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Author
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Topic: Negative Design: Eliminating Nonfunctionality
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John Bracht
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Member # 5
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posted 31. March 2002 21:43
The idea of "Negative Design" comes from a paper by H. W. Hellinga, PNAS 94:10015-7, Sept 1997:
quote:
To achieve specificity, the desired state (well-ordered core) has to have the lowest free energy of all possible states (ground state), and there has to be a large free energy difference between the next available state: the free energy of specifity (delta G spec). There are two ways to achieve such a free energy gap: raising the free energy of competing states, or lowering that of the desired state. One approach is to introduce specific features that prevent the formation of alternative conformations, thereby raising their energy ["negative design"].
[bold added]
This paper is talking about the details of protein folding. The basic idea is that for many laboratory-designed proteins, there are many equal-energy 3D structures the protein can take. But in biology, the proteins have only one lowest-energy (specific) configuration. Negative design involves actively choosing an amino acid sequence that makes alternative, formerly equal-energy structures less favorable. One way to do this is by "Constructing protein interiors out of sequences that increase the degree of geometric irregularity, making it less likely for alternative isoenergetic conformations to exist, [which] results in better-ordered cores." (quoted from same paper as above.) We can think of a protein as folding along a "landscape" which contains a free energy well; the protein is trying to reach the bottom of this jagged funnel. Negative design may be thought of as a way to engineer this free energy folding funnel in such a way that one, and only one, structure lies at the bottom. Notice that negative design is not required for proper folding (which we have from the beginning) but rather the negative design functions to eliminate competing, incorrect configurations, leaving only the desired one. Thus, I want to define a general engineering principle, "negative design," and see whether it applies to human inventions.
Negative Design: Any deliberate manipulation of an object done with the express purpose of preventing incorrect assembly of components into a whole.
Notice that as defined, negative design is an extra layer of design beyond just achieving functionality; it entails eliminating nonfunctionality.
Consider some examples of human design that fit this category. Think of a standard 110-volt electrical outlet, and a plug that goes into it. You may have noticed that some plugs can only go into the outlet one way; one prong is wider and one hole in the outlet is correspondingly wider to allow only one configuration. You may also have noticed that many older outlets and plugs have no such delineation and it is possible to insert the plug in two ways: they are "reversible". Newer plugs are not reversible, and they have been negatively designed (via the size of the holes) to prevent the incorrect configuration. Notice that the older plugs and outlets were perfectly functional, but the newer plugs incorporate an extra layer of engineering in the negative design (though I'm not really sure why only one orientation of plug is allowed in newer outlets).
Another example of negative design: my computer has a special plug for keyboard and mouse. Both of these plugs are round, with a round outlet in the back of the computer. Because they are round, they can be inserted in any number of ways--but only one way is correct. So there is a "notch" in the outlet on my computer that corresponds with a bump on the plug, and I think one of them incorporates a small rectangular piece of plastic in the center of the plug which can only insert in one way. This is another example of negative engineering, where any number of incorrect possibilities exist but are not "live options" due to clever design.
Now, how about an example (thanks to Micah Sparacio for this one) that does not incorporate negative design but probably would benefit from it. I occasionally use a debit card to purchase groceries at the local supermarket. I've noticed that whenever I run my card through the little card swiper I nearly always get it the wrong way the first time--I get the magnetic strip oriented the wrong way. There is no systematic constraint that rules out incorrect configurations, and since there are 4 possibilities (or, two with the magnetic strip on the bottom), it's easy to get it wrong.
So, I think negative design is a possibly useful design concept that exists in biology and has real counterparts in human engineering. It seems to come into play primarily in cases where multiple components interact in some way, to eliminate incorrect configurations.
Here's a thought-provoking question: is negative design a problem for natural selection, seeing as it goes "beyond" mere function to the elimination of nonfunction? In other words, once a basic functional protein arises, how does the quality of negative design get added to it via natural selection?
I'm not at all sure what the answer is to these questions, but I think negative design is a useful and interesting idea that provides insights from biology to engineering, and possibly vice-versa.
