This 10 April 2000 article in Business Week Online at http://www.businessweek.com/2000/00_15/b3676117.htm is an interesting overview of the challenge of deriving a proteome from a genome.

BUSINESSWEEK ONLINE : APRIL 10, 2000 ISSUE

Beyond the Genome: Biotech's Next Holy Grail
Now, companies are racing to decipher the human protein set

This summer, researchers will complete one of the greatest biological endeavors since Charles Darwin made his voyage of discovery on the HMS Beagle. Scientists will announce the completion of a rough draft of the human genome--a first attempt at decoding the entire set of about 100,000 human genes. Champagne corks will pop, backs will be slapped, and biologists will mark the start of a revolutionary era in biology.

After the first round of self-congratulation, however, researchers will find that their task has only just begun. The decoding of the human genome, as impressive as it is, means little until researchers find out what all those genes do. Sequencing the genome is just the first step in the long march toward an understanding of how humans grow and develop and how they get sick. ''Genes are powerful, but at the endyou want to get at the proteins'' that carry out bodily functions, says Dr. Randall W. Scott, chief scientific officer at Incyte Genomics Corp. (INCY).

At least seven biotech companies are racing to develop new tools to mine the proteome--the complete set of human proteins--for clues to human disease. The tools include powerful instruments that can sort and sequence thousands of proteins simultaneously. Even gene wizard J. Craig Venter--whose company, Celera Genomics Group (CRA), helped galvanize the human genome project--is jumping on the proteomics bandwagon. ''We are going to build the Celera proteomics facility. It's a major, major effort,'' says Venter.

Academic research centers and pharmaceutical companies are using proteomics to identify diagnostic indicators for a range of diseases, from breast cancer to heart disease to Alzheimer's. The National Cancer Institute and the Food & Drug Administration are funding a multimillion dollar ''Tissue Proteomics Initiative'' to identify proteins linked to early stages of colon, breast, and other cancers. Meanwhile, drug companies, including Bayer (BAYZY), Merck (MRK), and Pfizer (PFE), are starting collaborations on proteomic projects to complement their other research efforts.

Most genes serve as blueprints for the production of proteins, the workhorses of living cells. To understand disease, doctors need to survey the activities and functions of these proteins. Thus, genomics--the study of the genome--will soon give way to a far more challenging discipline: proteomics, or the study of proteins. And the human genome project may soon be replaced by a human proteome project, which would enable scientists to answer many fundamental biological questions.

Many critical functions of the cell are accomplished by a complex cascade of events in which one protein acts on another, which acts on another, and so on. Examples of the process include, say, the changes that occur when a neurotransmitter is dispatched from one brain cell to another. Understanding of these processes could be used to develop better drugs for cancer and diabetes. It should also help identify patients most likely to suffer side effects.

Even with the new genomics technologies, it takes 10 or 15 years to get a drug out of the lab--to evaluate it in animal and human trials and get it onto the market. Proteomics promises to shorten that to perhaps a few years by helping researchers identify safer, more effective candidate medicines earlier in the drug-development process. ''It will go so far beyond the genome project, we'll be delighted,'' predicts Arnold Oliphant, vice-president for functional genomics at Salt Lake City biotech company Myriad Genetics Inc. (MYGN).

This eagerness to understand the proteome isn't new. In 1980, scientists proposed a project called the Human Protein Index, which Congress seriously considered funding. But before the program got off the ground, the political tide turned in favor of a human genome project. The prevailing attitude was, it's so much easier to work with DNA, we shouldn't waste time fooling with proteins.

Compared with proteins, DNA molecules are simple. A DNA molecule is a long, spiraling ladder--the famous double helix--composed of just four basic constituents. Proteins, on the other hand, are highly complicated beasts that fold up into intricate and often unpredictable shapes. They are built from 20 building blocks, called amino acids, each of which has its own unique chemical properties. That alone makes sequencing proteins a much harder task. In addition, the set of proteins a cell uses is constantly changing. Some proteins are broken down and their components recycled in minutes, while others can persist in the cell for hours or even days. Their surfaces are constantly modified, too--a sugar molecule might be added to one, a phosphate molecule tacked on to another. These additions can activate or deactivate proteins. Scientists estimate that the 100,000 genes in the human genome can generate perhaps as many as a million different proteins.

