Downloaded by guest on September 30, 2021 www.pnas.org/cgi/doi/10.1073/pnas.1821284116 design Tapia-Rojo tweezers Rafael magnetic a force with under captured folding in states Ephemeral odn ehns fpoenLa eysottmsae.Deto Due timescales. short very we the at Finally, observing L directly protein instrument. by of to setup mechanism the by our folding dynamics of of approach potential folding dependency the this demonstrate talin force confirm the force-sensitive further We validate over extremely the instrument forces. of the our of description using calibrate range analytic to full wide us a a allows provide by which created we force, field (14), pulling magnetic head the of tape description a microsecond Karlqvist the a Accord- it. to on of ing control force impeccable an the maintaining while changing timescale, of capable spectrometer need the fields. motivating magnetic rapidly, fast-changing implement changes force to force applying magnets, the or quenches where of molec- force protocols early fast pair capturing upon impedes occurring the and events ms, of ular 100 to movement up is take mechanical can change which force the the by instruments, limited However, tweezers (10–13). magnetic days even standard to or access in hours gives several and of stability timescales, inherent long an cova- provides HaloTag with chemistry, combined lent which, exquisite force, magnetic an pulling the trap, and of conditions magnetic control force–clamp the (3–9). intrinsic of beads offer compliance tweezers extreme superparamagnetic the to to Due tethered technology forces biomolecules pulling this apply on to of force gradients tweezers use field Magnetic magnetic the uses spectroscopy. spectroscopy force explore to application we its that Hence, in 2). natural (1, time strong becomes in create rapidly it very to change evolved can that which fields heads, magnetic of features M state globule molten spectroscopy force dynamic a opening piconewton, dynamics protein a study to of force. protocols under fraction spectroscopy observed control force a of a been range below with never forces resolution interrogating has fast-changing of and under capability molten which molecules the ephemeral L, provides individual the instrument protein Our of in before. formation state the millisecond-long globule demonstrate out capture carrying We to by domain. instrument talin quenches our R3 and of the the sizes potential measuring the of step by properties unfolding/folding validated folding L is the protein theory cre- of This field dependence head. magnetic force tape the a of by approximation bead ated Karlqvist superparamagnetic a the on on acting based that force expression of magnetic analytical the bandwidth an predicts developing a by capability instrument with our unique force brate the the offers changing in which one of design, implement to tweezers heads magnetic tape of a use is conventional which this Here, frequencies, twist nowadays. we high demanded storage fluc- very high-density at into the for field signals essential magnetic electric the convert of tapes, to tuations VHS Their ability cards. old the credit on highlights as record bands design diverse magnetic and or as read disks, technologies hard-drive to modern 2018) in 13, used tapes December ubiquitously review magnetic for are (received on 2019 heads 8, March tape approved and Magnetic MD, Baltimore, University, Hopkins Johns Ha, Taekjip by Edited a eateto ilgclSine,Clmi nvriy e ok Y10027 NY York, New University, Columbia Sciences, Biological of Department nti ril,w rsn antctp edtezr,aforce a tweezers, head tape magnetic present we article, this In eae,rsligi epudrtnigo h physical the of understanding over deep a perfected in been resulting have decades, systems agnetic | a,1 antctp head tape magnetic dadC Eckels C. Edward , | rti mechanics protein a | n ui .Fern M. Julio and , rti folding protein 0kz ecali- We kHz. ∼10 | andez ´ 1073/pnas.1821284116/-/DCSupplemental ulse nieArl1 2019. 1, April online Published hsatcecnan uprigifrainoln at online information supporting contains article This 1 the under Published Submission.y Direct PNAS a is article J.M.F.This interest.y of and conflict paper. no y E.C.E., the declare wrote R.T.