Feyn­man Said "Just Look At The Thing!"

by Jan Spitzer

On Oc­to­ber 28, 2010, Elio posted this Tal­mu­dic Ques­tion: "Richard Feyn­man, the fa­mous physi­cist, said: It is very easy to an­swer many of these fun­da­men­tal bi­o­log­i­cal ques­tions; you just look at the thing! To take him up on it, imag­ine a mi­cro­scope that lets you ob­serve sin­gle mol­e­cules in a liv­ing cell at one Angström res­o­lu­tion. What's the first thing you would do with it?" Thank you, Elio for al­low­ing me to pro­vide some thoughts on the mat­ter from the per­spec­tive of a phys­i­cal chemist/chemical en­gi­neer.

Such a mi­cro­scope could in­deed help ad­dress some of the fun­da­men­tal is­sues in bi­ol­ogy to­day. I must say up­front that I am sur­prised that mi­cro­bi­ol­o­gists would want to look at (small) mol­e­cules ('pure' chem­istry), or at the chem­i­cal de­tails of 'big­ger things', as sug­gested by the very no­tion of us­ing a 'Schaechter-Feyn­man su­per­mi­cro­scope'. This hy­po­thet­i­cal in­stru­ment would have a res­o­lu­tion of 0.1nm with ex­po­sure times in the pi­cosec­ond range (mak­ing it a bit akin to an in­frared spec­tropho­tome­ter) and would op­er­ate in Feynman's quan­tum me­chan­i­cal world. It would look at the chem­istry of bi­ol­ogy, dis­sect­ing cells into their mol­e­c­u­lar com­po­nents that are then chem­i­cally char­ac­ter­ized in­di­vid­u­ally. But let me ex­plain this more...

What Should We Look At?

Feyn­man sug­gested that 'see­ing bet­ter' is 'bet­ter.' How­ever, what we see of­ten de­pends on what we are look­ing for. At such very high res­o­lu­tions, we risk fo­cus­ing on de­tails so small that we lose con­text and per­spec­tive, like look­ing at the leaves of in­di­vid­ual trees and los­ing sight of the sur­round­ing for­est. We will also miss the lakes, the mead­ows, and even the blaz­ing sunset—the red­dish light scat­ter­ing for­ward from the nu­cle­at­ing par­ti­cles of the nano-fog, the beauty of which, Feyn­man in­sists, a mere poet may miss (1). Sim­i­larly, a nar­rowly-fo­cused mol­e­c­u­lar re­searcher over­looks the re­la­tion­ship of the de­tail to the liv­ing whole (2,3). I would sug­gest ob­serv­ing at a slightly larger scale, say di­men­sions of 10 to 100 nm and du­ra­tions of a mil­lisec­ond, to see the 'metabolons', 'mod­ules', 'hy­per­struc­tures', and the many func­tional pro­tein com­plexes (sig­nal­somes, stres­so­somes, tran­scrip­tomes, di­vidi­s­omes, etc.) to find out if and how they ex­ist as dis­crete phys­i­cal ob­jects. We might then see these large, tran­sient bio­macro­mol­e­c­u­lar clus­ters ap­pear and dis­ap­pear, and see them in­ter­act at the sub­cel­lu­lar level. We could take full ad­van­tage of the atomic res­o­lu­tion of this su­per­mi­cro­scope to vi­su­al­ize ionic cur­rents of ATP, GTP, phos­phates, K and Mg ions, bi­car­bon­ate, glu­ta­mate etc., track­ing them from their ori­gins to where they sink and dis­ap­pear. Even the vec­to­r­ial ionic cur­rents gen­er­ated in the cell en­ve­lope with their move­ments through and around the large 'omic' bio­macro­mol­e­c­u­lar clus­ters could be vi­su­al­ized as the cell grows. What would we see when a bac­te­r­ial cell be­gins to die? Will the ionic cur­rents 'die'? (4–6). So many events to ex­plore with our su­per­mi­cro­scope!

What Did Feyn­man Ac­tu­ally Say?

Feynman's sug­ges­tion be­trays the physicist's 'weltan­schau­ung', the world view that Feyn­man-in­flu­enced bi­ol­o­gists fo­cused on. At the time, how­ever, nei­ther bi­ol­o­gists nor physi­cists un­der­stood what the 'thing' to look at was. The sit­u­a­tion then was sim­i­lar to one that chemists had to face 100 years ear­lier (circa 1860)—having to de­velop meth­ods of chem­i­cal analy­sis: first, qualitative—to know what kind of atoms and mol­e­cules one is deal­ing with, and then quantitative—how many, in what pro­por­tions, their struc­tures, the chem­i­cal re­ac­tions be­tween them, etc. It is worth­while to reprint that sec­tion of Feynman's ('nan­otech­nol­ogy') talk:

