The Kinesin Motor Machine.

How Tiny Cell Proteins Generate Force To 'Walk'

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ScienceDaily (Dec. 4, 2008) — MIT researchers have shown how a cell motor protein exerts the force to move, enabling functions such as cell division.

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Kinesin, a motor protein that also carries neurotransmitters, "walks" along cellular beams known as microtubules. For the first time, the MIT team has shown at a molecular level how kinesin generates the force needed to step along the microtubules.

Microtubules, quantum physics, and consciousness? Microtubules form tracks for neurotransmitters to be transported and can possibly act as quantum computational structures.

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The researchers, led by Matthew Lang, associate professor of biological and mechanical engineering, report their findings in the Nov. 24 online early issue of the Proceedings of the National Academy of Sciences. Because kinesin is involved in organizing the machinery of cell division, understanding how it works could one day be useful in developing therapies for diseases involving out-of-control cell division, such as cancer. The protein consists of two "heads," which walk along the microtubule, and a long "tail," which carries cargo. The heads take turns stepping along the microtubule, at a rate of up to 100 steps (800 nanometers) per second. In the PNAS paper, Lang and his colleagues offer experimental evidence for a model they reported in January in the journal Structure. Their model suggests — and the new experiments confirm — that a small region of the protein, part of which joins the head and tail is responsible for generating the force needed to make kinesin walk. Two protein subunits, known as the N-terminal cover strand and neck linker, line up next to each other to form a sheet, forming the cover-neck bundle that drives the kinesin head forward. "This is the kinesin power stroke," said Lang. Next, Lang's team plans to investigate how the two kinesin heads communicate with each other to coordinate their steps. Lead author of the PNAS paper is Ahmad Khalil, graduate student in mechanical engineering. Other MIT authors of the paper are David Appleyard, a graduate student in biological engineering; Anna Labno, a recent MIT graduate; Adrien Georges, a visiting student in Lang's lab; and Angela Belcher, the Germehausen Professor of Materials Science and Engineering and Biological Engineering. This work is a close collaboration with authors Martin Karplus of Harvard and Wonmuk Hwang of Texas A&M. The research was funded by the National Institutes of Health and the Army Research Office Institute of Collaborative Biotechnologies.

Few videos describing the motor:
Kinesin Transport Protein
Kinesin Explanation

Mice with a few kinesin mutations? Look like there is something wrong with their neurphysiology, almost like they are not interacting with the environment in the correct way?
Kinesin mutations in mice

Ever wondered how cellular machinery causes replication of cells?
Awesome video:
Inside the cell
And it does not even remotely cover the intricate mechanisms controllong the process.

Another video of mitosis:
Mitosis
Active cyclinB/cdc2 plays a part in nuclear envelope breakdown, and destruction of cyclinB and abolition of cdc2 activity allows nuclear envelope formation.

In real life it looks something like this:

Idle Control Units and Metabolomics

Well designed cars have well designed idle control units that spontaneously kick in to maintain the speed of the crankshaft within a pre-set range (usually >200rpm). An interesting study has demonstrated that 4 single celled organisms in two domains of life (bacteria and eukaryotes) uses the same number of biochemical reactions when optimizing growth.

Spontaneous Reaction Silencing in Metabolic Optimization
From the article:

Performing numerical optimization in glucose minimal media (Materials and Methods), we find that the number of active reactions in such optimal states is reduced by 21%–50% compared to typical non-optimal states, as indicated in the middle bars of Figure 2. Interestingly, the absolute number of active reactions under maximum growth is, 300 and appears to be fairly independent of the organism and network size for the cases analyzed. We observe that the minimum number of reactions required merely to sustain positive growth [7,8] is only a few reactions smaller than the number of reactions used in growth-maximizing states (bottom bars, Figure 2). This implies that surprisingly small metabolic adjustment or genetic modification is sufficient for an optimally growing organism to stop growing completely, which reveals a robust-yet-subtle tendency in cellular metabolism: while the large number of inactive reactions offers tremendous mutational and environmental robustness Papp:2004dn, the system is very sensitive if limited only to the set of reactions optimally active. The flip side of this prediction is that significant increase in growth can result from very few mutations, as observed recently in adaptive evolution experiments.

Reaction irreversibility and spontaneous cascading (article) of inactivity are described as built-in mechanisms that mediate these metabolic adjustments. The authors also point out that 638 out of the 931 reactions in the E. coli glucose metabolic network can be removed whilst maintaining a maximum growth rate in glucose. The mutational robustness as a result of inactive reactions under maximum growth thus act as a sort of preadaptation whereby different pathways can be spontaneously activated under shifting environmental conditions.

