From: ian@inf.ethz.ch (Ian)
Newsgroups: sci.nanotech
Subject: Re: What computer simulation can't do yet
Date: 29 Sep 92 03:39:06 GMT
Organization: Dept. Informatik, Swiss Federal Institute of Technology (ETH)

In article <Sep.27.20.56.35.1992.20078@planchet.rutgers.edu> kr%portal@cup.portal.com writes:
"ian@inf.ethz.ch (Ian) has written on 9/25/92:
"
"-"
"-"If  by  reverse  folding  you mean  "given  a  set  of  geometric 
"-"requirements  for the active-site of a protein, what sequence  is 
"-"required"  then (I think) the problem is a lot older  than  that.  
"-"After all people like Anfinsen were working on protein folding in 
"-"general  in 1973 (just the oldest reference that I had  to  hand) 
"-"and  I  don't  believe that they  never  considered  the  obvious 
"-"corollary.
"
"There are a lot of obvious things that nevertheless seem to require an
"unconventional intruder who brings them really to people's attention.
"An example is PCR, which was invented by Kary Mullis after the
"technological capabilities for implementing PCR have been hanging around
"(unused for this purpose) for well over a decade...

Well, *I* certainly thought of the inverse problem as soon as I heard of the
forwards problem but then I'm certainly unconventional (:-)

"Let me just quote from the published source itself
"("Molecular engineering: An approach to the development of general
"capabilities for molecular manipulation" by K. Eric Drexler, PNAS 78 (1981)
"p.5275-5278):
"
""Indeed, chemists cannot yet predict the three-dimensional conformation of
"a natural protein from its amino acid sequence, an ability that might seem
"requisite to the design of new proteins. Two considerations suggest that this
"obstacle is surmountable: first, the continuing improvement in protein science
"and, second, the difference between natural science and design engineering."

I'm sure it's surmountable, I work on it myself, but it's very, very hard.  The
big question is "Will we really need it ?".

""Regarding the second consideration, natural scientists seek a more general
"understanding than design engineers require. Science seeks the ability to
"predict the conformations of all natural peptides."

And unnatural ones, the real problem is a general one concerning all linear, 
non-repeating polymers.  (Or if you like, just the question of systems with
vast configurational (or even phase) spaces).

"", natural selection accepts any folding process so long as the chain reaches
"its natural conformation with essentially 100% yield."

1/2 true, there are a few other constraints as well (such as not binding to DNA
or other proteins, not costing too much in energy or limitting chemicals).

""Thus, it is little wonder that chemists cannot yet predict the conformations
"of natural proteins; they are not designed to fold predictably.
"Engineers (in contrast to scientists) need not seek to understand all proteins
"but only enough to produce useful systems in a reasonable number of attempts.
"An engineer designing a protein that has 1000 amino acids may choose among 
"some 10^1300 different amino acid sequences. It might be that only one in 10^9
"(or even 10^700) randomly selected sequences would yield a predictable
"conformation, yet this tiny fraction represents a vast number of proteins.
"Through use of strategically placed charged groups, polar groups, disulfide
"bonds, hydrogen bonds, and hydrophobic groups, the engineer should be able
"to design proteins that not only fold predictably to a stable structure
"(sometimes) but that serve a planned function as well. Even a low success rate
"will lead to an accumulation of successful designs. Thus, the difficulties
"encountered in predicting the conformations of natural proteins do not seem
"insurmountable obstacles to protein engineering."

There's a slight problem here, if only 1 in 10^700 sequences folds, we're still
never going to find one (there just aren't enough genetic engineers).  However,
the point is still valid as folding is not a clear-cut, on/off precedure,
successive approximation and over-design can radically reduce the search time.

Hmmm, perhaps I mis-read him.  It doesn't matter, I agree anyway.

"
"When he gives away copies of this paper nowadays, usually there is a note
"attached, speaking even more clearly:
"
""Protein Engineering:
"A 1988 view of some 1981 predictions
"
"A 1981 paper [1] discussed de novo protein design as part of a long-term
"{what a conservatively careful formulation:-) my commentary} strategy for
"developing complex molecular devices and systems. It presented arguments
"against the view that the fold-design problem is an extension of the classical
"(and still unsolved) fold-prediction problem (i.e., predicting folds from
"sequences without homologous models), a view which has discouraged efforts
"at design.
"Fold prediction is a scientific problem: it must deal with naturally evolved
"sequences, but natural selection's 'design goals' enforce only the physical
"reliability of folding - not its human predictability. This results in folds
"of only minimal stability. Fold design, in contrast, is an engineering problem.
"Protein engineers, exploiting their freedom of design, can work with sequences
"artificially selected for superior predictability and stability of folding.

This is very close to the 'bridge-building' approach to MD which has been 
mentioned elsewhere.  The general principle seems to be:

  "So what if we don't inderstand it, lets use the bits we do understand"

Which I find a very promising attitude.

"These observations indicated that "the difficulties encountered in predicting
"the conformations of natural proteins do not seem insurmountable obstacles to
"protein engineering"[1].

It depends what we want the engineered proteins to do, if they are for
test-tube use then anything will do.  If, on the other hand, they are for use
in a nanite or modified organism then sooner or later we are going to meet
some of the 'secondary constraints' which evolution has had to cope with.
(see above)

In particular, if we need proteins that can be degraded, proteins of restricted
energetic cost, proteins without unfortunate side-effects and/or proteins which
not only catalyse the right reaction, but only catalyse it the the desired
degree then the problem is rapidly becoming so convoluted that we won't be able
to use sequences which are 'simplified' sufficiently that they fold with
absolute certainty.  (nb This point is to some extent a general one.  For 
example, in the the MD discussion many people have said that the slight
failings of the force-field are irrelevant provided we put sufficient 
structural over-design into the system.  This over-design get harder and harder
the more other constraints we work with (such as catalysis, easy of synthesis,
stability vs known 'hostile' chemicals, functionality and control).  So we 
can't discard the difficulties altogether.)

This isn't an immediate problem, however, as far simpler systems come first.

"In accord with the implications of this argument, we have seen the successful,
"de novo design of a globular protein (alpha4) [2,3] while the classical fold
"prediction problem remains unsolved [4]. Likewise confirmed has been the 
"suggestion that design can increase protein stability beyond that enforced
"by natural selection. In recent years, deliberate single-residue modifications
"have raised protein stabilities through a variety of mechanisms [5,6]. Owing
"to design choices consistently biased towards stability, the protein alpha4
"has a stability of 22kcal/mole, substantially greater than the 4-9 kcal/mole
"of typical natural proteins of a similar size [3].

I can't remember, has anyone put a catalytic group on such a construct ?
(I know of a catalytic single alpha-helix, made artificially)