May 5, 2007

2007 week 18: Gentle Intro to Proteins & Folding


Some articles to help you get your feet wet...
Follow the links for the full article as usual.


Rensselaer Researchers Develop Approach That Predicts Protein Separation Behavior:
Applying math and computers to the drug discovery process, researchers at Rensselaer Polytechnic Institute have developed a method to predict protein separation behavior directly fromprotein structure. This new multi-scale protein modeling approach may reduce the time it takes to bring pharmaceuticals to market and mayhave significant implications for an array of biotechnology applications, including bioprocessing, drug discovery, and proteomics,the study of protein structure and function.
"Weintend to test the model against more complicated protein structures aspart of its further development," said Breneman. "The outcome of this work will yield fundamental information about the complex relationship between a protein's structural features and its chemical binding properties, and also aid in evaluating its potential biomedical applications."

Beyond Biology: Simple System Yields Custom-designed Proteins:
The diversity of nature may be enormous, but for Michael Hecht it is just a starting point.Hecht, a Princeton professor of chemistry, has invented a technique for making protein molecules from scratch, a long-sought advance that will allow scientists to design the most basic building blocks of all living things with a variety of shapes and compositions far greater than those available in nature.
Nearly all the internal workings of living things are built from proteins. While genes are the "blueprints" for organisms, proteins are the products built from those instructions. The molecules that transmit signals in the brain, carry oxygen in the blood and turn genes on and off are all proteins.
Scientists have long wanted to design their own proteins, but doing so has proved a major challenge. Proteins are strings of chemical units called amino acids and are often more than 100 amino acids long. When cells make them, these long chains fold spontaneously into complex three-dimensional shapes that fit like puzzle pieces with other molecules and give proteins their unique abilities. There are 20 different amino acids, so the number of possible combinations is enormous. However, the vast majority of these combinations are useless because they cannot fold into protein-like structures.
The advance reported by Hecht and colleagues involves a simple system for designing amino acid sequences that fold like natural proteins. First publishing the idea in 1993, Hecht realized that some amino acids were strongly "water-loving" while others were "oil-loving." The two types naturally separate from each other, with the oil-loving ones clustering in the protein core and water-loving ones forming the perimeter. He also saw that natural proteins with good structures tend to have certain repeating patterns of oil-loving and water-loving amino acids. For example, taking a string of water-loving units -- no matter which ones -- and inserting any oil-loving unit every three or four positions typically creates proteins that fold into bundles of helices.

Lasers Improve Scientists' Understanding Of Complex Proteins:
By shooting lasers at an RNA polymerase (RNAP) and a strand of DNA, scientists have learned a critical component of how a complex protein develops.
Using a system called fluorescence resonance energy transfer (FRET) on a single molecule, a researcher at the Lawrence Livermore National Laboratory’s Physical Biosciences Institute (PBI) in collaboration with UCLA scientists found that the procedure that regulates genes in a strand of DNA is a single process.
Earlier studies done with less precision resulted in scientists believing that the beginning and end phases of RNAP copying a DNA strand into RNA were two different processes. Using FRET, however, the recent study suggests that “there is no mechanistic difference between the start and finish,” said Ted Laurence of Livermore’s PBI. RNAP is the molecular machine that serves as a gene transcription tool. When it attaches to a strand of DNA, RNAP transcribes genes to RNA, which then is translated into a protein.

Livermore & NIH Scientists Create Technique To Examine Behavior Of Proteins At Single Molecule Level:
The work, presented in the Aug. 29 edition of Science, marks the first time protein-folding kinetics has been monitored on the single-molecule level. Proteins are long chains of amino acids. Like shoelaces, they loop about each other or fold in a variety of ways, and only one way allows the protein to function properly. Just as a knotted shoelace can be a problem, a misfolded protein can do serious damage. Many diseases, such as Alzheimer's, cystic fibrosis, mad cow disease and many cancers result from misfolded protein.