What are the most cen­tral and fun­da­men­tal prob­lems of bi­ol­ogy to­day? They are ques­tions like: What is the se­quence of bases in the DNA? What hap­pens when you have a mu­ta­tion? How is the base or­der in the DNA con­nected to the or­der of amino acids in the pro­tein? What is the struc­ture of the RNA; is it sin­gle-chain or dou­ble-chain, and how is it re­lated in its or­der of bases to the DNA? What is the or­ga­ni­za­tion of the mi­cro­somes? How are pro­teins syn­the­sized? Where does the RNA go? How does it sit? Where do the pro­teins sit? Where do the amino acids go in? In pho­to­syn­the­sis, where is the chloro­phyll; how is it arranged; where are the carotenoids in­volved in this thing? What is the sys­tem of the con­ver­sion of light into chem­i­cal en­ergy? It is very easy to an­swer many of these fun­da­men­tal bi­o­log­i­cal ques­tions; you just look at the thing! You will see the or­der of bases in the chain; you will see the struc­ture of the mi­cro­some.

For the record, the "mi­cro­somes" con­tained circa 20 nm 'gran­ules', which were even­tu­ally pu­ri­fied and char­ac­ter­ized as today's ri­bo­somes.

Ar­ti­facts, ar­ti­facts…

(Fig. 1.) The nanovi­sion of the scan­ning tun­nel­ing mi­cro­scope: 'Watch­ing mol­e­cules sit'. Source

Ob­vi­ously, look­ing at the 'thing' is not that sim­ple. The 'thing' could be an ar­ti­fact of sam­ple prepa­ra­tion. The his­tory of elec­tron mi­croscopy has pro­vided us with many such ar­ti­facts (7,8) and this re­mains an is­sue to­day for the new spec­tro­scopic in vivo meth­ods (9,10). There is also the sam­pling problem—the vari­abil­ity be­tween and within cel­lu­lar pop­u­la­tions. It is chal­leng­ing to main­tain pop­u­la­tions of 'model' bac­te­r­ial strains 'frozen in evo­lu­tion' and 'con­stant', there­fore re­pro­ducible be­tween dif­fer­ent lab­o­ra­to­ries. In ad­di­tion, the phys­i­o­log­i­cal state of a cell pop­u­la­tion varies with the time of sam­pling, as well as with en­vi­ron­men­tal and nu­tri­ent con­di­tions (11, 12). This would be a shock for Feyn­man, who thought about 'the thing' as some­thing sta­tic, pic­tur­ing bio­macro­mol­e­cules as 'sit­ting' some­where. This is im­por­tant for nano-tech­nol­ogy (Fig. 1), be­cause when things 'do not sit', 'mol­e­c­u­lar hell breaks loose' thanks to the sec­ond law of thermodynamics—the en­tropy (dis­or­der) seek­ing to max­i­mize it­self. In­deed the great­est achieve­ments of nan­otech­nol­ogy have been so far mainly in the ar­eas where mol­e­cules do 'sit', i.e. in the solid state.

The Sum of the Parts

Which brings me back to the 'reductionist's prob­lem' en­coun­tered when at­tempt­ing to re­duce ('soft mat­ter') bi­ol­ogy to chem­istry. Can we re­ally un­der­stand a 'liv­ing' sys­tem (a bac­te­r­ial cell) by tak­ing it apart, then iden­ti­fy­ing all its com­po­nents by their chem­istry and struc­ture? This, af­ter all, is what X‑ray crys­tal­log­ra­phy and NMR are achiev­ing. The bio­chem­i­cal and phys­i­o­log­i­cal func­tions of many pro­teins and nu­cleic acids have been elu­ci­dated through en­zy­mol­ogy and ge­netic stud­ies (mu­tant analy­sis, for the most part). But to re­it­er­ate Craig Venter's ob­ser­va­tion: No sin­gle cel­lu­lar sys­tem has all of its genes un­der­stood in terms of their bi­o­log­i­cal roles (13). In fact, a pro­tein may have two (or more) func­tions (moon­light­ing pro­teins), e.g., glu­ta­mate race­mase, es­sen­tial for pro­vid­ing the D‑glutamate needed for bac­te­r­ial wall syn­the­sis, also in­hibits DNA gy­rase. I would ar­gue that today's fun­da­men­tal chal­lenge in cel­lu­lar bi­ol­ogy is that RNA and pro­teins do not 'sit'; they move, as does ATP, ions, wa­ter mol­e­cules, DNA, the ri­bo­somes, even the cell it­self. They all move on time scales that span about 10 or­ders of mag­ni­tude, from pi­cosec­onds to sec­onds! And they move about in a crowded en­vi­ron­ment, where mol­e­cules and macro­mol­e­cules in very close mol­e­c­u­lar prox­im­ity in­ter­act with each other, break­ing and mak­ing chem­i­cal bonds, non-co­va­lently at­tract­ing and re­pelling one an­other with mol­e­c­u­lar forces rang­ing dur­ing the cell cy­cle from the ul­tra-weak (tran­sient, non-her­i­ta­ble) to the very strong (per­ma­nent, 'her­i­ta­ble') (4,5, 14–17).

