Current and Future Opportunities in Structural Dynamics of Macromolecules at LCLS

Mark S HunterSample Environment and Delivery DepartmentLinac Coherent Light Source

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Damage-free room temperature structures of Biomolecules

• Outrunning radiation damage allows room temperature measurements• Avoid site specific and global radiation damage

• LCLS allows crystals too small for conventional high-resolution structural analysis• Could save months to years in optimization

Small crystal size is one of the main bottlenecks to high-resolution GPCR structures

Initial hit (5x1x1 mm) (Suitable for SFX)

One year later (40x20x7 mm)Traditional Microcrystallography

Serial femtosecond crystallography:• Small crystal size• No harvesting• Room temperature

Optimization

Traditional microcrystallography: • Crystal harvesting• Freezing• Crystal alignment

Months-to-Years

Cherezov et al, 2009, J R Soc Interface 6, S587

Small crystal size is one of the main bottlenecks to high-resolution GPCR structures

Initial hit (5x1x1 mm) (Suitable for SFX)

One year later (40x20x7 mm)Traditional Microcrystallography

Serial femtosecond crystallography:• Small crystal size• No harvesting• Room temperature

Optimization

Traditional microcrystallography: • Crystal harvesting• Freezing• Crystal alignment

Months-to-Years

Cherezov et al, 2009, J R Soc Interface 6, S587

3

Damage-free room temperature structures of Biomolecules

• Outrunning radiation damage allows room temperature measurements• Avoid site specific and global radiation damage

• LCLS allows crystals too small for conventional high-resolution structural analysis• Could save months to years in optimization

Small crystal size is one of the main bottlenecks to high-resolution GPCR structures

Initial hit (5x1x1 mm) (Suitable for SFX)

One year later (40x20x7 mm)Traditional Microcrystallography

Serial femtosecond crystallography:• Small crystal size• No harvesting• Room temperature

Optimization

Traditional microcrystallography: • Crystal harvesting• Freezing• Crystal alignment

Months-to-Years

Cherezov et al, 2009, J R Soc Interface 6, S587

Liu et al. Science, 342, 1521 (2013)• Differences observed in cryogenic and room

temp structures of G-protein coupled receptors~40% of drugs target GPCRs

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Highly successful G protein coupled receptor structure determination program

Slide taken from Vadim Cherezov

t01 fs 1 ps 1 ns 1 µs 1 ms 1 s

10 fs 100 fst¥

Enzyme catalysis Protein synthesis

Photo-isomerization, charge separation, H+ / e- transferAmino acid

sidechain mot.

Domain motion Protein foldingEnzyme Transition

States Pro isomerization

Membrane Ion transport, signal cascades

Biochemical time scales

Chemical & Physical time

scaleselectron

transitions

Bond vibrations

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Time scales for select chemical and biochemical processes

• Unique properties of LCLS that will facilitate studying these dynamic processes:• Short, intense pulses à Diffraction before destruction à Room/Physiological Temperature Studies

• Short pulses à High Temporal Resolving Power à Follow time series of reactions (enzymes) or dynamics (general biomacromolecules)

LCLS temporal resolving power

Time scales taken from Allen M. Orville

LCLS provides the spatiotemporal resolving power to follow these processes.What can be done with this unique combination?

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Fluorescent protein design based on chromophore twisting in the excited state

Model of on-off switching: I*T(twisted intermediate) was not detected by previous Transient Absorption (TA) measurements

• Picosecond time-resolved crystallography on reversible photoswitching fluorescent protein rsEGFP

• Found bulky valine side chain (V151) interfered with twisted intermediate

• Twisted intermediate not detected via TA

• Doubled the quantum efficiency of this fluorescent protein via V151A mutation

Models showing the off (white), on (cyan), and twisted (pink) structures of the chromophore

Coquelle, N., Sliwa, M., Woodhouse, J. et al. Nature Chem 10, 31–37 (2018). https://doi.org/10.1038/nchem.2853

V151 V151

Photosystem II -- Following the catalytic cycle and oxidizing water

Structural dynamics of protein, cofactorsCrystallography

Chemical changes at the catalytic siteX-ray spectroscopy

1F

2F

3F

( )

Spin state

Taguchi et al. JACS 134, 1996 (2012)Kern et al. Nature (2018) 563, 421.

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Time scales for select chemical and biochemical processes

• MISC will allow for reaction of enzymes to be studied (theoretically) in µs to ms time scales• Caged compounds and photoswitchable ligands can allow laser-driven reactions or dynamics

• Shorter time points can be accessed• Utilize the great infrastructure at LCLS for laser-triggered experiments

LCLS temporal resolving power

Fast mixingCaged compounds

Photoswitchablecompounds

t01 fs 1 ps 1 ns 1 µs 1 ms 1 s

10 fs 100 fst¥

Enzyme catalysis Protein synthesis

Photo-isomerization, charge separation, H+ / e- transferAmino acid

sidechain mot.

Domain motion Protein foldingEnzyme Transition

States Pro isomerization

Membrane Ion transport, signal cascades

Biochemical time scales

Chemical & Physical time

scaleselectron

transitions

Bond vibrations

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Where do we go from here?

