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