The tremendous robustness of these systems raises an interesting question regarding the origins of these non-essential pathways under maximum growth rates. The authors provide a testable hypothesis:

An alternative explanation would be that in variable environments, which is a natural selective pressure likely to be more important than considered in standard laboratory experiments, the apparently dispensable pathways may play a significant role in suboptimal states induced by environmental changes. This alternative is based on the hypothesis that latent pathways provide intermediate states necessary to facilitate adaptation, therefore conferring competitive advantage even if the pathways are not active in any single fixed environmental condition.

This alternative is based on the hypothesis that latent pathways provide intermediate states necessary to facilitate adaptation, therefore conferring competitive advantage even if the pathways are not active in any single fixed environmental condition. In light of our results, this hypothesis can be tested experimentally in medium-perturbation assays by measuring the change in growth or other phenotype caused by deleting the predicted latent pathways in advance to a medium change.

Even more intriguing is the fact that metabolic adjustments are also controlled by anticipatory transcriptional reprogramming in response to environmental changes. It is posited to be as result an “associative learning” paradigm.

Looking at the motor industry again, anticipatory systems and structures have been designed in order to optimize the structure stiffness for a particular crash scenario. Pre-crash sensing is used to adjust structural stiffness and crumple zones in response to a particular deceleration scenario in order to maximize the crash worthiness of the vehicle. It seems this kind of anticipatory programming is an ancient invention, a few billion years old.

Using this information, another core element can be added to an initial state: Robust metabolic networks with tremendous adaptability that "idle" under maximum growth conditions.

Figure 1: An initial state. Reverse engineer ubiquitous core components of various life forms at present. Will it repeatedly produce similar endpoints after evolutionary processes, irrespective of its origin?

Protein folding, Nanotubes and Engineering

More on protein folding:
Many proteins have intricate folds and one of these fold types include the figure eight knot fold. A team of researchers tried to figure out how these proteins are folded. At present, it is only known that the knot is formed quickly soon after polypeptide chain formation, with an unknown mechanism. The researchers tentatively propose:
"an early threading event may be the defining feature of a polypeptide-knotting with the ensuing folding occurring in a similar fashion to unknotted proteins; the folding of a knotted protein differs only with an initial knotting event in a denatured-like state."

Perhaps an an as of yet undiscovered knot-folding machine?
Article: Exploring knotting mechanisms in protein folding


And nanotubes? Bah, old news... a few hundred million years old...
Tunnelling nanotubes: Life's secret network

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The closest animal equivalents to plasmodesmata were thought to be gap junctions, which are like hollow rivets joining the membranes of adjacent cells. A channel through the middle of each gap junction directly connects the cell interiors, but the channel is very narrow - just 0.5 to 2 nanometres wide - and so only allows ions and small molecules to pass from one cell to another. Nanotubes are something different. They are 50 to 200 nanometres thick, which is more than wide enough to allow proteins to pass through. What's more, they can span distances of several cell diameters, wiggling around obstacles to connect the insides of two cells some distance apart. "This gives the organism a new way to communicate very selectively over long range," says Gerdes. It is a previously unknown way in which cells can communicate over a distance Soon after they first saw nanotubes in rat cells, he and Rustom saw them forming between human kidney cells too. Using video microscopy, they watched adjacent cells reach out to each other with antenna-like projections, establish contact and then build the tubular connections. The connections were not just between pairs of cells. Cells can send out several nanotubes, forming an intricate and transient network of linked cells lasting anything from minutes to hours. Using fluorescent proteins, the team also discovered that relatively large cellular structures, or organelles, could move from one cell to another through the nanotubes

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Their work, published in May, shows that nanotubes are not just an artefact of the methods used to grow cells in culture, as some have suggested. And what they have seen is spectacular: some of the longest tunnelling nanotubes ever observed, more than 300 micrometres long, connecting dendritic cells in the cornea (The Journal of Immunology, vol 180, p 5779). "We can see them their whole course, spindling all the way through the cornea," says McMenamin. "It's fantastic." "I'll bet you that within weeks to months, people will start noticing them in other tissues. It's just a case of how you look," he adds. "You've got to know what you are looking for. It's a bit like being a good bird-watcher. A hundred people will see a flock of seagulls, and it's only a very good bird-watcher who will spot this one tern flying in that flock." Gerdes, meanwhile, continues to marvel at what is unravelling before his very eyes. "Whatever one can think of has been done by nature," he says. "It is unbelievable what the cell is able to do."