John Bracht
[ 02 April 2002, 12:32: Message edited by: Moderator ]
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Micah Sparacio
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posted 02. April 2002 12:41
Just a quick point:
I'm pretty sure that Walmart has negative design on their credit card machines. I know I was at some store where I couldn't get my card to slide through, and finally the lady at the cash register told me that there is only one way you can slide the card or else it gets "stuck" and won't slide all the way through.
I'll have a chance to check later today;)
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Art
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Member # 179
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posted 02. April 2002 17:44
Hi John,
I think that a few other ideas should go into your thinking on this subject.
For example, you said: quote:
This paper is talking about the details of protein folding. The basic idea is that for many laboratory-designed proteins, there are many equal-energy 3D structures the protein can take. But in biology, the proteins have only one lowest-energy (specific) configuration.
Actually, this is not true. Anyone who struggles with the production of recombinant proteins in E. coli knows this (or should). As often as not, the desired protein will take on a conformation (or configuration) that is not what one would call "native" or "functional"; instead, an aggregated state results. There is no reason to suppose why the aggregated state is not the lower-energy configuration, and I would argue that it is (generally speaking - each case is obviously going to be somewhat different).
In this light, it would appear as if "negative design" may not be widespread, or even existent, when it comes to proteins.
quote:
Here's a thought-provoking question: is negative design a problem for natural selection, seeing as it goes "beyond" mere function to the elimination of nonfunction? In other words, once a basic functional protein arises, how does the quality of negative design get added to it via natural selection?
I don't think that there's any conceptual difficulty here. There is a very large class of proteins (chaperonins) that accomplishes the "elimination of nonfunction".
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John Bracht
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posted 03. April 2002 11:59
Art,
I think we're talking about two very different things. Negative design refers to the engineering of the "folding (energy) landscape" such that only one sequence lies at the bottom of the funnel. This folding energy landscape is absolutely separate from the issue you raise, namely, the ability of a protein to traverse that landscape to find its optimum structure.
You bring up the question of protein aggregation. Aggregation occurs when unfolded proteins encounter each other and stick together, thereby preventing proper folding. This is a problem in a crowded cellular environment where there are necessarily many proteins chains around and some of them will be unfolded. However, as you point out later on in your post, there are certain proteins which exist purely to prevent this problem, in effect to keep unfolding protein chains from contacting each other before they are properly folded. These proteins are called "chaperones."
The effect of these chaperone proteins is precisely to allow the protein to drop into the deepest part of its energy well on the folding landscape. Notice that when the protein aggregates, it gets "stuck" at a suboptimal point on that landscape, i.e. a nonfolded state. The chaperone allows the protein to fall to the lowest-energy configuration. Negative design has to do with engineering that energy landscape, and nothing to do with whether the protein successfully traverses that landscape to reach the optimum structure.
Second, you point to the chaperones (specifically, the chaperonins) as molecules that do the negative design. I believe you are mistaken on this, for the reason cited above: chaperones only help a protein drop down its own energy gradient, they do not determine in any way how that gradient will be structured. Negative design is all about adjusting the sequence such that only one structure lies at the bottom of the gradient. Since chaperones have no control over the energy gradient, they cannot be the source of negative design.
Just to make it clear: chaperones are generic folding-enhancers, but they do not themselves contain any information about what that fold will be. Indeed, a recent issue of Science (March 8, vol 295, page 1852-8) has an article by F. Ulrich Hartl and Manajit Hayer-Hartl on chaperone proteins, and it states "The various chaperone factors protect nonnative protein chains from misfolding and aggregation, but do not contribute conformational information to the folding process."
So the information on how to fold is found in the protein itself, in its sequence of amino acids. It is this sequence that determines the energy landscape, and it is the chaperone that guides the protein down that landscape. Most importantly, the negative design work occurs at the level of protein sequence, and hence through an engineering of the energy landscape--not through molecular chaperones that guide the protein down that landscape.
I hope this helps; thanks for your comments.