-R., authors J.M.F. The and research; R.T.-R. and designed data; analyzed J.M.F. R.T.-R. research; and performed R.T.-R. contributions: Author magnetic the between distance the on pulling the only case, not and our magnets In depends the beads. force of magnetic magnet strength the This and of specifications geometry (4–6). the the bead for the specific vertical and is the law magnet with the force pulling between the distance relates that curve calibration a convert to molecules it tethered use to we applied 1B). here signals, (Fig. tape, force a into sig- on signals electric record convert electric magnetic to designed a is into head nals band- tape implementation; a a our as with with Just force kHz Appendix). the (∼10 16), kilohertz change 15, several to of (4, ability width the designs Com- gap highlights tweezers it. setup around narrow electromagnetic our wrapped other coil a a with has to pared supplied core is current The electric mate- an magnetic (1). of permeability core (25 head.) large toroidal tape a with the of rial of consists implementation head (See magnetic the 1). A on (Fig. details tape readers technical in card the used credit those or to identical recorders 902836), tape Industries; magnetic (Brush of micropositioned head pair a by the magnets substituting coil-mounted but voice (6), tweezers magnetic conventional Step L Protein Using Tweezers Sizes. Head Tape Magnetic the of Calibration Results which L, protein of milliseconds. well-defined state few to a globule in molten quenches forms the force capture ultrafast we out forces, carry to ability the owo orsodnemyb drse.Eal [email protected] or [email protected] Email: addressed. be may correspondence [email protected] whom To a,1 n eidcfre,sc stoeeprecdi cellular in experienced those as realistic such noise systems. under mechanosensing mechanical forces, dynamics example, periodic instru- protein for and Our combine, of L. that study protein perturbations the of the opens states capturing ment by globule speed dynamics molten its folding and ephemeral protein the talin measuring sub- ultrasensitive of by of with stability can spectrometer signals the that force demonstrate force this field We changing resolution. magnetic time rapidly a a millisecond and uses create that large to spectrometer deliver head force changing tape a slowly introduce commercial and we forces Here constant ramps. to limited is instru- spectroscopy mentation force rapidly Current timescale. change millisecond and the complex in are biology in signals Mechanical Significance nmgei wees h ple oc sdtrie yusing by determined is force applied the tweezers, magnetic In ) vrwihasrn antcfil sgnrtdwhen generated is field magnetic strong a which over µm), antctp edtezr olwtesm ceeas scheme same the follow tweezers head tape Magnetic NSlicense.y PNAS PNAS | pi 6 2019 16, April . y | o.116 vol. www.pnas.org/lookup/suppl/doi:10. | o 16 no. IAppendix SI | 7873–7878 for SI

APPLIED PHYSICAL AND SCIENCES COMPUTATIONAL BIOLOGY  g/2  1 1 I(mA) F (z, I ) = AI 2 arctan + BI , AB z  2  2 1 + z 1 + z Magnetic g/2 g/2 gap Head [1] F where A and B are constants built with the physical param- eters of our problem (SI Appendix). Our calibration strategy (μm) z hinges on determining A and B by relating the extension ∆L of the folding/unfolding steps of protein L—used as a Length (nm) Length molecular ruler—with F through the FJC model, ∆L(z, I ) = h   i F(z,I )lK kT ∆Lc coth − , where F (z, I ) takes the shape C Signal Micro-positioner kT F(z,I )lK Current of Eq. 1, ∆Lc is the contour length increment, lK is the Kuhn i) Magnetic length [∆Lc = 16.3 nm and lK = 1.1 nm for protein L (10)], and Fluid Head kT = 4.11 pNnm. This relation could have been equally derived ii) Chamber using a worm-like chain description of the step sizes. However, the FJC model has an explicit ∆L(F ) dependence that makes it iii) PIEZO analytically more convenient. Camera We carry out experiments on protein L octamers using the M- iv) µ Light 270 2.8- m beads as described previously (ref. 6 and Materials and Methods), working at different values of z and I and mea- Fig. 1. Magnetic tape head tweezers setup. (A) Photograph of the mag- suring the average step size ∆L(z, I ). Fig. 2A shows a typical netic tape head tweezers