Hi, my name is Olivier and I'm translating part of the website in French. I'm running into difficulties, not being a specialist in proteins :) ... could someone give me a quick (if possible) explanation of what a flexible backbone protein is? Is it, as the name suggests, a protein whose structure can freely change? Here is the part of the sentence I'm currently working on:

"... second, many problems of current interest, such as flexible backbone protein design and protein-protein docking with backbone flexibility, involve a combination of the different optimization methods..."

Any help would be greatly appreciated, thanks.

Olivier

"... second, many problems of current interest, such as flexible backbone protein design and protein-protein docking with backbone flexibility, involve a combination of the different optimization methods..."

Olivier

Hi Olivier,

welcome aboard!

Yes, that sentence gave me the creeps as well. It was hard to translate it before I read some web pages on the topic (which are rare).

Flexible backbone protein design means "designing a new protein while allowing its backbone to change its conformation during the relaxation phase".

If you are interested how it works, here it is (but it is not necessary to know that for the translation):

If you want to design a new protein you chose a structure for it that will later have properties that you desire. So you want to find the lowest energy sequence of amino acids for a known structure.

If you have calculated that sequence, you will in turn have to do a refinement of your structure. Therefor you look what exact structure your sequence will adopt when you allow more degrees of freedom. This means you are back in the stage of structure prediction: you calculate the lowest energy structure for a known sequence. Doing so requires you to make the backbone of the protein in your calculations flexible, so it can kind of wiggle in place to really adopt the lowest energy structure.

After that is done you will now have to look if your sequence still yields the desired structure accurately enough to perform its task or if you might have to change some side chains, i.e. exchange some amino acids in the sequence for others. This way you fine tune the structure.

What you are basically doing is go through that cycle several times until your result is acceptable: first find the sequence for the structure (fixed backbone design), then the refined structure for that sequence (prediction with flexible backbone) and so on. The result: you are designing a new protein and need to keep its backbone flexible to really be sure to get the correct structure => flexible backbone protein design.

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"I know that you believe you understand what you think I said, but I'm not sure you realize that what you heard is not what I meant." R.M. Nixon

?And a "flexible backbone design" is in contrast to a "fixed backbone"?

?And so is a fixed backbone a model where we ASSUME the backbone structure is a specific shape, and only the sidechains would be adjusted and testing energy levels of different shapes?

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Running Microsoft's "System Idle Process" will never help cure cancer, AIDS nor Alzheimer's. But running Rosetta@home just might!
https://boinc.bakerlab.org/rosetta/

?And a "flexible backbone design" is in contrast to a "fixed backbone"?

?And so is a fixed backbone a model where we ASSUME the backbone structure is a specific shape, and only the sidechains would be adjusted and testing energy levels of different shapes?

Yes, both are correct. The difference between the flexible and fixed backbone design (BD): In the fixed BD you just optimize the side chains until your structure is of the lowest energy for the chosen, inflexible backbone.

Only the following optimization takes the fact into account that the backbone is not really fixed but can change its conformation. That optimization uses exactly the same method that is applied for a structure prediction. Only both steps together are named flexible BD.

edited for some mismatches, time for bed I guess...

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"I know that you believe you understand what you think I said, but I'm not sure you realize that what you heard is not what I meant." R.M. Nixon

Hi Olivier,

welcome aboard!

Yes, that sentence gave me the creeps as well. It was hard to translate it before I read some web pages on the topic (which are rare).

Hey, thanks! That helps me a lot, I'll work on that tomorrow.

OK, I have a few more questions...

So the backbone of a protein is pretty much its main chain (or sequence) of amino-acids molecules ? I’m not sure I understand correctly the difference between the sequence and the structure: the sequence is the order of the amino-acids molecules within the protein (for a given structure), and the structure is the amino-acids’ 3D spatial configuration? If it’s not too much bother for you, could you give a simple example of the flexible backbone protein design process?

What are the residues of the sequences of a protein then? Are there really side chains, or are all chains equally important for the structure?

What is protein folding exactly ?

I woner about this sentence: « We used the physical model described above and a modification of our rotamer search-based computational design strategy to generate novel DNase-inhibitor protein pairs predicted to interact tightly with one another but not with the wild-type proteins »; what does the wild-type in that case mean? Proteins not of the type designed here?

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"I know that you believe you understand what you think I said, but I'm not sure you realize that what you heard is not what I meant." R.M. Nixon

Great stuff Christoph... I think if I read through this another 3 times I'll really be getting a handle on things.

If you want to post some pictures, you can scratch them off with paintbrush, save them as JPEGs, and upload them to a photo sharing site such as photos.yahoo.com and then post a link here.

How to post an image in a message.
How to post an image without making the message boards cumbersome to use.

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Add this signature to your EMail:
Running Microsoft's "System Idle Process" will never help cure cancer, AIDS nor Alzheimer's. But running Rosetta@home just might!
https://boinc.bakerlab.org/rosetta/

Thank you so much Christoph, I'm going to take a look at all that. I'm sorry if this info can be found somewhere else on the site, I haven't had much time to check it out, between that translation, real work, kids, etc... (not necessarily in the right order!) :-) Don't hesitate to point me to a link if you don't have time to answer all my questions! Thanks a lot again.

