How can we monitor dynamics of biomolecules in real-time?

It is a prob­lem that has plagued biol­o­gists for over fifty years: how is the helix struc­ture of pro­teins and DNA formed? Whilst var­i­ous exper­i­men­tal tech­niques do exist to deter­mine the pre­cise three-dimen­sion­al struc­ture of bio­mol­e­cules, mea­sur­ing their dynam­ics, which hap­pen in the range of sev­er­al fem­tosec­onds (10 x 10–15 sec­onds) to sec­onds, is still an exper­i­men­tal chal­lenge. This area of research is being explored by a team of French researchers, led by Pas­cale Changenet and François Hache at the Optics and Bio­sciences Lab­o­ra­to­ry (LOB) at École Poly­tech­nique, using time-resolved cir­cu­lar dichroism.

Pas­cale Changenet. It is a tech­nique based on chi­ral com­pounds, which absorb polarised light dif­fer­ent­ly depend­ing on whether it is right or left-hand­ed cir­cu­lar. A com­pound is chi­ral when it is not super­im­pos­able on its image in a mir­ror. Such objects can there­fore exist in two forms, called enan­tiomers in the case of mol­e­cules. “Chi­ral­i­ty” is an impor­tant com­po­nent of liv­ing organ­isms that can be found every­where at dif­fer­ent scales. Our hands are exam­ples of chi­ral objects. Pro­teins and DNA are chi­ral mol­e­c­u­lar assem­blies made up of ele­men­tary build­ing blocks (amino acids and nucleotides) which are them­selves most­ly chi­ral. Using cir­cu­lar dichro­ism, it is pos­si­ble to char­ac­terise their spa­tial heli­cal arrange­ment, espe­cial­ly when they are in equi­lib­ri­um, in solution.

How­ev­er, cir­cu­lar dichro­ism is still under­de­vel­oped for dynam­ic (or non-equi­lib­ri­um) mea­sure­ments, par­tic­u­lar­ly at ultra-short times. This requires the use of lasers deliv­er­ing fem­tosec­ond puls­es and the so-called “pump-probe” tech­nique. A first intense laser pulse, the pump, is used to ‘per­turb’ the bio­mol­e­cules, while a sec­ond, less intense pulse, the probe, is used to observe the response of the sys­tem to the light per­tur­ba­tion. It is a bit like the flash that pre­cedes the tak­ing of pho­tographs a few moments lat­er. By con­trol­ling the delay between the arrival of the pump and probe puls­es in the sam­ples, it is pos­si­ble to mea­sure the sequence of events trig­gered by the pump with a tem­po­ral res­o­lu­tion lim­it­ed by the dura­tion of the pump and probe puls­es. In addi­tion, by pre­cise­ly con­trol­ling the cir­cu­lar polar­i­sa­tion of the probe at each pump-probe delay, we can trace the film of the con­for­ma­tion­al change of bio­mol­e­cules over time.

The main advan­tage of cir­cu­lar dichro­ism is that it can access the con­for­ma­tion­al dynam­ics of bio­mol­e­cules down to extreme­ly short time scales, which is, for exam­ple, not pos­si­ble with oth­er com­pa­ra­ble tech­niques such as nuclear mag­net­ic res­o­nance (NMR). Although it is pos­si­ble today to make time-resolved X‑ray dif­frac­tion mea­sure­ments with very short time res­o­lu­tion, this requires the use of large instru­ments such as syn­chro­trons or free elec­tron lasers (XFEL) which are very expen­sive and have lim­it­ed access.

While cir­cu­lar dichro­ism gives less pre­cise infor­ma­tion on the struc­ture of bio­mol­e­cules than X‑ray dif­frac­tion, our exper­i­ments use com­mer­cial laser sources and can be per­formed direct­ly in our lab­o­ra­to­ry. The prin­ci­ple of these mea­sure­ments is extreme­ly sim­ple. How­ev­er, they require the abil­i­ty to detect extreme­ly small vari­a­tions in light sig­nals of the order of 1/10,000, which is prob­a­bly why very few peo­ple use it at the moment. With con­stant progress in the devel­op­ment of laser sources and optics, these exper­i­ments are set to become increas­ing­ly accessible.

François Hache. Our team is a pio­neer in the devel­op­ment of cir­cu­lar dichro­ism mea­sure­ments at very short times. The advan­tage of these mea­sure­ments is that they do not require spe­cif­ic mod­i­fi­ca­tions of the sam­ples stud­ied and require very small quan­ti­ties of pro­teins or DNA, com­pared to X‑ray dif­frac­tion mea­sure­ments which require the crys­talli­sa­tion of sam­ples and are destructive.

PC. It is becom­ing pos­si­ble to reli­ably pre­dict the three-dimen­sion­al struc­ture of pro­teins from their sequences, espe­cial­ly with the recent devel­op­ment of the Alpha Fold 2 algo­rithm. How­ev­er, the func­tion of bio­mol­e­cules is not only linked to their three-dimen­sion­al struc­ture, but also to their dynam­ic changes. Mea­sur­ing and mod­el­ling these dynam­ics – span­ning time scales of sev­er­al orders of mag­ni­tude – is still a major chal­lenge in mod­ern biophysics.

FH. Our stud­ies focus on bio­mol­e­cules in solu­tion in vit­ro. It is dif­fi­cult to envis­age stud­ies of this type in cells, tis­sues or even in vivo. Our work is pri­mar­i­ly aimed at under­stand­ing how helices are formed in pro­teins or DNA and iden­ti­fy­ing the deter­min­ing envi­ron­men­tal fac­tors, such as tem­per­a­ture, pH or the nature of the ions. Under­stand­ing the effects of the inter­ac­tion of cer­tain mol­e­cules specif­i­cal­ly tar­get­ing DNA on its struc­ture is an avenue that we are also explor­ing to pro­vide input for new research avenues in phar­ma­col­o­gy or medicine.

Julien Hernandez