Study Finds New Moves in Protein’s Evolution

27 September 2013  The Scripps Research Institute

 

Protein SourcesLA JOLLA, CA – Highlighting an important but unexplored area of evolution, scientists at The Scripps Research Institute (TSRI) have found evidence that, over hundreds of millions of years, an essential protein has evolved chiefly by changing how it moves, rather than by changing its basic molecular structure.

The work has implications not only for the understanding of protein evolution, but also for the design of antibiotics and other drugs that target the protein in question.

Proteins are machines that have structures and motions

 

“Proteins are machines that have structures and motions,” said TSRI Professor Peter E. Wright, who is the Cecil H. and Ida M. Green Investigator in Biomedical Research and a member of TSRI’s Skaggs Institute for Chemical Biology. “While we’ve known that proteins evolve via structural change, we haven’t really known until now that they also evolve via changes in their dynamics.”

Creating and studying proteins is a complicated business – in this video we take you behind the scenes with people who do it.

Backstage Science: Protein Crystals

The new study, which appears in Nature Structural and Molecular Biology on September 29, 2013, focuses on the enzyme dihydrofolate reductase (DHFR), which is so important for synthesis of DNA that it is found in almost all living organisms. DHFR is also a frequent target of medicines, including antibiotic, anticancer and antimalarial drugs.

Family Lineage

 

Human and E. coli DHFRs are structurally conserved but have different active site loop movements.

 

Wright and his laboratory have been interested in learning more about DHFR so scientists can target it more effectively and better thwart the emergence of drug resistance. In a study published in 2011 in Science, Wright and his colleagues demonstrated that the dynamics of the DHFR enzyme in the common bacterium E. coli are crucial to its catalytic function.

For the new study, the researchers analyzed and compared the dynamics of the E. coli DHFR enzyme with those of human DHFR: despite eons of separate evolution, the human and bacterial enzymes retain very similar atomic-level structures.

The team used a variety of techniques to characterize the two versions of the enzyme, including X-ray crystallography and nuclear magnetic resonance, analyses of DHFR amino-acid sequences and evaluations of the enzyme’s functionality in cells and in vitro under various conditions.

They also examined DHFRs from other species in addition to bacteria and humans to get a better idea of the evolutionary paths the enzyme took on its way to higher organisms.

“We didn’t imagine, when we started, how different the dynamics would turn out to be and that there would be an evolutionary pattern of atomic-level dynamics in the enzyme family,” said Gira Bhabha, who was first author of the study. Bhabha, a graduate student at TSRI during the study, is now a postdoctoral researcher at the University of California, San Francisco (UCSF).

E. coli DHFR uses relatively extended motions of flexible amino-acid loops in its active region to grip and release its binding partners. The human enzyme seems to move subtly and efficiently by comparison and essentially with a different mechanism. “The dominant motion in the human enzyme is a clam-shell-like movement with a twist, which allows opening and closing of its active site,” said Bhabha.

Looking Back to Chart a Path Forward

Bhabha and Wright suspect that these strikingly different dynamics of the E. coli and human DHFRs evolved as adaptations to very different cellular environments. Indeed, the human DHFR appears to be so well tuned for working in human cells that—as the researchers found—it cannot work properly in E. coli cells. “It seems that the much higher concentration of product molecules in E. coli cells effectively shuts down the human version of the enzyme,” Bhabha said.

Wright and his laboratory now plan further investigations of DHFR’s dynamics and hope eventually to elucidate the sequence of mutations that occurred to differentiate DHFR in humans and other mammals from the evolutionarily older, bacterial forms of the enzyme.

That evolutionary history should help scientists understand how evolutionary changes in DHFR lead to drug resistance. Knowing how human DHFR differs in its dynamics from its counterparts in bacteria and other disease-causing organisms also should enable researchers to design anti-DHFR drugs that are more specific for the target enzyme and have fewer side effects.