(Fig. 2.) The tran­si­tion from chem­i­cal evo­lu­tion of 'pre­bi­otic soups' to bi­o­log­i­cal (Dar­win­ian) evo­lu­tion at the ori­gin of life 3.5 BYA, and the po­ten­tial 'birth of the liv­ing state' from the 'bi­otic soup' of ex­tant bac­te­r­ial pop­u­la­tions.

The Humpty-Dumpty Prob­lem

The great achieve­ment of the re­duc­tion­ist par­a­digm, of the tak­ing apart of liv­ing things, is the demon­stra­tion that all we see there is just 'or­di­nary' chem­istry and physics. The chem­istry of the lipids, pro­teins, and par­tic­u­larly the nu­cleic acids (the ge­netic code) is the 'same' for all liv­ing things; unique liv­ing sys­tems are each a vari­a­tion on the same chem­i­cal theme com­posed from rel­a­tively few chemicals—the 'build­ing blocks of life' (12). These bio­mol­e­cules and bio­macro­mol­e­cules have been syn­the­sized by non-bi­o­log­i­cal meth­ods of or­ganic chem­istry, start­ing with Wöhler's syn­the­sis of urea in 1828 and cul­mi­nat­ing with the chem­i­cal syn­the­sis (and en­zy­matic as­sem­bly) of the chro­mo­some of the bac­terium My­coplasma my­coides (13). There can be lit­tle doubt that all such syn­thetic bio­macro­mol­e­c­u­lar prod­ucts will have the same prop­er­ties (chem­i­cal and bi­o­log­i­cal) as those syn­the­sized en­zy­mat­i­cally by cells. How­ever, it is when the mol­e­cules and macro­mol­e­cules come to­gether in a dy­namic en­vi­ron­ment that the 'liv­ing state' of mat­ter is born. Be­cause we do not yet know how the 'liv­ing state' of mat­ter comes about, we are as­ton­ished at see­ing bac­te­r­ial cells 'do what they do'—growing and di­vid­ing at an in­cred­i­ble speed, some­times in less than an hour! Even more as­ton­ish­ing is the fact that cel­lu­lar pop­u­la­tions of progenotes and uni­ver­sal an­ces­tors (18,19) emerged 3.5 bil­lion years ago! In my opin­ion, ef­forts should fo­cus on 'putting Humpty-Dumpty to­gether again' (20), syn­the­siz­ing 'life' (21), or coax­ing 'life' to emerge from a 'bi­otic soup' (22), mim­ic­k­ing the emer­gence of the chem­istry of life from the 'pre­bi­otic soup' (Fig. 2) about 3.5 bil­lion years ago (23–25). These 'syn­thetic' ap­proaches will have to take into ac­count the phys­i­cal chem­istry un­der­ly­ing the in vivo state of liv­ing cells i.e. the tran­sient in­ter­ac­tions of crowded bio­macro­mol­e­cules within them, and their cyclic non-equi­lib­rium na­ture (14–17,20). Here Richard Feynman's other, much later sen­ti­ment comes to the res­cue: What I can­not build (cre­ate), I can­not un­der­stand (Fig. 3). It is clear that mi­cro­bi­ol­o­gists are in a unique po­si­tion to de­vise new pro­to­cols to study and even cre­ate life, with­out hav­ing to chem­i­cally syn­the­size any­thing, by tak­ing bac­te­r­ial cells apart, 'killing them gen­tly', and then try­ing to re­vive the 'bi­otic soup' (Fig. 2). There are no fun­da­men­tal rea­sons why such pro­to­cols would not work, and they would re­move some of the mys­tery from Pasteur's dic­tum 'Omne vivum e vivo'. We just need to fig­ure out how to han­dle the spher­o­plasts, pro­to­plasts, the nu­cleoid, ri­bo­somes, and large pro­tein clus­ters! Play with them; make them as­sem­ble into a liv­ing con­di­tion. Ad­mit­tedly, this may not be as easy as just 'look­ing at the thing' but may well turn out to be quite valu­able.

(Fig. 3.) Feynman's en­dur­ing sen­ti­ment: 'What I can­not cre­ate, I do not un­der­stand'.