Olivier

Hi Olivier,

I did several attempts at writing this, it was very hard to put it into words without pictures and the screen saver. After all that is very difficult chemical stuff if you want to go through it in detail. Maybe we can exchange e-mail addresses, that would make exchanging links and texts much easier. Ok, but now for the answer:

You mistake the �side chains� as chains like those you see in your Rosetta graphics, but that is not correct. The Rosetta graphics only shows the backbone. The side chains are better called residues as they are part of the amino acids. Every amino acid has exactly one residue. Think of the amino acids as magnetic sticks with a flag on them. You will always connect north pole and south pole of the magnets � that is the backbone. The magnets themselves are indistinguishable: the always consist of the atoms

��.[-N-C-C-O-][N-C-C-O-][N-C-C-O]�..

On each magnet you have one flag (the residue). It is always hanging on the C atom that is directly to the right of the N atom. This flag determines the nature, the name and the chemical properties of the amino acid (as the part in the backbone is indistinguishable from its neighbour). It is important to be aware that it is the stick plus the flag that makes one amino acid. The order in which the amino acids follow one another is called the sequence. This sequence is called the �primary structure�.

There are 20 such flags with different chemical properties and thus there are 20 different amino acids. Some are similar and can form bonds: lets say the flags have numbers and it is the flags 1 to 7, 8 to 14 and 15 to 20 that can form bonds to flags from the same group.

Ok, now look at your Rosetta graphics. You see only the backbone, no residues at all, that is important. If you would stretch the backbone to a long ribbon, it would have a zig-zag shape which remains; you cannot stretch that to a straight line as the angles of the bonds in the backbone are more or less fixed. So you must rotate around those bonds to change the shape. Bends are only achieved by rotating a number of adjacent bonds, not by changing angles.

Now imagine that the different side chains are trying to get near to ones that they can form bonds with. Often they can do so with close neighbours. One of the results from that are the helices you see in the graphics: as the zig-zagging remains intact, one good way to get neighbouring parts together is to form these helices. Such neighbouring structures are called the �secondary structure�. There are different sorts, but they will not bother us here.

After that secondary structure is formed, the helices, sheets, hairpins an whatever will still have unbound residues and will now continue to bend in order to form bonds to adjacent parts. When finally all bonds are optimally formed and only a few side chains are still �dangling outside� of the protein without a partner you have the final form of the protein: its 3D structure, also called the �tertiary structure�.

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The process of reaching that structure is called folding as you kind of have a ribbon that folds together. Rosetta folds proteins by sifting through numerous possibilities of how they can bend and bond and calculating the overall energies of each structure. It does not say anything on the way that structure is reached step by step in nature, that is what Folding@home does.
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Describing protein design is very hard. I'll just try to give an impression. Think of it like you would have to take a motor out of a car and you needed a tool that takes apart all screwes at once, not one by the other. That is how proteins work: snap on and do.

Now you have several parts to assemble that tool from, your amino acids. Of course you will first put all wrenches in place and then build a rig aorund that that hold all those wrenches. The wrenches are the residues that do the job, the rig is the backbone. The overall shape of the rig is determined by the residues of the other amino acids that interact with each other, not with the motor.

There are two ways to construct that rig: you may just assemble it roughly by putting the wrenches in place and assemble the rest of the amino acids around that. The angles will then not all exactly be in accordance with the zig-zag of the backbone, so it will twitch here and there, there will be tensions, but it will fit and work. That is fixed backbone design.

If you now go through a second step in which you calculate the shape your rig really takes and after that redesign it a little by using some better fitting lengths of wrenches, so that there are less tensions and twitches, you do flexible backbone design. It is most efficient if you repeat it a few times, thus reducing the tensions to a minimum.

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On �wild types�: they are the types occurring in nature. What the sentence means is that they created two new proteins. These proteins were designed in a way that they should only react with one another and not with the natural occurring type anymore. So you now have two pairs of proteins that can be in the same cell without interfering with one another.

One application is this: you want to cut out a specific part of a DNA that contains a defective gene. So you design a protein that does exactly that. Then the DNA is repaired with a functional copy of that gene. After that you want to stop the protein from cutting out the gene again, as the task is completed. So you need to design a protein that exactly fits into the first one and remains in it, thus blocking its function.

But, as there are numerous specific proteins that cut out DNA and all of them have their own special, vital task, you need to prove that you can produce a protein that will definitely only stop the other artificial protein and not the natural ones too, which might be extremely harmful. That is what they showed: the wild types bond to one another and the designed types bond to one another, but no cross-bonding. It is to be hoped that someday that can become a pretty safe method if done carefully.

great explanations!

as soon as we get some time, we will add the sidechains to the screensaver. I get impatient watching sometimes during the fullatom relax stage because it doesn't seem like anything is changing, but that is because you can't see the sidechains moving around. also, it is fun to watch the sidechains changing conformations and identities in protein design calculations (we will start sending these out after CASP is over)

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Hi Olivier,

I did several attempts at writing this, it was very hard to put it into words ....