Other contributors to the study, “Divergent evolution of protein conformational dynamics in dihydrofolate reductase,” were Damian C. Ekiert, Madeleine Jennewein (who made substantial contributions to this research while working in the Wright lab as a high school and undergraduate intern), Gerard Kroon and Lisa M. Tuttle of TSRI (Ekiert and Tuttle, TSRI graduate students during the study, are now at UCSF and the Fred Hutchinson Cancer Research Center, respectively); Christian M. Zmasek and Adam Godzik of the Sanford-Burnham Medical Research Institute; TSRI Professor H. Jane Dyson, who co-supervised Bhabha’s research; and TSRI Professor Ian A. Wilson.

The study was supported by funds from the National Institute of General Medical Sciences (GM75995 and U54GM094586), the Skaggs Institute of Chemical Biology at TSRI and the Damon Runyon Cancer Research Foundation.

Attached files

  • Molecular biologist Peter Wright is a professor and Cecil H. and Ida M. Green Investigator at The Scripps Research Institute.

 

Brown Rice Protein: For Your Digestive And Athletic Needs

By Scarlett Blandon, MS RDN. 

Unless you were a vegan (or a yogi…or maybe a protein powder junky) the chances of you knowing about brown rice protein are slim to none.  Rice protein is a novel source of protein derived from the entire rice grain (including bran layer), and is available as a concentrate or isolate just like other protein powders.

It offers several benefits that other protein powders do not, but chances are those merits have been drowned out by:

(1) the negative stereotypes surrounding rice protein’s  plant-based origin;

(2) the popularity and media-hype of whey protein.

Sure, rice protein might not have all the looks and attractive qualities that whey does, but it’s those unique differences that make it an outstanding alternate protein source for athletes and sports enthusiasts alike.  Here are a few reasons why you might consider brown rice protein:

 

Easy on the stomach—and the immune system.

Good DigestionDairy- and soy-derived protein supplements might be wonderful for X number of reasons, but unfortunately, not everyone can digest these proteins, and some might even be highly allergic to them.  The Food Allergy Research & Education Organization states that about 15 million Americans have food allergies and this number appears to be on the rise1.  Milk and soy happen to be two of eight foods accounting for 90% of all food allergies1. Aside from dairy allergies, 30-50 million Americans are lactose intolerant2.  And despite lactose-free claims on dairy-derived protein supplements, many still anecdotally experience GI distress which can negatively interfere with training or performance (or number of friends).

Gluten is another, increasingly common immune-system offender. Some commercial protein supplements may contain ingredients derived from or made with gluten. This can be detrimental to the 3 million Americans diagnosed with Celiac disease and many more who remain undiagnosed3.  What’s worse is that damage to the microvilli from gluten can actually cause a person to develop lactose-intolerance, rendering them doubly restricted from those food groups4.

On the other hand, brown rice protein is derived from rice, a well-known hypoallergenic food source.  As a staple food in many cultures (for thousands of years!), rice is highly unlikely to elicit an allergic reaction (or intolerance) and is not surprisingly recommended as a first food for babies.  As such, rice protein is expectedly gentle on the GI tract and may offer greater benefit to those athletes or exercise enthusiasts with food allergies, intolerances or sensitivities.

Aids in recovery and builds muscle, much like whey.

Woman and man with six pack absUp until recently, nothing was truly known about the ability of rice protein as a sports nutrition supplement.  Despite this, rice protein withheld much criticism from the athletic and scientific community given previous literature on the generally inferior quality of plant-based proteins and other data showing that low doses of plant-based proteins (mainly soy- and wheat-derived) do not increase muscle protein synthesis compared to animal-based proteins5,6.

Yes, rice and thus rice protein is limited in lysine and apparently less digestible than dairy proteins. But do these apparent “weaknesses” in rice protein make it unsuitable for sports nutrition? One study to date, says no.