The Path To A Folded Protein, Long A Subject Of Debate, Appears In Many Cases To Be Long And Winding:
It's a long-simmering debate in the world of physical chemistry: Does the folding of proteins into biologically active shapes better resemble a luge run fast, linear and predictable or the more freeform trajectories of a ski slope? New research from the University of Pennsylvania offers the strongest evidence yet that proteins shimmy into their characteristic shapes not via a single, unyielding route but by paths as individualistic as those followed by skiers coursing from a mountain summit down to the base lodge. The new support for a more heterogeneous model of protein folding comes in a paper published today on the Web site of the Proceedings of the National Academy of Sciences.

"The traditional view has been that a protein passes through a series of fixed reactions to reach its folded state," said senior author Feng Gai, a Penn chemist. "Our work suggests quite strongly that folding is a far richer phenomenon. Like skiers, some proteins rocket down an energy gradient to their destination while others take their time, meandering indiscriminately."

Gai's work subtly shifts scientists' understanding of one possible remedy: molecular chaperones, promising compounds that "rescue" misfolded proteins and are believed capable of blocking the progression of neurodegenerative disease. Rather than giving sluggish proteins the oomph to finish folding, the Penn work indicates that chaperones may return misfolded proteins to an unfolded state so they can start all over again.

"In the skiing analogy, chaperones could be thought of as rescue helicopters that return wayward skiers to the summit so they can try to make their way down the mountain again," said Gai, an assistant professor of chemistry at Penn.

Untangling The Protein Folding Problem:
Protein folding research is "undergoing explosive growth," according to an editorial by Jay Winkler, Ph.D., and Harry Gray, Ph.D., both chemists at the California Institute of Technology and guest editors for the special issue. "Protein folding was once considered an almost intractable problem," write Winkler and Gray, but new efforts "are beginning to reveal the secrets of this prototypal spontaneous self-assembly process."

The special journal issue focuses on the chemical kinetics of the folding phenomenon -- the rate of change as the protein assumes its three-dimensional structure -- and includes a study on recent efforts to make "real time" observations. Folding happens very quickly, which makes it difficult to observe.

For some proteins, the change occurs in milliseconds (thousandths of a second); for others, it can be even faster. Despite recent progress toward understanding the mechanisms of protein folding, scientists still don't agree on exactly how it happens, as evidenced in the journal by the differing conclusions of several articles about the same protein.

Proteins are involved in many vital roles in humans, including metabolism, immunity and muscle movement. They are made up of amino acids, and it is the sequence of these amino acids that determines the eventual folded structures of the proteins, as well as the actual mechanism of the folding process.

Since a protein's structure is a key factor in how it functions in the body, the goal for researchers is to be able to predict the final three-dimensional structure based on the amino acid sequence.

How Proteins Fold Into Their Critical Shapes:
Experimental evidence provided by a Cornell researcher and colleagues at the Scripps Research Institute in La Jolla, Calif., support a long-held theory of how and where proteins fold to create their characteristic shapes and biological functions.
The theory proposes that proteins start to fold in specific places along an amino acid chain (called a polypeptide chain) that contains nonpolar groups, or groups of molecules without a charge, and continue to fold by aggregation, i.e., as several individuals of these nonpolar groupings combine. Using the same principle that separates oil and water, these molecules are hydrophobic -- they avoid water and associate with each other.

In the water-based cell fluid, where long polypeptide chains are manufactured and released by ribosomes, the polypeptide chains rapidly fold up into their biologically functional structure. The theory proposes that there are sites along the polypeptide chains where hydrophobic groups initially fold in on themselves, creating small nonpolar (hydrophobic) pockets that are protected from the water.

Research Answers Key Question In Biochemistry: How Proteins Fold Into 3-D Structures:
In research published in the July 29 issue of Nature, U of T post-doctoral fellow Dmitry Korzhnev and his supervisor, Professor Lewis Kay of the Department of Biochemistry, become the first researchers to characterize at an atomic level of detail the intermediate -- or substructure -- that forms as a protein folds to its 3-D state.