setup, highlighting the magnetic tape head. (B magnetic tape head tweezers trajectory for a protein L octamer. and C) Schematics of the magnetic tape head tweezers experiment. Elec- First, working at z = 275 µm, we unfold the octamer with a pulse tric current signals of any shape are converted to force signals, which are of 800 mA, and the unfolding of each domain appears as a step- directly applied on tethered . wise increase in the extension of 14.3 nm. Next, we decrease the current to 225 mA, and the polyprotein collapses, showing unfolding/refolding dynamics with steps of 7.9 nm. Finally, we head and the bead, but also on the electric current. Following unfold the remaining domains at 665 mA and move the head the strategy previously developed (6), we generate a current law away from the bead to z = 550 µm, which results in a decrease of by relating the pulling force (F ) with the distance from the head the pulling force, as the protein folds to a state where all eight to the bead (z) and the electric current (I ) through the unfold- domains are folded on average, showing steps of 5.6 nm. For ing/folding step sizes of protein L, which scale with the force each (z, I ) condition, we determine the step sizes as the distance following standard polymer physics models such as the freely between two consecutive length histograms, calculated over the jointed chain (FJC) model (17). However, to address this prob- smoothed trajectory (black trace). lem, we need a description of the dependence of the pulling force with z and I . In conventional magnetic tweezers, an empirical exponential dependence between force and distance is gener- ally assumed (6, 18). However, here we can rely on an analytical A relation based on the Karlqvist description of the magnetic field 150 14.3 nm 7.9 nm 5.6 nm created by a tape head (SI Appendix) (1, 14). 100 Under an external magnetic field B~ , the force act- ing on a superparamagnetic bead can be described as 50     V χb 0 F~ = B~ · ∇ B~ + ρV ∇ M~ 0 · B~ , where ρ is the density of Length (nm) µ0 ) 3 3 600 z=275 μm the bead (kg/m ), V its volume (m ), B~ the applied magnetic μm 300 z=550 μm

z ( 0 field (teslas), χb the magnetic susceptibility of the bead (dimen- 800 mA 665 mA −7 800 sionless), and µ0 = 4π × 10 Tm/A (19–21). This approxima- 400 225 mA tion of the bead’s magnetization is valid when working at weak 0

I (mA) 0 100 200 300 400 500 fields (B < 19 mT). See SI Appendix for further discussion on Time (s) this approximation. Despite using superparamagnetic beads, we BC include an initial magnetization term M~ 0, which has been demon- 14 1000 12 50 pN strated to be necessary when working with commercial beads 800 40 pN 10 30 pN and at weak fields (see ref. 19 and SI Appendix for a discussion 8 600 20 pN on this approximation). The magnetic force on a superparamag- 6 z=250 m 15 pN netic bead is often described in the literature only with the first 4 z=275 m 400 10 pN 2 z=300 m M~ z=325 m Current (mA) 5 pN term. However, in actual superparamagnetic beads 0 can be Step Size (nm) 0 200 comparable to the magnetization induced by weak fields (19) (SI 0 200 400 600 250 275 300 325 350 375 400 Appendix). Current (mA) z (μm) The lateral Fx and vertical Fz components of the magnetic force acting on the bead are obtained by using the Karlqvist Fig. 2. Calibration of the magnetic tape head tweezers. (A) Dynamics of description of the magnetic field on the force acting on the bead protein L at different values of the current and vertical distance z. Data are acquired at 1.4 kHz (gray trace), while the black curve is a filtered trace with SI Appendix (see for explicit derivation). In practice, we work a Savitzky–Golay algorithm (box size, 101). (B) Global fit of the step sizes to over the gap region (−g/2 ≤ x ≤ g/2) where the magnetic field the FJC model to determine the current calibration law. Data are collected is constant and the lateral component vanishes, Fx = 0. Thus, over >40 molecules and >300 events at each distance z.(C) Contour plot of the vertical component evaluated at x ≈ 0 can be written as the pulling force applied by the magnetic head to the superparamagnetic (dropping the z subscript) beads as a function of the vertical distance z and supplied current I.

