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Biomolecules: three techniques at the cutting edge of research

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

On April 20th, 2022 |
3 mins reading time
3
How can we monitor dynamics of biomolecules in real-time?
Pascale Changenet
Pascale Changenet
CNRS Research Director in biophysics at Ecole Polytechnique (IP Paris)
François Hache
François Hache
CNRS Research director and Professor of Physics at Ecole Polytechnique (IP Paris)
Key takeaways
  • Changes in the helix structure of proteins and DNA can happen in a few tens of femtoseconds (10 x 10-15 seconds). With such a short time window, observing such processes remains an experimental challenge.
  • Chirality is an important component of life. A molecule is chiral when it is not superimposable with its mirror image, meaning that the same molecule can have two possible shapes.
  • The technique known as circular dichroism plays on these 'chiral' shapes, which absorb polarised light in different ways. This can be used to create a unique signal for each possible conformation – like a barcode – to trace changes in its conformations over time.
  • With advances in AI, it is becoming possible to reliably predict the 3D structure of proteins from their sequences, notably with the new Alpha Fold 2 algorithm.
  • Understanding the effects of the interaction of certain molecules targeting DNA on its structure is an avenue to be explored for new research in pharmacology or medicine.

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.

What is 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.

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What are the advantages and disadvantages of time-resolved circular dichroism compared to other techniques?

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.

How have you responded to the rapid advances in artificial intelligence for predicting the conformation of biomolecules?

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.

What are the potential clinical applications that could result from your research?

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