John Bracht
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Nelson Alonso
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posted 03. April 2002 14:49
Dependent on the protein concentration, an aggregated state can represent the state of lowest delta G. Note, however, that aggregation is not a first-order reaction, and thus its energy landscape depends on the protein concentration.
At low protein concentrations, the native state is usually at the single global minimum of delta G.
[ 03 April 2002, 14:50: Message edited by: Nelson Alonso ]
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John Bracht
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posted 03. April 2002 15:07
Nelson,
That makes sense to me. I am thinking of the energy gradient as being the one that exists when the protein is an an aqueous environment, one that does not tend to stabilize unfolded, non-native proteins. It does make sense that a hydrophobic environment (like one dominated by unfolded protein chains) would tend to make aggregates the lowest energy state.
Thanks for the clarification--I think this is a caveat that I need to add to my above discussion. Under this view, chaperone proteins act to prevent the protein folding along the "wrong" energy gradient (the one caused by contact with other unfolded proteins) and isolates the protein so it can fold along the right energy gradient. I think the rest of the argument is still applicable.
John Bracht
[ 03 April 2002, 15:10: Message edited by: John Bracht ]
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Nelson Alonso
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posted 03. April 2002 16:15
John,
You also might want to take a look at a possible exception to this. For example,forget about aggregation reactions, that is irrelevant to your point, but even in the absence of aggregation reactions, a protein may be kinetically trapped in the native conformation,but this may not represent the global energy minimum. I think that these occur rare enough to not really dent your argument.
[ 03 April 2002, 16:44: Message edited by: Nelson Alonso ]
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Art
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posted 03. April 2002 19:31
Nelson said:
quote:
John,
You also might want to take a look at a possible exception to this. For example,forget about aggregation reactions, that is irrelevant to your point,
I would disagree. I don't think that we can ignore the myriad of situations in which newly-synthesized proteins associate immediately with partners of various sorts. The "nonfunctionality" in situations like this would be very different, depending on context.
quote:
but even in the absence of aggregation reactions, a protein may be kinetically trapped in the native conformation,but this may not represent the global energy minimum.
However would we really know this?
quote:
I think that these occur rare enough to not really dent your argument.
The argument is interesting enough. But I would suggest that it may be of limited relevance to real-life biology if we restrict our view of proteins as much as John may be doing.
Another example for John to mull over - how would your idea deal with prions?
And one more point, while I'm here. It seems to me that John's idea is grounded in a rather standard teleological POV, one that postulates positive action to avoid or eliminate "nonfunctoinality". While it may not be true here, this POV in general tends to cause one to miss the most prevalent (and, for many of us, obvious) way that living things deal with non-functionality - they destroy the offending entity. Macromolecule turnover is so pervasive that it is likely that the need for "negative design" that John envisages may be rather minimal.
Or, to put things in a more positive light, the actualization of "negative design" that is intended to minimize nonfunctionality may take on a form much different than the tailoring of proteins (to assume one and only one configuration) that John is suggesting. Rather, several conformations may actually be possible, but only one may be relatively immune to the ongoing degradative processes in the cell.
[ 03 April 2002, 23:49: Message edited by: Art ]
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Nelson Alonso
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posted 03. April 2002 20:25
Aggregation reactions are irrelevant for the reasons John and I have pointed out. They are too condition-dependant to be relevant.
Same for prions. Prions are dependant on the pre-existence of another prion protein in its native form that can then be converted. And the native prion protein is synthesised from mRNA, mRNA processed from a primary transcript, and this transcribed from DNA. From a Darwinian perspective, prions may be proteins which are on their way out.
Everytime you bring up aggregation reactions, or things like prions, you leave open the situation where aggregation reactions do not occur (low protein concetrations.
[ 04 April 2002, 13:19: Message edited by: Nelson Alonso ]
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Art
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posted 03. April 2002 23:57
Nelson said: quote:
Aggregation reactions are irrelevant for the reasons John and I have pointed out. They are too condition-dependant to be relevant.