One striking feature that makes us different from other primates is our innate ability to develop a theory of mind at around 4-6 years. We start to develop the remarkable ability to simulate what other people are thinking and understand their thoughts through our own thinking. We also create our own model of the world in our own minds that leads to understanding of concepts.

Attempting to unravel nature's mysteries by trying to look at it from another engineering Mind's eye seems to make sense when one marvels at the engineering feats in cells. Why not?

Replication Machinery and Transcription Factories

How DNA Is Unwound So That Its Code Can Be Read

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ScienceDaily (Nov. 24, 2008) — Researchers at The Scripps Research Institute have figured out how a macromolecular machine is able to unwind the long and twisted tangles of DNA within a cell's nucleus so that genetic information can be "read" and used to direct the synthesis of proteins, which have many specific functions in the body.

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"This is a fundamental processes that takes place countless times inside each of our cells every day, but how it happens had not been understood." says the study's lead investigator, Francisco Asturias, Ph.D., associate professor in the Department of Cell Biology at Scripps Research. "The structure we have solved provides important clues into one of the first steps in gene expression regulation."

Anybody interested in some of the 3D-structure, go to rcsb.org.

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"Remarkable Unpacking and Repacking" To understand the complexity of the process, it is important to know that if the DNA in each cell were stretched out, it would be more than three feet long—and given the trillions of cells within a human body, it has been calculated that a single individual's DNA could stretch to cover the distance to the sun and back many times over. So DNA must be packaged into tidy little chromosomes. The DNA in each gene first assembles into what looks like a string of beads: the string is the DNA and to compact its length, it is wrapped two times around a spool-like bead of histone protein, to form a nucleosome. But there is so much DNA in a single gene that each gene is packed into a necklace of nucleosomes on a DNA string. These beads then become further compressed into twisted ropes that eventually form chromatin, in which DNA is compacted about 10,000 times from its extended length. What the Scripps Research scientists set out to do is to understand how the RSC complex unwinds DNA from the many histone beads within a gene so that other molecular machines can read the genetic code. RSC is a huge complex of 13 different proteins and the scientists first found that it holds an individual nucleosome in what looks like a vise grip. They then found that RSC creates a little bulge in the DNA that can be propagated around the nucleosome and make possible translocation of the DNA with respect to the histones, exposing the DNA so that it can be read. "Imagine a rubber band wrapped twice around a water glass. The easiest way to move the band is to pull a little of it away from the glass and then slide it" Asturias says. "By using energy from an external source (ATP hydrolysis) RSC can repeatedly pull DNA away from the histones and eventually expose all of the DNA." The researchers believe that by translocating a nucleosome along the DNA, RSC eventually slides into the next adjoining nucleosome, causing the histones to be ejected and exposing the DNA. "Interestingly, although its DNA is gradually exposed, the nucleosome to which RSC is bound remains intact," Asturias says. The structure RSC interacting with a nucleosome explains how previously observed DNA bulges formed by chromatin remodeling complexes are formed, and why a single intact nucleosome appears to be left on a fully activated gene before other cellular machinery scoop up the histones and repack the DNA until it needs to be read again. "Every time your cell expresses a gene, it goes through this remarkable unpacking and repacking," he says. "We are happy to have provided some clarity to the process."

Published article:
Structure of a RSC–nucleosome complex and insights into chromatin remodeling

Nice video showing DNA wrapping.

An interesting blog entry (and an interesting blog worth a read) describes how the transcription machinery is assembled and disassembled in a few sites in cells.

Yet another twist in the world of gene expression - transcription factories

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First of all I will ask you, did you know that transcription only happened at a few sites within the nucleus? In mouse cells from the animal there are between 100-300 of these but in cultured cells such as HeLa cells there are many more. Transcription factories also known as RNAPII foci are where most, if not all mRNA is produced. This amazed me and raises the obvious questions of why and how. The why may be obvious. It is a good idea to keep the nucleus as I see it ‘tidy’ but in more technical terms it is a way of keeps gene expression organised and regulating it (see below). The how this work I don’t think has been addressed! All I can say is watch this space.

There is more there.