The completion of the human genome project will speed proteomics research. ''Without genomics, proteomics is so much harder,'' says Keith Williams, chief executive at Proteome Systems Ltd., a proteomics company based in Sydney, Australia. But even armed with critical genetic data, researchers need to develop instruments powerful and sensitive enough to detect and characterize extremely small quantities of a protein--as little as a billionth of a gram. That's because many of the most important proteins are present in only fleeting amounts. The mighty gene sequencers used by Celera, developed by PE Biosystems in the mid-1990s, ensured that gene sequencers had the capacity to tackle 100,000 genes. Those instruments are one of the reasons the sequence of the human genome will be finished five years ahead of schedule.

With potentially a million proteins to unravel, even more powerful technologies are needed for proteomics. Those instruments are coming. Already, Oxford GlycoSciences in Oxford, England, Large Scale Biology in Rockville, Md, and Williams' Proteome Systems have developed sophisticated, reliable methods of miniaturizing the sheets of jello-like polymer that are used to separate individual proteins from the many thousands contained in a serum or urine sample. They have even found ways to sequence the 20 different building blocks rapidly but accurately.

CHURNING OUT DATA. The current separation and sequencing tools may not be as fast or as automated as researchers would like, but they are churning out more information about novel proteins than the companies' computer systems can handle. That has intrigued pharmaceutical companies, which are always hungry for new drug targets. Large Scale Biology has signed contracts with 24 pharmaceutical and biotech companies, while competitor Oxford Glycosciences has done at least six deals. Proteome Systems has orchestrated its own share of contracts, including one with Indianapolis-based Dow AgroSciences to list and classify all of the proteins made by various crop plants.

But cataloging proteins is only one way to harness the power of proteomics. Pharmaceutical companies really want to understand the protein changes that occur when a normal cell becomes diseased. Pfizer Inc. has teamed with Oxford GlycoSciences PLC to use proteomics to unearth biological markers that define the various stages of Alzheimer's disease. In practice, this entails analyzing hundreds of samples of spinal fluid taken from patients with mild, moderate, and severe forms of Alzheimer's and comparing them with samples from healthy people (diagram).

The goal is to find key differences among the samples and use that knowledge to chart the progression of the disease at the cellular level. Right now, to diagnose Alzheimer's conclusively, doctors must conduct a lengthy series of memory tests. But if doctors could use proteomics to find a unique set of proteins that distinguishes the early stages of the illness from later ones, or even from no disease at all, then they would have a rigorous and objective means of diagnosis. For instance, patients with proteins A and B might have a case of Alzheimer's with only mild memory loss. Patients with proteins X and Z, however, might have such a severe form of the disease that they need special treatment. Knowing that information would allow physicians to correlate subjective symptoms with real biological changes, says proteomics proponent Dr. B. Michael Silber, the director of pharmacogenomics and clinical biochemical measurements at Pfizer.

Cancer is another area where researchers believe proteomics will yield big payoffs. In 1998, Dr. Emanuel F. Petricoin, a senior fellow at the FDA, and Dr. Lance A. Liotta of the NCI sponsored a program designed to identify protein markers that are specifically linked to early onset of ovarian, prostate, and other cancers. The hope is that these early markers will lead to better diagnostic tests that can identify lesions at the pre-malignant state, when they are just beginning to grow and spread throughout the body. So far, the initiative has uncovered nearly three dozen new protein markers that are consistently seen in pre-malignant cells, including several new markers--in addition to the already widely used PSA--for one of the most common cancers, prostate cancer.