7874 | www.pnas.org/cgi/doi/10.1073/pnas.1821284116 Tapia-Rojo et al. Downloaded by guest on September 30, 2021 Downloaded by guest on September 30, 2021 ai-ooe al. et Tapia-Rojo tatmsaeo ilscns oti i,w einthe design pulse fingerprint we the aim, After protein this 4A. of To Fig. mechanism milliseconds. in folding depicted of the protocol timescale force explore a the to at sample it resolution. L unprecedented directly use an to we with protein thus Here, folding and a of forces pathway well-defined to Pro- of pulses Pathway Folding the L. in tein States Ephemeral of Detection Direct superparamagnetic a and on head acting tape (19). force bead the the by for created approximation field the magnetic the Karlqvist the for of adequacy description the of range demonstration a direct over a providing data experimental the follows accurately 3C Fig. in differ- line at talin) Eq. on inverting probability folding of values (50% ent pN 8.7 generate to setup S10B). tweezers considerable Fig. magnetic Appendix, in inde- standard (SI been our result has with force dependency measured This the pendently probability. of folding the value how of illustrating the shifts series, in are time states changes the both of subtle pN histograms 8.7 length at from while lated folded 3 pN, 9 the Fig. at occupying populated. 20% equally of to pN) probability 8.2 80% (at pN, state an 0.8 from just transitions Over sensitivity. it force aforementioned its highlights (see forces sensi- different 3A and extreme three Fig. its (23). at given force monomer we domain, the so, talin to do mutant predict To tivity IVVI to bead. R3 superparamagnetic used the a we use confirming on framework by force theoretical force magnetic the the pulling of the adequacy of the description Sensitivity. our Fine-Force validate Talin Using We Law Calibration the of Verification with extended chambers. be thinner can range produce This to pN. designs 50 alternative Appendix), of of (SI distance force maximum design minimum a a chamber ates at fluid work our we can range With the we on. on work depending force, to the wish of control the in versatility r cl rmylo odr e stefreicess h force The increases. force the as on red depends dark to yellow from scale ors gradient. a magnetic within same forces the pulling given reproducible error, size; 5% highly in generate dispersion to of us 1.4% M-270 allows (just the small in demon- very variation we bead-to-bead is As the 22). (11), (10, previously measured strated S9), previously Fig. those Appendix , match (SI which L the protein reproducing of by properties calibration folding/unfolding our error of any goodness the as demonstrate precision, micrometer establishing with in head magnetic the with ing force of pulling scale length the a of over dependence the to that law reveals current our where establish we Thus, sizes. be step of tribution 1.564 ue tfu ifrn itne n lte safnto of function yielding algorithm, a as plotted B and distances different head I four magnetic the at since sured mA, 1,000 at and saturates mA 200 between between distances rents at L protein xeietldt r te lblyt h J with FJC the to globally fitted are data Experimental . epo h au fteeeti urn necessary current electric the of value the plot we 3C, Fig. In i.2C Fig. olwn hssrtg,w esr odn/nodn tp of steps folding/unfolding measure we strategy, this Following steol reprmtr sn h Levenberg–Marquardt the using parameters free only the as F i.S10A Fig. Appendix, SI (z Nm.Tefi a egtdwt h Do h dis- the of SD the with weighted was fit The pN/mA. u ntuetalw st ar u eybifquench brief very out carry to us allows instrument Our F , I 0 = ) hw otu lto h oc vs. force the of plot contour a shows sgvni ioetn and piconewtons in given is z I z 1 and ,200 1, = .386I ecndsrb hsdpnec nltclyby analytically dependence this describe can We . n obtaining and z Eq. Appendix, (SI ilrsl napo eemnto of determination poor a in result will I A 2 nannierwy rvdn convenient a providing way, nonlinear a in g 0 = arctan 25 = A i.2B Fig. mA. .386 B hw h odn rbblt calcu- probability folding the shows ,ilsrtn h edo position- of need the illustrating µm, o h oeua osrc) which construct), molecular the for I ± 12.5 (z z 3 pN 0.131 hw rjcoyo hstalin this of trajectory a shows 250 hc epo sasldblack solid a as plot we which ),  .Oraayi expression analytic Our S23). 