I would maintain that, if John's ideas here are to be applicable to real living things (rather than artifical polypeptides in test tubes), then aggregation is very important. For example, would negative design function to promote the biosynthesis of soluble rubisco small subunits, or of polypeptides that can only occur as a complex (aggregate) with rubisco large subunits?
quote:
Same for prions. Prions are dependant on the pre-existence of another prion protein in its native form that can then be converted. And the native prion protein is synthesised from mRNA, mRNA processed from a primary transcript, and this transcribed from DNA. From a Darwinian perspective, prions may be proteins which are on their way out.
Sorry for being so cryptic. The issue I allude to is not the origination of prions, nor of their alleged autonomous nature. Rather, I was trying to get John to think about the fact that prion proteins can exist in either of two rather different, but apparently stable (and thus low-energy) conformations. This helps us to better accept that the matter of configurational possibilities may not be as simple as John is suggesting.
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Nelson Alonso
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posted 04. April 2002 00:07
Art:
Sorry for being so cryptic. The issue I allude to is not the origination of prions, nor of their alleged autonomous nature. Rather, I was trying to get John to think about the fact that prion proteins can exist in either of two rather different, but apparently stable (and thus low-energy) conformations. This helps us to better accept that the matter of configurational possibilities may not be as simple as John is suggesting.
Nelson:
But you are still running into the problem. The prion protein emphasizes protein folding as kinetically trapped intermediates. Perhaps prion diseases are most unique in that the aggregated form is resistant to proteolysis by housekeeping processes that otherwise clean up after proteins that have folded past their useful structures. The problem is, whenever you bring aggregate reactions and examples like prion, John can simply point you to where these situations do not occur, these situations literally cry out for an explanation. John Bracht has been most gracious in giving us one.
[ 04 April 2002, 00:09: Message edited by: Nelson Alonso ]
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John Bracht
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posted 04. April 2002 01:25
Nelson:
quote:
You also might want to take a look at a possible exception to this. For example,forget about aggregation reactions, that is irrelevant to your point, but even in the absence of aggregation reactions, a protein may be kinetically trapped in the native conformation,but this may not represent the global energy minimum. I think that these occur rare enough to not really dent your argument.
I'm not really sure what you mean by this. It is my understanding that whatever the lowest-energy configuration for a protein is, that is the native conformation. Perhaps I'm mistaken, but I think your example would be a case where a protein got stuck in a side pocket but not in its native conformation. Perhaps you could clarify your idea?
Art:
quote:
Or, to put things in a more positive light, the actualization of "negative design" that is intended to minimize nonfunctionality may take on a form much different than the tailoring of proteins (to assume one and only one configuration) that John is suggesting. Rather, several conformations may actually be possible, but only one may be relatively immune to the ongoing degradative processes in the cell.
This tailoring of proteins to specific conformations is not something I've just made up. The idea is well-supported by the scientific literature. Remember, scientists have found that natural proteins don't take on multiple configuraitons--they have negative design, orderly packing of side-chains in the hydrophobic cores, and this is why our attempts to design stable, native-like proteins often fail (our proteins often have disordered hydrophobic cores). So you're not just arguing with me, you're arguing with the literature. Perhaps you can cite some literature where your idea is supported?
Furthermore, it's my understanding that cellular degradation of proteins targets unfolded proteins. But negative design refers to elimination of competing folded states, not the elimination of unfolded states. The energy landscape itself manages to drive the folding, and it's the specificity of that fold that we're talking about here. Once more, what Art is talking about (the degradation of unfolded proteins) is vastly different from what I am talking about (the number of stable folds a protein can assume).
Art:
quote:
Sorry for being so cryptic. The issue I allude to is not the origination of prions, nor of their alleged autonomous nature. Rather, I was trying to get John to think about the fact that prion proteins can exist in either of two rather different, but apparently stable (and thus low-energy) conformations. This helps us to better accept that the matter of configurational possibilities may not be as simple as John is suggesting.