The study, published in Nutrition Journal in June 2013, found that 24 collegiate athletes were able to obtain significant gains in muscle, power and strength whether they were taking a 48g rice protein or whey protein supplement following resistance training for 8 weeks7. They also experienced similar increases in acute recovery. Despite differences in amino acid profile, digestibility and digestion rate of the proteins, there were no statistical differences between groups.

The authors assert that protein type or composition is of less significance, when key nutrients are adequately provided. In this case, one key nutrient: leucine.  The leucine levels provided by the brown rice protein supplement appeared to be within or above the 2-3g threshold needed to maximize muscle protein synthesis8-11.

Although more research is needed in this arena for rice protein, these results indicate that at 48g, rice protein can serve as a substitute for whey protein for building muscle and strength.  This would be especially beneficial for those athletes who follow a vegan/vegetarian lifestyle, are unable to digest dairy-based proteins and/or are looking for an alternate protein source.

Suits vegan or other plant-based lifestyles.

Vegan Menu SelectionsAlmost 16 million people consider themselves vegetarian and another 6 million consider themselves vegan in the US alone according to one 2012 survey12.  Motives behind these plant-based lifestyles might include views on animal welfare, religious and cultural beliefs and/or environmental concerns.  However, 47% of vegans indicate that their major reason for following this diet is actually health, followed by animal welfare (40%)13.  While exercise is a health-related activity, it is not unreasonable to consider the inclusion of some athletes or sports enthusiasts in this population.

Although vegans & vegetarians are able to consume all essential nutrients from plant-based foods alone, it can be a challenge for athletes when certain nutrients like protein are needed in higher quantities.  Supplements like rice protein offer a convenient and concentrated source of protein to help meet their needs. Unlike soy, rice protein does not contain phytoestrogens which can potentially interfere with hormones.

Rice protein is also an excellent option for those trending on natural or other plant-based lifestyles.  Unlike many dairy- or soy-based protein supplements, rice proteins are predominantly processed using only water and natural enzymes rather than toxic solvents like hexane. Certain rice proteins may also offer value over other protein sources since it is not from a genetically modified source, does not come from an animal known to be treated with growth hormones (rbST/bGH), anabolic steroids (AAS), estrogens and other hormones, antibiotics or other chemicals known to, suspected of, to affect or have an impact upon human health.

Whether you are looking to rotate your protein source, give your stomach a break, trend on a novel and natural product all while building muscle and gaining strength, rice protein might be the choice for you.

 

About the Author: Scarlett Blandon is the in-house nutrition scientist for Axiom Foods, the worlds’ leading manufacturer of hexane-free rice protein among other plant proteins, and for Growing Naturals, a consumer brand specializing in hypoallergenic plant proteins and natural lifestyle products. At Axiom and GN she oversees all research-related ventures and nutrition communications. Having worked closely with renowned researchers in the past, she is dedicated to expanding the literature on rice- and other plant proteins while cultivating the knowledge of consumers and manufacturers alike.