Biomolecular machines utilizing thermal fluctuations

Article related to the unique operations of biological molecular machines
Fluctuation as a tool of biological molecular machines
Abstract:

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The mechanism for biological molecular machines is different from that of man-made ones. Recently single molecule measurements and other experiments have revealed unique operations where biological molecular machines exploit thermal fluctuation in response to small inputs of energy or signals to achieve their function. Understanding and applying this mechanism to engineering offers new artificial machine designs.

The article continues:

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Biological machines are different from man-made artificial ones in many ways. One primary difference is the amount of energy supplied. For example, a supercomputer playing chess with a champion uses much larger amounts of energy than its adversary. A computer unit element, or IC chip, uses energy much larger than thermal energy (500 times greater) to avoid the disturbance caused from thermal noises whereas biological machines use the energy released from the hydrolysis of ATP, which is only approximately 10 times greater than thermal energy. Large excess energy inputs in computers result in far less efficiency at converting their energy inputs although they are more precise at their task than biological machines. Computers err once per 1060 trials, while basic biochemical reactions underlying biological machines err as often as once per 103 trials. For this reason, computers are in some respects superior to one of nature’s greatest machines, the human brain. Computers make calculations much faster as IC chips work on the order of nanoseconds (10−9 s), while the time scale for basic biochemical reactions in biological machines is milliseconds (10−3 s). They also have superior memory capacity and data transfer rate. The computer rate is on the order of 109 bites/s while in brain it is estimated to be only 400 bites/s. However, biological machines are more flexible, readily responding to changes in their environment. In contrast, man-made machines are designed to maintain their. function regardless of environmental changes. Therefore, the fundamental mechanisms between the two machines are different. Biomolecules and their assemblies, biomolecular machines, are in the order of nanometer in size meaning the effects of thermal noises are large. Nevertheless, biomolecules and molecular machines execute their roles despite these noises. But how? Recent experimental data suggest that biological molecular machines harness thermal fluctuation to achieve their functions. Thus, thermal fluctuation seems to play an important role from the molecular level to cellular and organism level. We have developed measurement systems that trace these thermal fluctuations in biomolecular machines when eliminating measurement noise. Our model biomolecular machine of choice is the molecular motor. Molecular motors are composed of a motor protein, which move using the chemical energy of ATP, and protein tracks, which the motors move along. Molecular motors and protein tracks share unique characteristic properties such as enzymatic activity, molecular recognition, energy conversion and self-organization with other typical molecular machines. Thus the results obtained for molecular motors may be extended to other systems.

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Thermal fluctuation is involved in function at different hierarchies of biological systems (Fig. 3). In the mechanism for molecular motor motility, it has been shown that thermal fluctuation is involved and biased to generate directional movement. In live cells, a mechanism that utilizes thermal fluctuation is also likely.

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Lastly, in addition to the stochastic nature at the molecular and cellular levels, visual perception has shown stochastic dynamics. Visual perception processes are explained by equations similar to formulae that govern the behavior of biomolecules (Murata et al., 2003). Thus the mechanisms obtained at molecular and cellular levels likely apply at even higher levels. These mechanisms offer blueprints to engineer artificial machines that utilize fluctuations.

Again, showing the carbon based nanotechnology in cells can be utilized and adopted by our engineers for our own designs seeing that these machines are able to harness even thermal fluctuations.

Efficiency of enzymes

How long will a reaction take to complete without enzymes?
Well, some reactions will only complete in about 2.3 billion years without them...
Without Enzyme, Biological Reaction Essential To Life Takes 2.3 Billion Years

But enzymes don't just pop into existence, not even under the BEST pre-biotic synthesis scenarios. Enzymes are produced and folded into the correct conformation by.... other enzymes... controlled by.... biomolecular machines and a genetic code..

More about the efficiency of enzymes:
Biochemistry: Enzymes under the nanoscope

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Small-scale interactions of substrates with an enzyme's active site — over distances smaller than the length of a chemical bond — can make big differences to the enzyme's catalytic efficiency. When Richard Feynman died in 1988, he left behind the following words on his blackboard: "What I cannot create, I do not understand." His message certainly resonates with protein engineers.

Enzymes are guided into their correct 3D configuration by other enzyme complexes known as chaperones (part of the heat-shock protein family). What happens if the configurations are not right? Scientists determined that some enzymes, as in the case of ketosteroid isomerase, are so precisely folded for their particular ligand/substrate that if it the 3d conformation was off by even 10 picometers (10^12 meters) it would lose its efficiency. Firstly, the conformation has to be just right to tightly bind the ligand into the pocket of the protein, then another mechanism (built in property of the enzyme) is responsible the transfer of electrons in order to catalyze and complete the enzymatic reaction. Our current best efforts at designing artificial enzymes are (from the article) “still tens of billions of times smaller than those of many enzymes.”