Prions are complicated. A situation where a misfolded protein interacts with a native structure and "teaches" it how to misfold is definitely different from the phenonmenon of a protein achieving its (unique) native fold purely on the merits of its own amino acid sequence. However, for the sake of argument let's assume that prions could fold improperly without guidance from a misfolded protein (even though we know this isn't correct). In this case, we have an example where negative design is lacking--and we see the detrimental effects! The lack of specificity is precisely why there is a problem. Furthermore, proper negative design would presumably eliminate this problem by making the undesirable fold too energetically costly to be stable. It would raise the energy of this competing conformation so the protein would tend to drop past it (not getting trapped by the local minima) into the correct configuration on the energy landscape.
Finally, I want to address your tendency to take on a negative, patronizing tone, and your comments that I'm somehow "oversimplifying" protein folding. First, I want to emphasize that I didn't invent these ideas--they are straight from the scientific literature itself!
I'm not claiming that negative design or protein folding is simple, in any way. However, to understand complex things like biology we tend to build models, and those models will utilize some amount of simplificiation from the complexities of biology. We have to start with some simple models and build from there. That's what I'm trying to do. However, you have consistently taken a very negative stance, raising niggling technical questions that aren't very important and sometimes deal with separate matters altogether. I feel like I'm trying to build a positive model using design concepts, and you're just trying to tear down what I'm building. I think this concept may be useful, and beneficial to explore, although I'm not at all certain about that and I may end up abandoning it in the end if it doesn't hold up to scrutiny. I'm not committed to seeing it hold up, but I'm going to try to develop this idea and see where it leads. That's the scientific method and many of you Darwinists critique ID-'ers for being too negative. But here I am the one with a positive argument and you seem just to want to shoot holes in it by any means you possibly can--using the same negative technique you are so quick to criticize in the other side.
Look, any model or idea of how biology works is of necessity going to be simplified to some extent. But let's work together to come to a better understanding of whether this model is correct and how it can be improved. Come over and think positively with me about how we can improve this idea or gain insight from it. If it doesn't capture some element of biocomplexity, then give me a positive idea of how to improve the model, a way to advance my understanding and the understanding of those who read our discussion. If it's totally wrong, show me why (and why the literature is wrong too!).
I'm just asking you to avoid the negative tone and the tendency to obfuscate or obscure the real issues. Let's work together toward a clearer understanding. Negativity doesn't help anyone's understanding, and it doesn't contribute to the spirit of this discussion board.
Thanks,
John Bracht
[ 04 April 2002, 01:35: Message edited by: John Bracht ]
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James A. Barham
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posted 04. April 2002 10:09
John Bracht:
I think you have a better grasp of the literature on proteins than I have, so I wonder if I could get your reaction---and possible correction---to the way I understand a couple of points.
First, is your concept of "negative design" simply a way of stating the well-known Levinthal paradox or what is simply known as the "protein folding problem"---namely, that it is impossible to understand protein folding simply as a stochastic "search" through all possible configurations for the the lowest energy configuration, in the normal statistical-mechanical way? Or is there more to "negative design" than just that? (I feel like there's something I'm missing.)
If your main point is indeed the one about the Levinthal paradox, then what about the attempts to explain the paradox by means of physical models of higher-order organization ("funnels") and also quantum tunneling? Would such ideas, if successful, disprove "negative design," or would they on the contrary confirm it?
Second, although I acknowledge the importance of the protein folding problem, I am even more interested in the fact that the minimum-energy or native state of proteins is highly "degenerate." That is, far from lying still in this energy well, the protein is constantly writhing back and forth among a large number of nearly isoenergetic conformational substates. (It has been described by condensed-matter physicists as "kicking and screaming.") Hans Frauenfelder and others believe that functionality is intimately related with this aspect of proteins---called "frustration."
I believe that in order to explain the functional properties of proteins we are going eventually to have to extend quantum field theory in such a way as to explain the fact that internal protein motions seem to visit only a condensed portion of their conformational phase space, in accordance with what Frauenfelder calls a "minimum frustration principle." That is, there appears to be something going on in protein function that eludes ordinary statistical mechanics. I was just wondering if you had given any thought to this aspect of protein function, and if so, how it might fit in with your negative design idea.
