Stanford Synchrotron Radiation Lightsource

The enzyme 3-Hydroxyanthranilate-3,4-dioxygenase (HAO) is critical for the metabolism of the amino acid tryptophan and the synthesis of the important coenzyme nicotinamide adenine dinucleotide (NAD+). Although the role of this enzyme has been long known, its mechanism and regulation have remained a mystery, because it is difficult to study. HAO creates the unstable product 2-amino-3-carboxymuconic semialdehyde (ACMS) that can self-cyclize into quinolinic acid (QUIN). Because excess amounts of QUIN have been implicated in neurologic diseases, HAO is a potential drug target. A group of scientists have determined the mechanism of the HAO enzyme by applying crystallographic techniques.

The bacterium Clostridium difficile (often called C. diff) can cause severe intestinal infections, responsible for about 500,000 cases and 29,000 deaths in the United States per year. While infections are more often found in ill and elderly people, infection rates are increasing in young and healthy people. The bacterium makes and secretes two related toxins, TcdA and TcdB. Understanding the structure of these molecules is a critical step to developing treatment. Unfortunately, since these toxin proteins are huge and flexible, scientists have been unable to determine the entire molecular structures until now.

CRISPR, a powerful new tool that can target and change specific sequences of DNA, is based on a prokaryotic immune system response. The first step of bacterial immunity via CRISPR is placing sequences of foreign (viral) DNA between specific palindromic DNA repeats in the bacterial genome. The enzyme complex Cas1-Cas2 must target the correct DNA locus for integration, since insertion of the viral DNA into other areas of the genome may cause damage to the bacteria.

Creating novel enzymes to perform specific chemical reactions is a field of great promise, but it is still in its early stages. Efforts usually involve using well-studied protein structural and functional domains to create new active sites. Scientists have recently developed a different approach, creating the active site in the interface between proteins in a multi-protein complex. They started with a well-researched, natural protein that, in its natural state, does not form complexes with other proteins, and nor does it catalyze the desired reaction.

Botulinum neurotoxins (BoNTs) invade motor neurons at their junctions with muscular tissue, where the toxins disable the release of the neurotransmitter acetylcholine and subsequently paralyze the affected muscles. Accidental BoNT poisoning primarily occurs through ingestion of food products contaminated by Clostridium botulinum, the bacterium that produces BoNTs. However, BoNTs by themselves are fragile and sensitive to low pH environments and digestive proteases. So how do they survive the harsh environment of the host’s gastrointestinal tract?

Understanding the physical underpinnings of how proteins interact specifically with one another and not with the myriad other molecules that coexist in every cellular compartment is a major goal of molecular biology. The broad outlines of an answer were suggested by Linus Pauling in the 1940’s: the aggregate effect of numerous weak and nonspecific van der Waals, hydrogen-bonding, and electrostatic interactions underlie high specificity and affinity. Since Pauling’s days thousands of co-crystal structures have provided concrete examples for how molecular recognition is achieved in different biological contexts. Yet, the ultimate proof for understanding a natural phenomenon lies in recapitulating it; in the words of Thomas Edison, ‘until man duplicates a single blade of grass, Nature laughs at his so-called scientific knowledge’.

A team of researchers working at SSRL has determined the atomic structure of an assemblage of fiber-forming proteins found in the cell membranes of many dangerous types of bacteria. The protein, called pilin, assembles into filamentous organelles called Type IV pili found on the surfaces of most Gram-negative bacteria. Type IV pili plays a central role in how these bacterial pathogens infect a host and are involved in cellular functions such as motility, adhesion, microcolony formation and uptake of DNA and specific filamentous phage.

Cocaine abuse remains a major public health problem despite ongoing research aimed at developing therapies to counter its harmful effects. Immunopharmacotherapy is one proposed therapy which would block cocaine in the blood stream before it reaches the central nervous system. Cocaine-binding antibodies seem likely candidates for soaking up drugs in the blood stream, but their only binding abilities are not sufficient to withstand high concentrations of the drug. What is needed is a monoclonal antibody with high binding characteristics and sufficient catalytic ability to metabolize cocaine.

Life as we know it depends on turning on and off the proper genes at the correct time. This process of gene expression starts when an RNA message is copied from DNA. Scientists have long known that an enzyme called RNA polymerase II plays the central role in this delicate transcription process. But the exact mechanism by which RNA polymerase II selects specific nucleotides and catalyzes the reaction that incorporates them into a growing RNA strand has not been well understood.

Ion channels in our cells generate the nerve impulses that enable the heart to beat, the body to move, and sensation and thought to occur. Scientists from the Salk Institute for Biological Studies have identified a tiny flexible gateway that controls the rapid-fire opening and closing of a family of ion channels through which nerve-triggering potassium ions flow in and out of cells of the body. Malfunctions in the channels leads to several human diseases, including epilepsy, cardiac arrhythmias and muscle disorders.

The Botox^® face lifts and botulism disease are both caused by a neurotoxin from the bacterium Clostridium botulinum. The toxin, often described as the most lethal substance known, is a member of the clostridal neurotoxins (CNTs) group, which block muscle contractions. When injected into someone's face, the effect is a lessening of wrinkles. When ingested, the toxin paralyzes muscles, including those of the internal organs, causing sickness and death. The toxin is also used in medicine for conditions such as uncontrolled blinking, lazy eye, and involuntary muscle contractions.