Proteomics will also give medical researchers important information about the potential side effects of new and existing drugs. ''Right now, one of the main quandaries...is that many drugs fail at late stages of clinical development because of unforeseen toxicities,'' says Petricoin. He believes that proteomics could be used to eliminate potentially life-threatening compounds before pharmaceutical companies invest tens of millions of dollars bringing them to market. Scientists might, for instance, test how different experimental drugs affect the proteins of a given tissue--say, the liver or the kidney. The drugs that produce the fewest changes are likely to be safest, says Petricoin.

MANAGEABLE CHUNKS. Because it is difficult to coordinate a massive effort to physically isolate and measure all of the proteins in a cell or tissue, some companies, including Curagen (CRGN), Myriad Genetics, and Hybrigenics, have opted for a simpler approach. They are fishing out specific groups of new proteins related to, say, insulin levels, and then determining the proteins' functions. That effort can have practical and beneficial outcomes. In a study with a pharmaceutical company, Myriad scientists took 10 proteins known to be involved in a particular disease and used them to uncover 200 additional proteins--and one of them looked like a promising drug target. In just a few months of work, Myriad scientists were able to provide the pharmaceutical company not only with critical biological information but also with valuable clues about a potential blockbuster medicine. ''That is phenomenal speed to identify a new drug target,'' says Oliphant.

Researchers at biotech companies and universities have clearly made a good start at unfolding the secrets hidden in the proteome. But there are still many hurdles to clear. Denis F. Hochstrasser, a Swiss chemist who helped found proteomics company GeneBio, cautions that the proteome is so difficult to analyze ''that no one technology is sensitive enough [yet] to find the needle in the haystack. It's going to require many years and many new innovations'' to crack the protein code (sidebar).

It's also going to take massive amounts of computing power and storage capacity. ''If you look at the complex network of [protein information] we will build on top of the genome...it will be in the petabyte range,'' says Celera's Venter. (A petabyte is a billion megabytes.)

These are technology problems that can and will be solved. Proteomics may be in its infancy, but already, a new post-genomic age has dawned.

By Ellen Licking in New York, with John Carey in Rockville, Md., and Amy Barrett in Philadelphia

Here is an article dated 14 November 2005 in Science Daily at http://www.sciencedaily.com/releases/2005/11/051114111031.htm

If this method can be adapted to study how proteins fold, it might lead to revolutionary developments (both in lab studies and in computational biology).

Source: Stanford University
Date: 2005-11-14

New Microscope Allows Scientists To Track A Functioning Protein With Atomic-level Precision

A Stanford University research team has designed the first microscope sensitive enough to track the real-time motion of a single protein down to the level of its individual atoms. Writing in the Nov. 13 online issue of the journal Nature, the Stanford researchers explain how the new instrument allowed them to settle long-standing scientific debates about the way genes are copied from DNA--a biochemical process that's essential to life.

(image omitted)

In a second paper published in the Nov. 8 online issue of the journal Physical Review Letters, the scientists offer a detailed description of their novel device, an advanced version of the "optical trap," which uses infrared light to trap and control the forces on a functional protein, allowing researchers to monitor the molecule's every move in real time.

"In the Nature experiment, we carried out the highest-resolution measurement ever made of an individual protein," says Steven Block, professor of applied physics and of biological sciences. "We obtained measurements accurate to one angstrom, or one-tenth of a nanometer. That's a distance equivalent to the diameter of a single hydrogen atom, and about 10 times finer than any previous such measurement."

Block co-authored the papers in Nature and Physical Review Letters with three current members of his Stanford Lab--graduate students Elio Abbondanzieri and William Greenleaf and postdoctoral fellow Michael Woodside--together with former graduate student Joshua Shaevitz, now at the University of California-Berkeley, and longtime collaborator Robert Landick at the University of Wisconsin.

Central dogma

In the Nature study, Block and his colleagues tackled a fundamental principal of biology known as the central dogma, which states that in living organisms, genetic information flows from DNA to RNA to proteins.

The process begins with DNA, the famous double helix, which stores genetic data. DNA is often compared to a twisted ladder consisting of two strands connected by molecular rungs called "bases," which are known by the abbreviations A, T, G and C. Lengthier DNA sequences code for genes, which contain explicit instructions for building a specific protein.