"Understanding how proteins fold is one of the Holy Grails of biochemistry," says Kay. "The intermediates that we can study make up only one or two per cent of the population of protein molecules in solution. It's hard to study them because they are present at such low levels. This is the first time we have been able to characterize an intermediate state at this level of detail."

If scientists can understand the pathway a protein takes from one state to another, they may be able to predict protein structure, something that can't be done very reliably at present. The ability to accurately predict protein structure has implications for drug design, as well as for improving commercial products.

Understanding the pathway a protein follows will also help scientists understand errors in folding, a problem linked to diseases such as cystic fibrosis and Alzheimer's.

Most Stable Parts Of Protein Are The First To Fold, Study Finds:
Like a 1950's Detroit automaker, it appears that nature prefers to build its proteins around a solid, sturdy chassis.

A new study combining advanced computational modeling and cutting-edge experiments by molecular biologists at Rice University and Baylor College of Medicine suggests that the most stable parts of a protein are also the parts that fold first.

Nature refuses to choose between form and function when it comes to protein folding; each protein's function is directly related to its shape, and when proteins misfold -- something that's known to occur in a number of diseases like Alzheimer's and Huntington's -- they don't function as they should.

In the new study, scientists designed and tested a new computational approach that aimed to study proteins with known shapes in order to ascertain which of their parts were the most stable in the face of chemical and thermal fluctuations.

"As far as we know, no one has ever used this type of knowledge-based, statistical approach to predict the stability cores of proteins," Ma said. "Our results suggest that thermodynamics and kinetics are closely correlated in proteins and appear to have co-evolved for optimizing both the folding rate and the stability of proteins."

Quantum Leap In Protein Folding Calculations:
Applying techniques derived from classical and quantum physics calculations may radically reduce the time it takes to simulate the way that proteins fold.

It's vital to understand the shapes that proteins take on as they fold up because the shapes determine how they function, both in keeping cells running and in leading to various diseases.

Rather than calculating the motions of a protein molecule step by step, as most simulations do, a team of Italian and French physicists studied the evolution of a molecule using variational principles. The technique allowed the physicists to evaluate all the possible paths that the molecule's parts would follow and then pick out the most likely one.

As a result, they expect to streamline protein folding calculations from trillions of steps to hundreds. The improvement is significant because conventional protein folding simulations that currently require supercomputers or large PC farms could instead be solved with individual desktop PCs running variational principle calculations.

Comprehensive Model Is First To Map Protein Folding At Atomic Level:
Scientists at Harvard University have developed a computer model that, for the first time, can fully map and predict how small proteins fold into three-dimensional, biologically active shapes. The work could help researchers better understand the abnormal protein aggregation underlying some devastating diseases, as well as how natural proteins evolved and how proteins recognize correct biochemical partners within living cells.

The technique, which can track protein folding for some 10 microseconds -- about as long as some proteins take to assume their biologically stable configuration, and at least a thousand times longer than previous methods -- is described this week in the Proceedings of the National Academy of Sciences.

"For years, a sizable army of scientists has been working toward better understanding how proteins fold," says co-author Eugene I. Shakhnovich, professor of chemistry and chemical biology in Harvard's Faculty of Arts and Sciences. "One of the great problems in science has been deciphering how amino acid sequence -- a protein's primary structure -- also determines its three-dimensional structure, and through that its biological function. Our paper provides a first solution to the folding problem, for small proteins, at an atomic level of detail."

Fiendishly intricate, protein folding is crucial to the chemistry of life. Each of the body's 20 amino acids, the building blocks of proteins, is attracted or repulsed by water; it's largely these affinities that drive the contorting of proteins into distinctive three-dimensional shapes within the watery confines of a cell. The split-second folding of gangly protein chains into tight three-dimensional shapes has broad implications for the growing number of disorders believed to result from misfolded proteins or parts of proteins, most notably neurodegenerative disorders such as Alzheimer's and Parkinson's diseases.