Researchers have literally unearthed clues as to why the 1918 influenza pandemic was so deadly. The 1918 influenza pandemic ranks as the largest and most destructive outbreak of an infectious disease, killing 20 to 40 million people worldwide. Using fragments of the flu genome from Alaskan victims preserved by permafrost and army autopsy tissues, James Stevens and Ian Wilson of the Scripps Research Institute in La Jolla, California and their collaborators have assembled genes from the 1918 flu virus.

Professor Roger D. Kornberg and his group in the Stanford University School of Medicine have devoted more than 20 years to the study of the process by which genetic information encoded in all living things by DNA is processed into a message (a process called transcription that produces messenger RNA) that then directs the synthesis of proteins. A breakthrough paper detailing the structures of the core RNA Polymerase II protein was published in Science in April 2000 and followed by two more papers in Science a year later.

SSRL has played an important role in characterizing a family of enzymes that detoxify heroin and cocaine, and have the potential to metabolically eliminate the nerve poisons sarin, soman, and tabun, which have claimed thousands of lives. Using x-ray crystallographic data, the Redinbo group at the University of North Carolina at Chapel Hill has uncovered the specific and general ways the carboxylesterase enzymes bind to those dangerous substances. 

Iron plays an integral role in many biochemical processes essential for life. However excess iron leads to the production of highly reactive hydroxyl radicals by Fenton chemistry (1). These free radicals are deleterious to cells as they react indiscriminately with proteins, DNA and lipids. Hence, iron homeostasis is a highly regulated process and is critical to human health (2). Disorders in iron metabolism, are however, surprisingly common.

Scientists have recently identified a family of human antibodies that can take out an unprecedented number of different types of flu viruses, including H5N1 'bird flu' and the 1918 H1N1 'Spanish flu', which killed millions around the world during World War I, as well as seasonal flu. Using SSRL's Beam Line 9-2, Dr. Robert Liddington from the Burnham Institute for Medical Research led a team of scientists that determined the crystal structure of one such antibody, F10, in complex with the hemagglutinin H5 to unveil the molecular mechanism of virus neutralization. Results were published online 22 February 2009 in the journal Nature Structural and Molecular Biology.

Medications can be rendered ineffective through cells developing multidrug resistance. This is the case in many forms of cancer cells that fail to respond to chemotherapy. The ability of these cells to avoid the effects of drugs can be due to the actions of P-glycoprotein (P-gp). This protein sits in the membranes of cells and acts like a pump. It ushers a wide range of potentially harmful molecules from inside the membrane to outside the cell. Unfortunately, it can also mediate the removal of life-saving medications.

UC Irvine researchers have unveiled the mystery behind one of the deadliest toxins that causes liver cancer. Aflatoxins are common contaminants of foods such as nuts and grains, which make up the staple diets of many developing countries. These toxins are produced by moldy fungi during food production, and are considered by the FDA to be an unavoidable food contaminant. Aflatoxin molecules are characterized by the presence of multiple aromatic rings. Chronic ingestion of aflatoxin B1 leads to liver tumors that are a major cause of death in Asia, Africa, and Central America. This toxin wreaks havoc of p53, an important gene in our body that prevents cancer. Without the protective effect of p53, then, aflatoxin further compromises immunity, interferes with our body metabolism, and causes severe malnutrition. It is urgently important to find inexpensive strategies that help protect the world population from aflatoxin food contamination.

An unusual property of the last year's H1N1 "swine flu" virus pandemic is that it disproportionately affected the young. People over the age of around 65 showed much less vulnerability than to more typical flu strains, suggesting that they might have been exposed to a similar virus over three decades ago. Another atypical property of the 2009 H1N1 strain is that its hemagglutinin (HA) subunit is the same subtype as the regular seasonal strains, whereas most pandemics are caused by viruses with novel HA domains.

HIV protease is a common and critical drug target for combating HIV infection and AIDS. As HIV develops resistance to anti-viral drugs, new therapies are required. Since most of the virus's mutations that confer drug resistance cluster in the active site of the protease, scientists are interested in molecules that may bind other places on the enzyme. Computer simulations aid the design of drugs and fragments, which are smaller than typical drugs, to bind the enzyme's surface in a way that compliments the activity of traditional active-site binding drugs.

Semaphorins are a group of proteins known for their critical role in nerve and vascular development and are bound by signaling receptors called Plexins. Some Semaphorins, including Sema7A, are involved in a variety of immune responses. Vaccinia virus, which is used in the smallpox vaccine, has a Sema7A homologue called A39R, which binds PlexinC1, Sema7A's receptor. 

It took nature billions of years to evolve proteins that can selectively bind to certain metals. Researchers are now seeking to create such proteins synthetically in the lab, with the end-goal of creating new metal-based functions.

While much is known about how the acquired immune system recognizes and responds to pathogens, the innate immune system, which also fights off infections and disease, is much less well understood. The inflammatory responses of the innate immune system can be activated by toll-like receptors (TLRs), which often bind to elements from pathogens that have a regular repeat, such as double-stranded RNA.

The Research group of Douglas Rees at the California Institute of Technology collected X-ray crystallographic data to a resolution of 1.16 Å at SSRL Beam Line 9-2 using the new Quantum-315 CCD detector from crystals of Nitrogenase MoFe-Protein, an extremely efficient enzyme found in bacteria that catalyzes the production of ammonia from dinitrogen. Bacteria produce about half of the world’s bio-nitrogen available for agriculture, the rest comes from nitrogenous fertilizer produced chemically at extreme temperature and pressure, consuming about 1% of the world's total annual energy supply.