A typical DNA ladder carries thousands of genes that encode thousands of proteins, which keep the organism alive and functioning. A single misplaced letter in gene's DNA sequence--a G substituted for a T, for example--can produce a defective protein that may cause a serious disease.

Transcription

The Block team focused on a crucial step in the central dogma, a process known as "transcription," where each gene is copied from DNA onto RNA. Transcription begins when an enzyme called RNA polymerase (RNAP) latches onto the DNA ladder and pulls a small section apart lengthwise. The RNAP enzyme then builds a new, complementary strand of RNA by chemically copying each base in one of the exposed DNA strands. RNAP continues moving down the DNA strand until the gene is fully copied.

For the Nature experiment, Block and his colleagues used DNA and RNAP extracted from E. coli bacteria, which is remarkably similar to RNAP in more complex organisms, including humans. "RNAP is one of the most important enzymes in nature," Block says. "Without it there would be no RNA messages, no proteins and no life."

Inchworms and scrunching

Exactly how transcription works at the molecular level has been intensely debated among scientists.

"People for years have known that RNA is made one base at a time," Block says. "But that has left open the question of whether the RNAP enzyme actually climbs up the DNA ladder one rung at a time, or does it move instead in chunks--for example, does it add three bases of RNA, then jump along and add another three bases." The latter process, called discontinuous elongation, is like reading a book, he explains: "When you read, you don't advance your eyes one letter at a time. You 'chunk': You read it in pieces."

Two basic hypotheses have been proposed for discontinuous elongation:

* Ihe inchworm model, in which RNAP moves along DNA like an inchworm, with the front end of the enzyme always ahead of the rear.
* The scrunching model, whereby RNAP pulls in ("scrunches") a loop of DNA, copies each base in the loop, then grabs another loop farther up the ladder.

Determining which model is correct has been a difficult challenge, because until now, no instrument was sensitive enough to track each microscopic step taken by RNAP along DNA during transcription. That's because conventional optical traps can't measure anything smaller than about 10 angstroms (1 nanometer). However, each base in the DNA ladder--A, T, G or C--is only separated by about 3.4 angstroms. "My lab has been working very hard for the better part of a decade to break the nanometer barrier and attain angstrom-level resolution," Block says.

Light and motion

To achieve that goal, the Block team had to overcome two inherent problems with conventional force clamps: fluctuating signals and bending light waves.

"When you shine a laser through the air, the light beam wiggles around a bit, for the same reason that stars twinkle in the sky," Block explains. "But we want to use that beam to measure the position of something to within the size of an atom, so if the beam moves just 1 angstrom, that's the end of the story. We took all the optics external to the microscope, enclosed them in a sealed box and replaced the air with helium gas, which has a refractive index that's 10 times closer to that of a vacuum than air. So you get, roughly speaking, 10 times less twinkling and an instrument with angstrom-level stability."

In addition to stabilizing the light, the researchers also had to improve the method for detecting force and displacement. Optical force clamps use tiny forces from an infrared laser beam to trap DNA and other molecules. In a conventional force clamp experiment, microscopic beads are attached near the opposite ends of a long DNA molecule--an arrangement that resembles a weight lifter's dumbbell. A single RNAP enzyme attached to the surface of one bead then moves along the DNA and churns out a complementary strand of RNA, drawing the ends of the dumbbell closer together as it advances. The two beads that form the dumbbell are usually held near the center two separate optical traps. But graduate student William Greenleaf discovered that if one of the two beads in the dumbbell was placed near the outer edge of its trap, the force on it would remain constant, allowing angstrom-level measurements to be made quickly and efficiently.

"That's just what you want--a clamp that allows RNAP to move with impunity, but the force itself doesn't change," Block says. "Normally the bead is inside the trap in the center, but right at the edge of the trap we have this magical property where the force is constant."

Unlike conventional instruments, the new force clamp requires no time-consuming computer computations to correct for competing forces. "This new technique is entirely passive, like a thermos that just sits there and keeps something cool," Block says. "All we have to do is shine light on the system and everything takes care of itself. As a result, we were finally able to resolve the minuscule, 3.4-angstrom steps taken by E. coli RNAP as it transcribes a bacterial gene."