The model developed by Shakhnovich and colleagues faithfully describes and catalogs countless interactions between the individual atoms that comprise proteins. In so doing, it essentially predicts, given a string of amino acids, how the resulting protein will fold -- the first computer model to fully replicate folding of a protein as happens in nature. In more than 4,000 simulations conducted by the researchers, the computer model consistently predicted folded structures nearly identical to those that have been observed experimentally.


What Mutations Tell Us About Protein Folding:
Scientists continue to be puzzled by how proteins fold intotheir three-dimensional structures. Small single-domain proteins mayhold the key to solving this puzzle. These proteins often fold intotheir three-dimensional structures by crossing only a single barrier.The barrier consists of an ensemble of extremely short-lived transitionstate structures which cannot be observed directly. However, mutations that slightly shift the folding barrier may provide indirect access to transition states.

The reliable folding of proteins is a prerequisite forthem to function robustly. Mis-folding can lead to protein aggregates that cause severe diseases, such as Alzheimer's, Parkinson's, or the variant Creutzfeldt-Jakob disease. To understand protein folding,research has long focused on metastable folding intermediates, whichwere thought to guide the unfolded protein chain into its foldedstructure. It came as a surprise about a decade ago that certain smallproteins fold without any detectable intermediates. This astonishingly direct folding from the unfolded state into the folded state has been termed 'two-state folding'. In the past few years, scientists have shown that the majority of small single-domain proteins are 'two-state folders', which are now a new paradigm in protein folding.

The characteristic event of two-state folding is the crossing of a barrierbetween the unfolded and folded state. This folding barrier is thought to consist of a large number of extremely short-lived transition state structures. Each of these structures is partially folded and will either complete the folding process, or will unfold again, with equal probability. Transition state structures are thus similar to a ball ona saddle point, which has the same probability, 0.5, of rolling toeither side of the saddle.

Since transition state structures arehighly instable, they cannot be observed directly. To explore two-statefolding, experimentalists instead create mutants of a protein. Themutants typically differ from the original protein -- the wild type --in just a single amino acid. The majority of these mutants still foldinto the same structure, however the mutations may slightly change thetransition state barrier and, thus the folding time; that is, the timean unfolding protein chain on average needs to cross the foldingbarrier.

The central question is: can we reconstruct the transition state from the observed changes in the folding times? Such a reconstruction clearly requires experimental data on a large number of mutants. In the traditional interpretation, the structural information is extracted for each mutation, independent of the other mutations. Ifa mutation does not change the folding time, then the mutated amino acid traditionally is interpreted to be still unstructured in the transition state. In contrast, if a mutation changes the folding time,the mutated amino acid is interpreted to be partially or fully structured in the transition state, depending on the magnitude of the change.

In a recent article in PNAS, a research team from the MaxPlanck Institute of Colloids and Interfaces and the University of California, San Francisco has suggested a novel interpretation of the mutational data. Instead of considering each mutation on its own, the new interpretation collectively considers all mutations within acooperative substructure, such as a helix. In case of the ±-helix ofthe protein CI2, this leads to a structurally consistent picture, inwhich the helix is fully formed in the transition state, but has not yet formed significant interactions with the ²-sheet.

In the future, the Max Planck researchers hope to construct complete transition states from mutational data. An important step is to identify the cooperative subunits of proteins, which requires molecular modelling. In a similar way to how a mountain pass shows us how to cross the landscape, the transition states eventually may help us to understand how proteins navigate from the unfolded into the folded structure.

