a random bit of the specification they decided to put in, but can anyone explain how excess nucleic acid causes gout?

Not what you're looking for? Try&hellip

(Original post by rm_27)
Hey guys, I guess you can say this is quite a random bit of the specification they decided to put in, but can anyone explain how excess nucleic acid causes gout?

No one actually knows the mechanism by which gout ultimately arises. It's so unfair when they ask these ridiculous questions to A-level students.

Nucleic acids, more specifically purines (rather than pyrimidines purines are adenine and guanine) are metabolised into uric acid. When uric acid crystallises, for as of yet unknown reasons, it causes gout. The more nucleic acid available, the more uric acid available, the likelier that it could crystallise and cause gout.

Still though, those are assumptions. Nonetheless, an association has been found between higher nucleic acid (protein) consumption from meat and seafood, and the incidence of gout.

(Original post by rm_27)
Hey guys, I guess you can say this is quite a random bit of the specification they decided to put in, but can anyone explain how excess nucleic acid causes gout?

Like Flying Cookie said, no specific basis for gout has been found. However, to add a few details, the main culprit in gout is high level of uric acid. This can be caused either by under-excretion or over-production of uric acid. Purines, which are double ring nucleotides, ultimately catabolize to form uric acid in your body.
One proposed mechanism is that uric acid crystallizes to form monosodium urate crystals. In a case of hyperuricemia (increased levels of uric acid), the monodsodium urate crystals may get deposited in different places. One common site for deposition is joints. When this happens, there is some sort of complex signalling (which we can ignore for present) that causes inflammation. This is characterized by increased presence of macrophages and other phagocytic cells such as neutrophils. So uric acid deposition in joints leads to "gouty arthritis".
Sometimes, over a long period of time, monosodium urate crystals may get deposited in your soft tissues in the form of nodules which are also called tophi (you can skip this if you find it difficult). The form of gout that results is called chronic tophaceous gout.

SO, to return to your original case: excess nucleic acid increases production of uric acid, which crystallized as monosodium urate in joints and soft tissues, leading to an inflammatory response which is called gout.
If you have any questions, ask away!


We have developed an extended project for an upper division biochemistry laboratory course that uses Mb as a central theme to investigate several important experimental techniques, as well as the relationship between the structure and function of a protein. Over six 3-h laboratory periods, the students isolate Mb from muscle tissue, spectroscopically characterize the oxidation state of its heme moiety and its ligand binding, analyze the protein's structure via molecular modeling programs, and track its thermal denaturation with FTIR. We have expanded and combined previously described experiments [29−32], and also updated procedures to reflect changes in the availability of structure analysis software like PyMOL 2, 37, 38 . This project has the advantage of fulfilling two sometimes disparate goals in laboratory curriculum development: It introduces students to a wide variety of biochemical techniques, while at the same time organizing the project around a central, biologically relevant theme.

Clear, Structured Daily Lesson Videos

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Experienced teachers guide you through every concept and example in your Matrix books.

Easy-to-follow lessons taught by subject matter experts

Gain in-depth knowledge and understanding with easy to follow theory explanations.

Learn at your own pace, online

Pause, replay or speed up and watch as many times as you need.

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Exam-style questions will be answered in detail so you don't miss a thing!

Honors College

When Phil McFadden talks about protein structures, it’s hard not to get swept up in his enthusiasm. The Associate Professor of Biochemistry and Biophysics still remembers the excitement he felt upon his first glimpse of the protein lysozyme. He seeks to instill that same feeling for the beauty of chemical form in the popular and innovative UHC colloquium Protein Portraits, which culminates in students creating a unique protein structure of their own design for a standing display.

These structures are not what you would normally find in science textbooks but are, rather, “portraits,” creative representations of proteins and their actions constructed from unconventional materials. In its last iteration this past spring, one student depicted the prion proteins linked to mad cow disease with colorful ribbons spiraling out of a ceramic cow’s head. Another used a halved globe and photograph collage to describe the proteins involved in HIV. At the end of the course, students display their work publicly in an exhibit that is part science fair, part art show. Some pieces – especially the edible ones – don’t last very long, but the protein portraits that can withstand the test of faculty and student hunger are kept in the Francis Cripps Reading Room of the Agricultural and Life Sciences Building on rotating display.

“In the everyday world we individualize our homes with house paint and landscaping, we decorate our rooms with rugs and furnishings, and we decorate our bodies with clothing and a hair style. Why not add a few artistic flourishes to a favorite protein? If we give ourselves something unique to remember it by, perhaps we will better bring an invisible protein into our mind’s eye where imagination can grab hold,” says Professor McFadden.

There are certainly plenty of proteins to keep the imagination busy. Nearly 77,000 protein structures are currently catalogued by the Protein Data Bank, and that number increases by a couple hundred each day. In its three iterations, the course has produced 36 protein portraits integrating scientific fact with artistic creativity.

The humanities and science find common ground in exploring the tantalizing question of what it means to be human. Students in Protein Portraits have the opportunity to use both scientific and artistic methods to describe a piece of the human puzzle that helps us resist disease, holds us together, and allows us to breathe: proteins. As Salvador Dali’s paintings have changed the way many people look at clocks, protein art from the course allows people to approach an essential component of our makeup from unexpected angles that uncover new meanings, resonances, and significance.

Anna Vigeland, now a graduate student in cellular and molecular biology at the University of Hawaii in Manoa, took the course this past spring and said she enjoyed viewing proteins in artistic terms that related form and function. “It was a different way to see them as descriptions in some science classes can be dense, it was helpful to take a step back and appreciate them.”

In the near future, perhaps more people will be able to appreciate proteins in a similar way, at least this is Professor McFadden’s hope. Just as iPhones and nanotechnology made the leap from imagination to devices and tools that have altered our lives, he predicts that structures in the protein world will also make the transformation from fantasy to fact, fact to art, and finally slip into the vernacular. His dream is to have a protein portrait emerge from the course that becomes a piece of the mood of the times, perhaps as a favorite toy of children who will learn while they play. “The protein portraits are works of art they are expressions of the heart,” says Professor McFadden. By forming personal bonds with natural building blocks, his students will continue to uncover the art of science and find science in art.

Watch the video: Lysozyme Presentation (August 2022).