Settling the debates

With these innovations in place, the research team appears to have settled some of the fundamental arguments over DNA-RNA transcription. "Quite simply, our experiment rules out both discontinuous-location models," Block says. "Neither the inchworm nor the scrunching model is consistent with our data, and the idea that some have held all along--that RNAP climbs the DNA ladder one base pair at a time--is probably the right answer."

The Stanford group also weighed in on another controversy concerning the actual mechanism that allows RNAP to advance. "RNAP is a molecular motor that starts at one end of the DNA and walks down to the other end," Block explains. "It gets its energy from the chemical reaction that occurs when it copies A, T, G or C. It's as if a machine that lays down asphalt could somehow be powered by the asphalt itself."

Scientists have come up with two different models to explain what drives this molecular motor:

* The power stroke model, in which pent up energy thrusts the enzyme forward--like a loaded spring that's periodically released.
* The Brownian (or thermal) ratchet model, whereby random thermal energy causes the RNAP enzyme to jiggle back and forth. Each incoming DNA base then locks the enzyme into the forward position so that it cannot jiggle backwards. "It would be as if you were repeatedly bouncing off a wall, and every time you happened to bounce a bit farther away, somebody came in and moved the wall up behind you, so you couldn't bounce so far back. You'd wind up drifting forwards, even though your own motion was mostly random," Block explains.

In the Nature study, Block and his colleagues concluded that the Brownian ratchet model is probably correct for RNAP, even though several other motor proteins are believed to move instead by the power stroke mechanism. "We've certainly come down hard in favor of the Brownian ratchet camp and against the power stroke camp," Block says. "But does that mean all power stroke models have been ruled out and that all Brownian ratchet models are acceptable? No."

Molecular folding

The Block team also applied the new force clamp technology to one the hottest fields in biomedical research--molecular folding. For a protein to function properly, it has to fold into a specific, intricate three-dimensional shape. Diseases such as Alzheimer's, Mad Cow and Parkinson's may result when proteins do not fold into their correct 3-D conformation. Medical researchers are trying to solve the mystery of how proteins fold in hopes of some day curing these and other diseases.

In the experiment published in Physical Review Letters, the Block group addressed certain aspects of the general folding problem on a simpler scale by focusing on single DNA hairpins--folded structures that can form when a single strand of DNA pairs with itself instead of with the opposite strand. "Hairpins are wonderful models," Block says. "By keeping the force constant, we were able to measure the folding and unfolding transitions of a single DNA hairpin at the angstrom scale. In the future, this may help us understand and predict what shape a more complex linear protein will assume in three-dimensional space."

Major advance

The development of an ultra-stable optical trapping system with angstrom resolution is "a major advance," says Charles Yanofsky, the Morris Herzstein Professor of Biological Sciences at Stanford and a pioneer of modern molecular genetics. The new device is like "adding movies to stills in understanding enzyme action," he says.

"This technical achievement will no doubt lead to new information about the molecular machinery that carries out basic cellular processes, particularly those related to replication, transcription and translation," adds Catherine Lewis, a program director in biophysics at the National Institute of General Medical Sciences (NIGMS).

"If I look in my crystal ball and see where this is going, I think this blows open the field of single-molecule biophysics," Block says. "We have achieved a resolution for a single molecule comparable to what a crystallographer typically achieves in a millimeter-sized crystal, which has 1,000 trillion molecules in it. Not only are we doing all this with one molecule at one-angstrom resolution, we're doing it in real time while the molecule is moving at room temperature in an aqueous solution."

Block notes that it took "years of careful instrument development, sponsored by the National Institutes of Health, and the construction of a special laboratory built by Stanford University to make this possible, along with the simply outstanding efforts of some incredibly bright and hard-working graduate students and postdocs here at Stanford. I am especially proud of this work."

The Physical Review Letters and Nature papers were supported by NIGMS and by Stanford University.