REFERENCES

  1. Facts and statistics. Food Allergy Research and Education. Available at:  http://www.foodallergy.org/facts-and-stats
  2. Lactose intolerance. Wexner Medical Center, Ohio State University. Available at: http://medicalcenter.osu.edu/patientcare/healthcare_services/digestive_disorders/lactose_intolerance/Pages/index.aspx\
  3. Case S. Celiac disease, gluten sensitivity and the gluten-free diet. July 2011. Available at: http://www.nutrition411.com/component/k2/item/28313-celiac-disease-gluten-sensitivity-and-the-gluten-free-diet
  4. Lactose intolerance and celiac disease. Celiac Disease Foundation. Available at: http://www.celiac.org/images/stories/PDF/lactose-intolerance.pdf
  5. Wilkinson SB, Tarnopolsky MA, Macdonald MJ, et al. Consumption of fluid skim milk promotes greater muscle protein accretion after resistance exercise than does consumption of an isonitrogenous and isoenergetic soy-protein beverage. Am J Clin Nutr. 2007; 85: 1031-1040.
  6. Norton LE, Layman DK, Bunpo P, et al. The leucine content of a complete meal directs peak activation but not duration of skeletal muscle protein synthesis and mammalian target of rapamycin signaling in rats. J Nutr. 2009; 139: 1103-1109.
  7. Joy J, Lowery RP, Wilson JM, et al. The effects of 8 weeks of whey or rice protein supplementation on body composition and exercise performance. Nutrition J. 2013; 12:86
  8. Norton LE, Layman DK. Leucine Regulates Translation Initiation of Protein Synthesis in Skeletal Muscle after Exercise. J Nutr. 2006; 136(2): 533S-537S.
  9. Norton L, Wilson GJ. Optimal protein intake to maximize muscle protein synthesis. Agro Food industry hi-tech. 2009; 20: 54-57.
  10. Paddon-Jones D, Sheffield-Moore M, Zhang XJ, et al. Amino acid ingestion improves muscle protein synthesis in the young and elderly. Am J Physiol Endocrinol Metab. 2004; 286: E321-E328.
  11. Tipton KD, Ferrando AA, Phillips SM, et al. Postexercise net protein synthesis in human muscle from orally administered amino acids. Am J Physiol. 1999; 276: E628-E634.
  12. Newport F. In the US, 5% consider themselves vegetarian. Gallup Wellbeing. 2012. Available at: http://www.gallup.com/poll/156215/consider-themselves-vegetarians.aspx

Scientists Clarify Structural Basis For Biosynthesis Of Mysterious 21st Amino Acid

Researchers at the RIKEN Systems and Structural Biology Center and the University of Tokyo have clarified the structural basis for the biosynthesis of selenocysteine (Sec), an amino acid whose encoding mechanism offers clues about the origins of the genetic alphabet. The findings deepen our understanding of protein synthesis and lay the groundwork for advances in protein design.

One of the most remarkable aspects of translation, the process whereby genetic information is converted into proteins in cells, is its universality: nucleotide triplets (“codons”) encode a set of twenty amino acids that form the building blocks for all living organisms. Selenocysteine, the “21st amino acid” whose antioxidant properties help prevent cellular damage, is a rare exception to this rule. Structurally similar to the amino acid serine (Ser) but with an oxygen atom replaced by the micronutrient selenium (Se), selenocysteine is synthesized through a complex juggling of the cell’s translational machinery whose mechanisms remain poorly understood.

Central to this multi-step process is a Sec-specific transfer RNA (tRNASec) with an unusual structure that enables it to hijack the “stop codon” UGA to allow incorporation of selenocysteine during protein synthesis. In earlier work, the researchers identified features of tRNASec that differentiate it from other tRNA, notably the peculiar structure of a domain called the D-arm, which appeared to act as an identification marker for recognition by the selenocysteine synthesis machinery. This time, the team analyzed the D-arm’s role in the interaction of tRNASec with O-phosphoseryl-tRNA kinase (PSTK), a protein whose selective phosphorylation is essential for selenocysteine encoding.

Using X-ray crystallography, the team showed for the first time that it is the unique structure of the tRNASec D-arm which enables PSTK to distinguish tRNASec from other tRNA. Reported in the August 13th issue of Molecular Cell (online August 12th), the discovery clarifies a pivotal step in selenocysteine biosynthesis, shedding new light on the mysterious 21st amino acid and the elaborate process by which it is created.

Attached files

Figure 3:  Structure of the tRNASec・PSTK complex. (a) Two PSTK molecules (colored blue and green) interact with each other to form a homodimer. Each PSTK molecule binds a tRNASec molecule. PSTK consists of two independent, linker-connected domains, the N-terminal catalytic domain (NTD) and the C-terminal domain (CTD). These domains independently bind tRNASec. (b) A close-up view of one of the PSTK molecules bound to tRNASec. The N-terminal domain (NTD) and the C-terminal domain (CTD) of PSTK interact with the acceptor arm (colored pink) and the D arm (light blue), respectively. PSTK does not interact with the tRNASec anticodon complementary to the UGA codon.