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Nelson Alonso
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posted 04. April 2002 13:16
James:
First, is your concept of "negative design" simply a way of stating the well-known Levinthal paradox or what is simply known as the "protein folding problem"---namely, that it is impossible to understand protein folding simply as a stochastic "search" through all possible configurations for the the lowest energy configuration, in the normal statistical-mechanical way? Or is there more to "negative design" than just that? (I feel like there's something I'm missing.)
Nelson:
Thats what I'm thinking.
quote:
The existence of intrinsic bias resolves this paradox by prejudicing the ensemble of available folding trajectories toward the native minimum. Thus, a folding protein need not discriminate among an astronomical number of conformations, because intrinsic bias "steers" the molecule toward a high degree of preorganization.
I would replace intrinsic with extrinsic here, unless I am misunderstanding John.
James:
That is, there appears to be something going on in protein function that eludes ordinary statistical mechanics. I was just wondering if you had given any thought to this aspect of protein function, and if so, how it might fit in with your negative design idea.
Nelson:
I thought that we were just beginning to understand folding through statistical mechanics.
John:
I'm not really sure what you mean by this. It is my understanding that whatever the lowest-energy configuration for a protein is, that is the native conformation. Perhaps I'm mistaken, but I think your example would be a case where a protein got stuck in a side pocket but not in its native conformation. Perhaps you could clarify your idea?
Nelson:
We may never know for sure whether proteins attain their global minimum in their native fold. However, in almost every known case there is no reason whatsoever to assume that the Anfinsen hypothesis is false. So I apologize if I muddied the waters a bit.
[ 04 April 2002, 14:29: Message edited by: Nelson Alonso ]
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John Bracht
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posted 04. April 2002 14:55
James,
You're almost there. Yes, negative design has to do with protein folding and this concept of an energy landscape with a funnel that the protein can drop into as it folds. Negative design is all about engineering this energy funnel so that one, and only one, structure lies at the bottom. The reason it's called "negative design" is because it involves raising the free energy of competing structures thereby leaving only the desired one at the bottom. It's a way to work against what you don't want to leave what you do want. The problem with many human-designed proteins is that there are many different structures at the bottom of the energy well; these are competing alternatives that the protein will tend to oscillate back and forth between. Thus, while these proteins may be very stable, they are not very specific.
Here's an image that really helped clarify the idea for me:
(from the Hellinga article, http://www.pnas.org/cgi/content/full/94/19/10015):
"Fig. 1. The requirements for specificity. Three different hypothetical sequences are shown along the x-axis. Each sequence can adopt many different states (in a disordered core, for instance). The free energy of each state is given by a horizontal line. The target state is shown in gray. Sequence A is nonspecific, because all the states are approximately isoenergetic. Sequence C has the incorrect specificity, because there is a competing state of lower free energy. Sequence B is specific, because the target state corresponds to the ground state, and there is a large free energy gap, Delta Gspec, between it and the next available state. Note that to improve A by moving to B, the free energy of the target state was lowered (Target State Optimization) and the competing states were raised (Negative Design)."
It's important to realize that Sequence B represents both positive design (the lowering of free energy of the native state relative to A) and negative design in the raising of the free energy of competing states relative to A. I think C is something like Nelson was talking about.
I think the "kicking and screaming" idea you're talking about refers to the idea that even once a protein achieves its specific, native conformation (which often requires negative design), it still tends to vibrate and oscillate at the atomic level (though it's not really changing overall conformation). I have heard of this (I think that's one reason it's difficult to get good X-ray crystallographic data on proteins and why the crystals must be cooled drastically: to minimize this extra motion). But I have no detailed knowledge of this phenomeon and I'm certainly open to correction if I'm way off base here. My idea of negative design refers to achieving the overall fold, not the tiny vibrations occurring at the atomic level within that fold.
I hope that helps. Thanks for your comments.
John Bracht
[ 04 April 2002, 14:58: Message edited by: John Bracht ]
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