1+ hw h tpszsmea- sizes step the shows and µm ( 12.5 1 ∼ z z /mA aeil n Methods and Materials 250 nmcoees This micrometers. in ) 2 9 + z .This Appendix). SI 2 ,wihgener- which µm, 325 and and .462I n cur- and µm I B hr col- where , >250 9 = z 1+ changes ( F .462 A 12.5 1 We . z µm, and ) 2 ± , t20 bakdse ie.W esr eaaeytestep 4D, (Fig. the stable separately and measure time 4D) We (Fig. threshold weak line). a the dashed setting of (black sizes events, s of 2.01 kinds at two on between unfold atically that solid states ephemeral (red τ of exponential two set at double of first average a presence a evi- identify the an to we revealing line), exhibits Fitting 4C), events kinetics. (Fig. unfolding different shape of bimodal distribution dent Our peak. a h vrg nodn time unfolding average expo- the an identi- since systematic written as kinetics—is timescales, a first-order of characteristic for plot distribution—signature nential different allows and of representation events fication This unfolding histogram, 4C). (square-root all binning Fig. of logarithmic with kinetics histogram the unfolding the several over sure unfold state arrowheads). native black 4A, the (Fig. seconds acquired when which However, domains pN. 4 (Fig. 10 timescale at second level same 250 the exten- the the in reaches and conformation, W than sion stable 60 any less head, acquire not in the does to protein and When current precision supply implementation. great we current which with at done power is the pN 1 100 to pN electric at the 10 sampling head by the obtained across force, current pulling the of surement and field the changes head long. magnetic milliseconds fold, the not protocol because This does in (iv), only kinetics. quench L again unfolding possible the pN possible protein during is slowest 10 formed which the structure to with at any force time observe force the to lowest increase us allowing we the exten- times Next, is folding reference (iii). varying which a a s during to set 10 pN subsequently to to and 1 pN (ii) of 10 protein force unfolded to the force of the sion decrease we (i), of values prediction at analytic different the sampled at with tape pN pN), together (C magnetic 8.7 9 molecules. the apply four and with to with necessary measured measured pN, talin are 8.7 Data of tweezers. pN, probability head Folding (8.2 (B) forces kHz. different 1.3 three at jectory 3. Fig. BC A N Force (pN) Length (nm) ocaatrz hsfligmcaimi eal emea- we detail, in mechanism folding this characterize To i.4B Fig. 10 10 20 30 40 50 g 47.07 = ∼100 Folding Probability 8 9 (t s eosresm peea ttsta nodi sub- a in unfold that states ephemeral some observe we ms, 0.0 0.2 0.4 0.6 0.8 1.0 .Ti iecntn eed on depends constant time This S7). Fig. Appendix, (SI µs 020 40 ∆t x [ exp = ) yia ai oanR tra- R3 domain talin Typical (A) magnetometer. molecular a as Talin 250 = 6 s ow a ar u otoldple nyfew only pulses controlled out carry can we so ms, ihihsteqec us for pulse quench the highlights ± τ MG 8.2 pN 7 14.511 Force(pN) x s(e) i.4B, Fig. (red). ms − 0 = 8 x 0 PNAS .49 9 − .Ti losu odsrmnt system- discriminate to us allows This s. exp(x ± 10 A | 0. 100 and pi 6 2019 16, April 01 11 Time (s) x − 0 8.7 pN n oesal n where one stable more a and s e rohas,wiethose while arrowheads), red B, I H.Tesews hnefrom change stepwise The kHz. x lnτ = (z ∆ 0 ). )] t Lower 2)(being (24) 10 = Current (mA) 200 400 600 800 ∆ 0 sesl eonzbeas recognizable easily is | 0 t agn rm00 s 0.01 from ranging , z o.116 vol. s(lc rc) the trace), (black ms mnfse as (manifested hw ietmea- direct a shows ∆t 100 Talin Pf=0.5 Prediction 10 = 200 x domains. Inset) 9 pN z() z( | lcrccurrent Electric ) lnt = 300 μm o 16 no. s(black) ms ,where ), 400 P f | = ∆ 500 7875 0.5), t

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0

APPLIED PHYSICAL BIOPHYSICS AND SCIENCES COMPUTATIONAL BIOLOGY A B

C D E

Fig. 4. Molten globule state formation at a millisecond timescale. (A) Pulse protocol for characterizing the folding mechanism of protein L, sampled at 1.3 kHz. (B) Details of the quench pulse, highlighting the formation of molten globule states over a few milliseconds. (C) Square-root histogram of the unfolding kinetics of all observed unfolding events at 10 pN, demonstrating the existence of two different populations. (D) Histogram of the unfolding step sizes of the weak domains (unfolding within less than 2.05 s) and the native states (Inset). Histogram was built from the aggregated data at every quench duration. (E) Probability of the molten globule (red) and native states (black) as a function of the quench time ∆t, globally fitted to a first-order kinetics model with one intermediate. Data were collected with 14 molecules and a total of 546 events.