Figure 1:  Comparison of selenocysteine (Sec) to the structurally similar amino aids serine (Ser) and cysteine (Cys).


Figure 2:  Selenocysteine biosynthesis. (Top) tRNASec is first ligated with serine to form Ser-tRNASec. The seryl moiety of Ser-tRNASec is then phosphorylated by PSTK to yield P-Ser-tRNASec, which is converted to Sec-tRNASec and used on the ribosome to insert Sec into a specific site in a nascent polypeptide of selenoproteins. (Bottom) In the case of the standard amino acid serine, tRNASer is ligated with serine and directly used for translation. Ser-tRNASer is not a substrate of PSTK.


Figure 4:  Interaction between the unique D arm of tRNASec and the PSTK CTD. (Top) Comparison of the secondary structure of tRNASec to that of a canonical tRNA. The tRNASec D arm consists of a six base-pair stem (D stem) and a four-nucleotide loop (D loop), in contrast with the 3–4 base-pair D stem and the 7–11 nucleotide D loop of the canonical tRNA. (Bottom) The D arm of tRNASec (colored light blue) snugly interacts with the PSTK CTD (green), whereas the D arm of the standard tRNA (blue) does not fit the PSTK CTD.


Figure 5:  tRNASec recognition by PSTK. The enzymatic activity of PSTK is governed by the specific interaction between its CTD and the unique D arm of tRNASec. The tight binding of the CTD to the D arm ensures that the N-terminal catalytic domain binds to the end of the acceptor arm, where the phosphorylation reaction occurs. In contrast, the CTD does not fit the D arm of canonical tRNAs, and thus PSTK does not act on them, segregating the Sec insertion pathway from the normal amino-acid translation process.


 

Poor Amino Acid Breakdown Increases Risk Of Heart Disease

homocysteine levels predictors of heart diseasePeople with a genetically determined, reduced breakdown of the amino acid homocysteine have an increased risk of coronary heart disease compared to healthy people. This is revealed in doctoral research carried out by Mariska Klerk at Wageningen University.

The researchers from Wageningen have demonstrated that people with a genetically determined, specific form of reduced homocysteine breakdown have a 16 percent higher risk of developing coronary heart disease. People with the aforementioned reduced breakdown have on average a 25 percent higher homocysteine concentration in their blood from birth onwards, compared to other people.

Homocysteine has been associated with cardiovascular disease for a long time. However, until recently it was not clear whether an increased concentration of this amino acid in the blood was the cause or consequence of cardiovascular disease. This research into people with a genetic predisposition for a high homocysteine concentration in the blood supports the argument that this high concentration is a cause of cardiovascular disease.

Homocysteine is an amino acid (protein building block) which is formed in the body during the breakdown of another amino acid (methionine) obtained from food. The body regulates the homocysteine concentration in the blood with the aid of several B vitamins, including folic acid. Apart from a genetic predisposition, a shortage of these vitamins can also lead to an increased concentration of homocysteine in the blood. Folic acid supplementation is therefore an effective strategy to reduce the quantity of homocysteine in the blood.

The research has also revealed that people with genetically determined, reduced homocysteine breakdown only had an increased risk of heart disease when the folic acid concentration in the blood was low. This result suggests that reducing the homocysteine concentration in the blood by means of folic acid supplementation will reduce the incidence of cardiovascular disease.

The researchers also investigated whether a reduction in the homocysteine concentration in the blood as a result of vitamin B supplementation had a favourable effect on blood clotting in healthy volunteers. The assumption was that homocysteine increases the risk of cardiovascular disease by overstimulating blood clotting (which could eventually result in thrombosis). Despite a considerable reduction in the homocysteine concentration in the blood of people who had received extra vitamin B, no clear effect on blood clotting could be demonstrated.

The research was funded by the Netherlands Organisation for Scientific Research (NWO).

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