Ref­er­ences

1. http://www.goodreads.com/quotes/127643-poets-say-science-takes-away-from-the-beauty-of-the
2. A Let­ter to the Blog by Franklin M. Harold: Let's Put Or­gan­isms Back in the Pic­ture!
3. Harold FM (2005) Mol­e­cules into cells: spec­i­fy­ing spa­tial ar­chi­tec­ture. Mi­cro­biol. Mol. Biol. Rev. 69:544–564
4. Spitzer JJ, Pool­man B (2005). Elec­tro­chem­i­cal struc­ture of the crowded cy­to­plasm. Trends Biochem Sci 30:536–541
5. Spitzer J (2011) From wa­ter and ions to crowded bio­macro­mol­e­cules: in vivo struc­tur­ing of a prokary­otic cell. Mi­cro­biol Mol Biol Revs 75:491–506
6. Davey HM (2011) Life, death and in-be­tween: mean­ings and meth­ods in mi­cro­bi­ol­ogy. Appl En­v­i­ron Mi­cro­biol 77:5571–5576\
7. Heuser J (2002) What­ever hap­pened to the 'mi­cro­tra­bec­u­lar con­cept'? Biol Cell. 94:561–96.
8. Pow­ell K (2005) "Porter­plasm" and the mi­cro­tra­bec­u­lar lat­tice. J Cell Biol. 170:864–865.
9. Mar­golin W (2012) The price of tags in pro­tein lo­cal­iza­tion stud­ies J. Bac­te­riol. 194: 6369–6371
10. Swulius MT and Jensen GJ (2012) The he­li­cal MreB cy­toskele­ton in E. coli MC1000/pLE7 is an ar­ti­fact of the N‑terminal YFP tag. J. Bac­te­riol. 194:6369–6371
11. Mika JT, Kras­nikov V, van den Bo­gaart G, de Haan F, and Pool­man B (2011) Eval­u­a­tion of Pulsed-FRAP and Con­ven­tional-FRAP for de­ter­mi­na­tion of pro­tein mo­bil­ity in prokary­otic cells. PLoS One 2011; 6(9): e25664.
12. Nei­d­hardt FC, In­gra­ham JL and Schaechter M (1990) Phys­i­ol­ogy of the Bac­te­r­ial Cell. Sin­auer As­so­ciates Pub­lish­ers, Sun­der­land MA.
13. Gib­son et al. (2010) Cre­ation of a bac­te­r­ial cell con­trolled by a chem­i­cally syn­the­sized genome. Sci­ence 329 (5987): 52–56
14. Zim­mer­man SB and Minton AP (1993) Macro­mol­e­c­u­lar crowd­ing: bio­chem­i­cal, bio­phys­i­cal, and phys­i­o­log­i­cal con­se­quences. Annu Rev Bio­phys Bio­mol Struct 22:27–65
15. Mik­los AC, Li C, Sharaf NG, Pielak GJ (2010) Vol­ume ex­clu­sion and soft in­ter­ac­tion ef­fects on pro­tein sta­bil­ity un­der crowded con­di­tions. Bio­chem­istry 49:6984–91.
16. Mika JY and Pool­man B (2011) Macro­mol­e­cule dif­fu­sion and con­fine­ment in prokary­otic cells. Curr. Opin­ion in Biotech­nol. 22:117–126.
17. Wang Q, Zhu­ravl­eva A, and Gierasch LM (2011) Ex­plor­ing weak, tran­sient pro­tein-pro­tein in­ter­ac­tions in crowded in vivo en­vi­ron­ments by in-cell nu­clear mag­netic res­o­nance spec­troscopy. Bio­chem­istry 50:9225–9236
18. Woese CR (1998). The uni­ver­sal an­ces­tor. Proc. Natl. Acad. Sci. USA 95:6854–6859
19. Fox GE (2010) Ori­gin and evo­lu­tion of the ri­bo­some. Cold Spring Harb Per­spect Biol 2:a003483
20. Gierasch LM and Ger­shen­son A (2009) Post-re­duc­tion­ist pro­tein sci­ence, or putting Humpty Dumpty back to­gether again. Na­ture Chem Biol 5:774 – 777
21. Szostak JW, Bar­tel, DP and Luisi, PL (2001) Syn­the­siz­ing Life. Na­ture 409:387–390
22. http://abscicon2012.arc.nasa.gov/abstracts/abstract-detail/can-a-living-system-self-construct-from-a-biotic-soup/
23. Bada JL and Laz­cano A (2003) Pre­bi­otic soup: re­vis­it­ing the Miller ex­per­i­ment. Sci­ence 300:745–746
24. Sul­li­van III, W.T. and Baross, J.A., ed­i­tors (2007) Plan­ets and Life. Cam­bridge Uni­ver­sity Press, Cam­bridge, UK.
25. Spitzer J and Pool­man B (2009) The role of bio­macro­mol­e­c­u­lar crowd­ing, ionic strength and physic­o­chem­i­cal gra­di­ents in the com­plex­i­ties of life's emer­gence. Mi­cro­biol Mol Biol Revs 73:371–388

Jan Spitzer is R&D man­ager of Mal­lard Creek Poly­mers in Char­lotte, North Car­olina.