While the weak domains exhibit a broad step size distribu- ∆t = 250 ms and compare the formation of the molten globule tion, the stable ones show a Gaussian distribution centered at at forces of 0.5 pN, 1 pN, 2 pN, and 4 pN. All of these forces pro- 10.5 nm. However, nearly 60% of the weak events fit onto an duce observable molten globule-like states, which unfold within analogous step size distribution about 10.5 nm (solid red line), the first 2 s of the 10-pN probe pulse. However, the distribu- which suggests that they correspond to an immature conforma- tion of their step sizes is surprisingly dependent on the quench tion that eventually evolves to the stable native state (molten force. Fig. 5 shows histograms of step sizes at 0.5 pN, 1 pN, 2 pN, globule-like). and 4 pN, normalized to facilitate their comparison. At forces Our results suggest that protein L folds through a sequential equal to or below 2 pN, we find a broad distribution, with a large reaction from the unfolded state (U) to a molten globule-like fraction of steps that exceed a single native protein L. However, state (MG) that matures eventually to the native conformation when we quench to a higher force of 4 pN, the distribution gets (N). Fig. 4E shows the probability of appearance of the molten significantly narrower, and we observe very few large steps. Fit- globule and native states as a function of ∆t. The molten glob- ting Gaussian distributions around 10.5 nm (red solid lines), we ule is manifested optimally in a timescale of ∼100 ms, decaying obtain respectively that at 0.5 pN, 1 pN, and 2 pN, 45%, 55%, and quickly when we quench during seconds. Oppositely, the native 48% of the steps are short, while at 4 pN this fraction increases domains show a sigmoid-like behavior, which saturates after a to 75%. This observation suggests that mechanical forces may few seconds. This dependence can be described analytically by assist folding by facilitating the formation of molten globule-like a kinetic model where the molten globule is an intermediate of states that lead to the native state, in contrast to the longer col- the native conformation (see SI Appendix for derivation). Solid lapsed structures that hamper the folding reaction. Indeed, our lines show the global fits of the molten globule/native probability data indicate that the probability of reaching the native state as a function of ∆t which yield the microscopic rates of forma- after a brief 250-ms-long quench increases with the force; while −1 tion of the molten globule (rMG = 10.97 ± 1.42 s ) and the at 0.5 pN, 1 pN, and 2 pN only 25% of the domains reached the −1 native state (rN = 1.28 ± 0.24 s ). The fraction of native states native conformation, a higher force of 4 pN allowed 50% of the does not saturate to 1 even when ∆t = 10 s, but rather to ∼0.8, domains to fold (SI Appendix, Table S3). and similarly the fraction of unfolded states plateaus at ∼0.2 (SI Appendix). This is due to the residual formation of stable nonna- Discussion tive conformations with large step sizes (Fig. 4D, Inset) that can It is now well understood that upon quenching the force be related to domain swap-like structures (25). on a mechanically unfolded/extended protein, the polypeptide Unlike previous observations of molten globule-like states in rapidly collapses, greatly increasing its entropy. For ubiquitin protein folding under force (26, 27), our instrumental design polyproteins studied with force–clamp atomic force microscopy maintains a fine control of the pulling force and allows measur- (AFM), this collapse was fast, taking place in the submillisec- ing its evolution at different folding forces. With the protocol ond timescale (26, 28, 29). The collapsed polypeptide was then described in Fig. 4A, we keep the quench time constant at observed to progress over a timescale of ∼1 s to its native

7876 | www.pnas.org/cgi/doi/10.1073/pnas.1821284116 Tapia-Rojo et al. Downloaded by guest on September 30, 2021 Downloaded by guest on September 30, 2021 ai-ooe al. et Tapia-Rojo narrowing the a as conformation, showed native pN the 10 of step at that unfolding states approaching globule of distribution molten Histograms the sig- force. of varied step quench sizes states of the globule distribution molten with note- weak the nificantly mechanically One that the timescale. observation of shorter earlier the sizes much observed was a result those on worthy to but mechanically (26), similar and ubiquitin 4), for short-lived (Fig. very intermediates through weak folds L discover protein we been Here, that has 13). (10, show- L intermediates without Protein folds mechanical not that obvious (33). protein tech- ing is weak state mechanically that spectroscopy a globule folder be molten to force two-state shown a classical our have a to applied L, known protein have study We to nique kHz. better bandwidth 10 a with pulling combined than the range of piconewton control the in exquisite force an demonstrating by globule issues these molten imple- of be evolution studied. not be time/force these not could the could in states changes thus piconewtons, evident and force few were mented, a method rapid lim- of trapping where the force optical Again, experiments state. a the