Penn Scientists Show How Mistakes In Protein Folding Are Caught By "Protein Cages" Called Chaperonins:
It's imperative for all biological processes that proteins correctly maneuver from a simple string of amino acids to their pre-destined three-dimensional structure. This transformation -- called protein folding - is one of the most active areas of molecular biological research, and has taken on even more importance with the growing knowledge that misfolding can lead to such disorders as Alzheimer's disease, Huntington's disease, and prion-related neurodegenerative diseases. Recently, researchers at the University of Pennsylvania Medical Center have discovered how proteins called chaperonins protect cells from harm by sequestering and unfolding misshapened proteins. A report on this study appears in the April 30 issue of Science.

"Proteins should know how to fold by themselves, but they sometimes get into trouble," says senior author S. Walter Englander, PhD, a professor of biochemistry and biophysics at the University of Pennsylvania School of Medicine. In times of stress, cells produce chaperonins, which are huge protein molecules that police other proteins that have misfolded as a result of any number of stressors -- including heat, heavy-metal poisoning, and ultraviolet radiation. If left unchecked, the misfolded proteins tend to clump, which can be harmful to normal cellular functions.

The Penn study looked specifically at a chaperonin called GroEL. "GroEL grabs the misfolded protein, engulfs it, pulls it open, and then throws it back out into the cytoplasm of the cell to fend for itself, " explains Englander. "The protein then takes its chances -- it may fold successfully, or it may get into trouble again." This entire process takes place within 13 seconds.

GroEL -- a sandwich of two circular proteins with a large central core into which average-sized proteins can fit - is able to capture thousands of different types of misfolded proteins. Its cavity is ringed with sites that bind nonspecifically to the hydrophobic, or water-avoiding, portions of proteins, which are normally found tucked deep inside a properly folded protein. "When a protein is misfolded and its hydrophobic insides are exposed, chaperonins snatch them up and help them to fold correctly by forcing them to unfold, so that they can try again," notes Englander.

Mechanism Of Protein Folding Unraveled, With Eventual Implications For Treating Diseases Caused By Folding Errors:
How a protein manages to fold is a seemingly impossible problem, suggests S. Walter Englander, PhD, a professor of biochemistry and biophysics at the University of Pennsylvania School of Medicine: "Even with a small, 100-amino-acid-long protein, the number of possible three-dimensional structures that the protein might manifest is larger than the number of molecules in the universe." Protein biologists believe that the amino-acid sequences laid out by the genetic machinery contain chemical instructions for the pathway that carries each protein to its final structure.

The Penn experiments show that the amino-acid chain progresses through a series of pre-determined, intermediate arrangements. Englander's lab has demonstrated that the protein cytochrome c builds its structure in steps by first making helices at either end that lay at right angles to each other. Then, strands, loops, and other helices build up against that initial foundation until the final arrangement is reached. All this can occur in less than one second, but trouble can arise along the way. "A few years ago we showed that on the complicated journey to their final structure, proteins have a large tendency to make mistakes that greatly slow them down," notes Englander. "Proteins need to fold fast because if they spend too much time in one intermediate state, they're vulnerable to aggregation with other proteins in the midst of folding, which can be very destructive to the cell."

The recent work straightens out misinterpretations about how fast this process can proceed. "Numerous papers published in the past two years all conclude that when you initiate folding in a rapid reaction experiment, you see some very fast sub-millisecond optical signal changes, as well as some slower ones" explains Englander. "This has always been interpreted as a rapid formation of some real structural intermediates. It is crucially important to understand which of these signals represent real protein behavior and which give you misleading clues that simply depend on the kind of experiment you are doing. Understanding the folding process and the real time scale of events begins to give you some idea of what you can do to fight diseases like Alzheimer's."

Englander's lab performed experiments that convincingly showed that these exceedingly fast initial signals are not real intermediates, but simply represent the protein stretching and pulling in the denaturing solution used in the experiment. The researchers made two copies of the same amino-acid chain, one that couldn't fold and one that could, and observed that both versions displayed the same initial ultra-fast burst of optical activity.

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