Cheers Christoph

Thank you for taking the time to make such a clear explanation.

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Bonjour Olivier,

Merci pour tes questions et pour traduire notre site en francais! Je pense que tu comprend le biochimie bein mieux que je comprend francais (excuse ma mauvaise grammaire et ortho, svp), mais quand meme j'aimerais bien lire ton site de web en francais, quand tu a fini faire la traduction.

Thanks Christoph for taking time and effort to explain things so well! I'm well behind, and there's not much left to explain, nonetheless, I thought I'd chime in to add a little more detail to the discussion of amino acids, residues, sequence and structure.

OK, I have a few more questions...

So the backbone of a protein is pretty much its main chain (or sequence) of amino-acids molecules ? I’m not sure I understand correctly the difference between the sequence and the structure: the sequence is the order of the amino-acids molecules within the protein (for a given structure), and the structure is the amino-acids’ 3D spatial configuration?

Christoph's diagram explains the difference between the main chain (the backbone) and the side chains (the "R" moieties). A further explanation that may help is that protein structure can be described at 4 different levels.

The PRIMARY STRUCTURE is given by the sequence of amino acids (the order of different R groups). It consists of the chemical structure of the protein and the only thing you need to add to the diagram below would be to replace the R symbols with the actual atoms that they represent. So for example a simple 3 residue peptide represented in text like this: Alanine-Serine-Cysteine, would have a primary chemical structure comprised of 3 of the monomer units in the diagram below, where the first R would be replaced by a methyl group (a carbon and 3 hydrogens), the 2nd R would be replaced by a carbon and an alcohol group, and the 3rd would be replaced by a carbon and a sulfur.

The SECONDARY STRUCTURE describes the local conformations of the primary structure: the helices mentioned below are an example of secondary structure. You need more than just the chemical formula to visualize the secondary structure, you need to see interactions between residues.

The TERTIARY STRUCTURE describes interactions between residues that are far from each other in sequence. In the linear chain below tertiary contacts can occur between residues that are separated in chain by 5 peptides bonds, or more (up to hundreds).

Together, the primary, secondary, and tertiary structures, if known, define and describe the 3D structure of a protein.

The QUARTENARY STRUCTURE is the final, highest order description of structure, and this describes the interactions between protein molecules - eg two proteins that are not linked to each other by bonds like the ones described by lines in the figure below, but which nonetheless come together to form a "dimer". The quartenary structure is often important in biology, as little "molecular machines" are built from complexes of proteins. There is a mode of Rosetta which examines quartenary structure, and tries to predict which proteins will interact and how they interact.

If it’s not too much bother for you, could you give a simple example of the flexible backbone protein design process?

Je pense que Christoph a donner un tres bon example du FlexBB design. Moi je essaie de developer les methodes de faire le FlexBB design, et c'est tres difficile! Il y a plusieres methodes qu'on essaie, mais le methode qui a eu le plus de succes est celle qui est decrit par Christoph. Si tu te sent un peu brave, tu peu aussi lire le papier du Brian Kuhlman sur le design d'un proteine completement nouvelle, qui a jamais etre vue en nature, et qui a ete cree par le FlexBB design. Le pdf du papier se trouve sure le site de web du Baker lab. Le papier a ete publis en 2003:

Kuhlman, B., Dantas, G., Ireton, G. C., Varani, G., Stoddard, B. L., Baker, D. (2003). Design of a novel globular protein fold with atomic-level accuracy Science 302, 1364-8.

What are the residues of the sequences of a protein then? Are there really side chains, or are all chains equally important for the structure?

Yes the side chains are important for all aspects of protein structure. The chemical nature (sequence) of sidechains is the full description of the primary structure. The size of the side chains restrict which secondary structures are allowed. The non-covalent chemical interactions between side chains specifies the tertiary structure. Also, you may look at a previous post, as well as the science FAQ. Mais je pense que les explications donner par Christoph sont bein meilleure que les miens ;-)

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Bonjour Olivier,

Merci pour tes questions et pour traduire notre site en francais! Je pense que tu comprend le biochimie bein mieux que je comprend francais (excuse ma mauvaise grammaire et ortho, svp), mais quand meme j'aimerais bien lire ton site de web en francais, quand tu a fini faire la traduction.

Bonjour, et merci. Malheureusement, je pense que tu comprends mieux le français que moi la biochimie! Mon truc c'est plutôt la physique nucléaire, et encore, comme je suis ingénieur, le temps où je faisais de la physique nucléaire pure est... très loin... mais toutes les réponses apportées très gentiment ici avec beaucoup de patience m'aident beaucoup. Merci! Je ne sais pas trop quelle sera l'adresse du site final, mais j'essaierai de le poster, si jamais notre travail est utilisé.

Thanks Christoph for taking time and effort to explain things so well! I'm well behind, and there's not much left to explain, nonetheless, I thought I'd chime in to add a little more detail to the discussion of amino acids, residues, sequence and structure.

Yes, thanks again Christoph, and thanks Vanita, that helped me some more. I'm going to try that paper you pointed me to.