stable at of mechanically itations that, its to H globule- slowly RNase molten matured the in on-pathway observed state labile also mechanically like (27) noting a al. beyond of et states, existence Cecconi intermediate Similarly, an weak existence. prevented the their limitations of These in study precision 28). with accurate (26, ascertained range be piconewton not was could the apparatus force force–clamp studies. quench the those the and in of evident response were time AFM The force–clamp lim- were the instrumental The of states states. itations weak globule-like states molten mechanically folding be these to transient proposed (30–32), those bulk to in Akin observed sizes. wide step a showed of and labile distribution mechanically mechani- were of that ensemble states from weak an cally through progression proceeded This native to (26). collapsed conformation stable mechanically molecules. eight of 113 total pN), a (1 in and 62 events pN), pN) (0.5 (4 56 71 and with pN), 75% measured (1 to 61 were ephemeral increases pN), distributions the fraction (2 of The this 48% pN. while and 4 nm, force 55%, 10.5 at 45%, the pN, around as 2 distributed decreases and are pN, steps states 1 large pN, distribution finding 0.5 the At force of globule-like increases. quench probability molten the the the increase to we and fit As narrows, nm. Gaussian 10.5 a ( B), at is pN peaked line states, 1.0 Red 4D. (A), Fig. pN from 0.5 quenches of (C forces pN quenching 2.0 at measured states ephemeral 5. Fig. C A

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(34, are spectral noise in physiology different change in with that remains signals forces forces it to to or However, respond time proteins magnitude. fast different how respond of unexplored proteins forces how investigate pulling to to used spectroscopy traditionally Force ramps. been or has pulses more square protocols, classic force than additional complex of possibility the that open head spectrometers tape tweezers magnetic force Furthermore, by mechanically. force introduced pro- otherwise the time be change in would dead and state the milliseconds in globule few a hidden molten in the forms which observe L, tein directly has to microseconds few us The a in forces. allowed force of the range changing ana- large of a described capability over unique be and can accuracy and head great accuracy the with lytically by high generated maintaining a force with while The force stability. kHz, the 10 change above to us bandwidth enables which spectrometer, force states ephemeral weak such now of folding. here studies protein detailed in demonstrate for we path that a instrument assemble opens The to polypeptide state. the difficult native of lowest more positioning the it the correct makes the at that dis- out entropy form sort high Highly to that a state. such those the have native as of may the forces, such formation to states, the lead collapsed for that ordered force states optimal results globule an These molten is conformation. there native prob- higher that the a narrower suggest to and much maturation 4) a of (Fig. in states ability results globule molten forces of higher distribution to strik- quenching is It that a states. drives ing disordered 5A) sug- Fig. more results pN; toward Our 0.5 polypeptide 5). (e.g., collapsing forces (Fig. lower pN to 4 quenching to that gest up increased was force quench ntemnsrp.Ti okwsspotdb I rnsGM116122 Grants F30-HL129662. NIH Grant financial by NIH comments for by supported Areces supported and was Ramon was E.C.E. discussions Fundacion mem- support. acknowledges work valuable the R.T-R. of the This HL061228. all and for manuscript. thank laboratory We the J.M.F. us. on with the plasmid of talin bers the sharing for Liverpool ACKNOWLEDGMENTS. in timescale. globule described fast are Molten their analysis to experimental trace. due on trace, filtered details raw Further the the using of analyzed algorithm were histograms experiments were Savitzky–Golay sizes length Step a analysis. using and using visualization measured facilitate filtered to box, subsequently 101-points a and with kHz protein a 1.7 contain- using EDTA) and damage, nM oxidative 1 avoid NaCl, to of 7.3) mM concentration (pH 150 acid 7.2, ascorbic pH mM Hepes, 10 ing mM (10 buffer Hepes in Poea n -emnlAia o itnlto.Frdtie expres- detailed For see biotinylation. methods purification for enzyme AviTag and HaloTag sion C-terminal N-terminal an a by and flanked (Promega) were constructs polyprotein Appendix All (SI chambers fluid made Measurements. Single-Molecule .Frfrhrdtissee details further For fernandezlab.biology.columbia.edu/). nsmay ehv mlmne antctp edi a in head tape magnetic a implemented have we summary, In 0n.Dt eeaqie tart ewe . kHz 1.2 between rate a at acquired were Data nM. ∼50 PNAS etakD.Io askvfo nvriyof University from Barsukov Igor Dr. thank We | l xeiet eecridoti custom- in out carried were experiments All pi 6 2019 16, April ,fntoaie sdsrbdbfr (6). before described as functionalized ), IAppendix SI l xeiet eecridotin out carried were experiments All l xeiet eedone were experiments All . | o.116 vol. IAppendix SI | o 16 no. IAppendix SI | . 7877 .

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