The ribosome is a super complex of enzymes, a molecular machine responsible for the building of polypeptide chains which in turn are folded into active proteins by chaperone complexes.
Problems do occur, but checks and balances are present. For example:
Side-chain recognition and gating in the ribosome exit tunnel
At the exit tunnel of the ribosome, it is hypothesized that there are gate and latch mechanisms with active valves controlling the exit of polypeptides. The researchers conclude that these mechanisms play a role in the regulation of "nascent chain exit and ion flux". Sort-off like a final checkpoint.



As already seen, without enzymes, reactions that are crucial to life might take billions of years to complete. Small changes (10 picometers) in the 3D structure of an enzyme can also negatively affect the function of an enzyme.
So how are enzymes folded into their active conformation?

Chaperonins: Two-stroke, two-speed, protein machines

Article:
Setting the chaperonin timer: A two-stroke, two-speed, protein machine
From the article:

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Protein machines and their man-made, macroscopic counterparts share several common attributes, e.g., concerted, coordinated movements, cyclical operation, and energy transduction. These machines are seldom reversible because each cycle generally involves at least one irreversible step, e.g., the consumption of fuel. Often these machines operate at variable speed, a plethora of timing devices adjusting the cycle speed in response to demand. An exemplary bipartite protein machine is the chaperonin system, typified by GroEL and GroES from Escherichia coli. GroEL is composed of 2 heptameric rings, stacked back to back, which, in the presence of GroES, operate out of phase with one another in the manner of a 2-stroke, reciprocating motor (1, 2). Driven by the hydrolysis of ATP, the chaperonin proteins function as a biological simulated annealing machine (3, 4), optimizing the folding of their substrate proteins (SPs) whose passage to biologically functional conformations is thus assured.

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The picture of the chaperonins that emerges from our work is that of a machine equipped with a timer, the trans ring, poised to respond to the appearance of SP [substrate protein inside the cavity] but otherwise idling in a quiescent state. We note that Nature’s design of this 2-speed protein machine minimizes the hydrolysis of ATP in the absence of SP. However, it maximizes the number of turnovers and minimizes the residence time available to the encapsulated SP to reach the native state, design principles well suited to the operation of an iterative annealing device.

Partial part and dynamics of the system.
Nice video of how it operates

Machines folding machines into place. Beautiful...

Trash Removal

How Cells Take Out The Trash To Prevent Disease

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ScienceDaily (Nov. 10, 2008) — Garbage collectors are important for removing trash; without them waste accumulates and can quickly become a health hazard. Similarly, individual cells that make up such biological organisms as humans also have sophisticated methods for managing waste.

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The researchers, including postdoctoral fellows Jason MacGurn and Chris Stefan, identified nine related proteins in yeast, which they named the "arrestin-related trafficking" adaptors or ARTs. Each of these proteins identifies and binds to a different set of membrane proteins. Once bound, the ART protein links to an enzyme that attaches a chemical tag for that protein's removal. The ARTs are found in both yeast and humans, suggesting the fundamental nature of their function. Once the protein is tagged, the piece of membrane with the targeted protein forms a packet, called a vesicle, that enters the cell's cytoplasm. There, the vesicle enters a larger membrane body called an endosome, which in turn dumps it into another compartment called the lysosome, where special enzymes break apart big molecules to their core units: proteins to amino acids, membranes to fatty acids, carbohydrates to sugars and nucleic acids to nucleotides, and those basic materials are then reused. The paper in Developmental Cell, co-authored by Emr with postdoctoral fellows David Teis and Suraj Saksena, describes for the first time how a set of four proteins assemble into a highly ordered complex. This complex encircles membrane proteins that must be disposed of in the lysosome. Emr's lab was the first to identify and characterize these protein complexes (known as ESCRTs). The Developmental Cell paper describes the order of events in which the ESCRT complexes encircle and deliver "waste" proteins into vesicles destined for recycling in the lysosome.

Lysosomal, autophagic, chaperone mediated autophagy processes...etc. taking out the trash: All highly regulated, exquisitely controlled processes carried out by biomolecular machines. And these same processes are so ancient... present in yeast...