• THz and M/FIR can trigger dynamics throughout the timeline shown• Allows for measurements on samples grown in extremely viscous media

LCLS temporal resolving power

Mid and Far IR and THz

t01 fs 1 ps 1 ns 1 µs 1 ms 1 s

10 fs 100 fst¥

Enzyme catalysis Protein synthesis

Photo-isomerization, charge separation, H+ / e- transferAmino acid

sidechain mot.

Domain motion Protein foldingEnzyme Transition

States Pro isomerization

Membrane Ion transport, signal cascades

Biochemical time scales

Chemical & Physical time

scaleselectron

transitions

Bond vibrations

ARTICLESNATURE CHEMISTRY

yield only very limited structural information about the underly-ing atomic ensemble. In contrast, the application of temperature jumps to time-resolved X-ray scattering and diffraction has been very limited. Nearly two decades ago, Hori et! al.37 used tempera-ture-jump Laue crystallography to study the initial unfolding step of 3-isopropylmalate dehydrogenase. That study explored only a single pump–probe time delay, which allowed them to observe laser-induced structural changes but precluded kinetic analysis. Within the past two years, the laser temperature-jump method has been paired with X-ray solution scattering to explore the oligomer-ization of insulin under non-physiological conditions25,26 and hae-moglobin27. The results and analysis we present here expand the role of the temperature-jump method in structural biology, by showing that temperature-jump X-ray scattering experiments can be used as a general method to explore the functional, internal dynamics of proteins in solution. Additionally, we provide a detailed outline of a data reduction and analysis procedure suitable for tempera-ture-jump small-angle X-ray scattering (SAXS)/wide-angle X-ray scattering (WAXS) experiments.

The temperature-jump SAXS/WAXS experiments we describe here used human cyclophilin A (CypA)—a proline isomerase enzyme that functions as a protein-folding chaperone and as a modulator of intracellular signalling pathways. CypA has been the subject of many NMR experiments that have identified two primary dynamic features of interest (Fig. 1b). First, the active site-adjacent loops (covering approximately residues 60–80 and hereafter referred to as the ‘loops’ region) are mobile on the millisecond timescale38. This region is especially interesting because evolutionarily selected mutations within these loops perturb the dynamics of the loop39, alter the binding specificity of CypA for substrates such as immu-nodeficiency virus capsids40, and restrict the host range of these

viruses41,42. Second, a group of residues that extends from the active site into the core of the protein (hereafter referred to as the ‘core’ region) has also been shown to be mobile on a millisecond times-cale38. Subsequent work incorporating multi-temperature X-ray crystallography43, mutagenesis44 and further NMR experiments45 has established a relationship between catalysis and conformational dynamics of a group of side chains in this region. Motivated by the sensitivity of the conformational state of the active site–core net-work to temperature43, we performed infrared laser-driven temper-ature jumps on buffered aqueous solutions of CypA and measured subsequent, time-dependent changes in SAXS/WAXS. While our measurements provide only low-resolution structural information, we were able to measure the kinetics of protein conformational changes in CypA. We identified two relaxation processes, and by performing temperature-jump experiments at a range of different temperatures, were able to calculate thermodynamic properties of the transition states for the underlying conformational transitions. Specific mutants in the ‘loops’ or the ‘core’ regions of CypA show that the two processes are independent, each representing a distinct and uncoupled reaction coordinate on a complex conformational landscape. Collectively, our measurements and analysis show that a wealth of information about a protein’s conformational landscape can be obtained by pairing laser-induced temperature jump with time-resolved X-ray scattering.

ResultsA method for simultaneous measurement of structural and kinetic details of intrinsic protein dynamics. To measure protein structural dynamics, we utilized a pump–probe method that pairs an infrared laser-induced temperature jump with global measure-ment of protein structure via X-ray solution scattering (Fig. 1c).

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Repeat 1 Repeat 2

X-ray image number

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10 1.0

0.8

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IR

cb

"T (

°C)

Structural relaxation

Time delay (ns)

Heating Cooling

Conformationalchanges

t1

t4

t3

t2

IRpump

X-rayprobe

10 ns

500 nsSAXS/WAXS

Sampleflow

!t

Fig. 1 | Overview of temperature-jump SAXS/WAXS experiments. a, During a temperature-jump experiment, an infrared (IR) laser pulse, several nanoseconds in duration, vibrationally excites the water O–H stretch and rapidly heats an aqueous solution of protein molecules (red curve). Heating is fast, but kinetic barriers to protein motions cause a lag in the structural relaxation to a new thermal equilibrium of conformational states (grey curve). b, Ribbon model depicting a single CypA molecule, with the ‘core’ dynamic residues (linked to catalysis) coloured red and the ‘loop’ region adjacent to the active site (determines substrate specificity) coloured blue. Sites of key mutations (S99T) are identified by spheres at the corresponding C! positions (red: S99T; blue: D66N/R69H). c, Schematic of the temperature-jump SAXS/WAXS instrumentation, with key features highlighted. Liquid sample flows through the interaction region, where it interacts with mutually perpendicular IR pump and X-ray probe beams. Both pump and probe sources are pulsed, with a defined time delay between arrivals at the sample. SAXS/WAXS patterns are recorded on a single detector panel. d, Data collection sequence used for the temperature-jump experiments. For each pump–probe time delay, a pump–probe measurement (‘laser on’) was performed, followed by a measurement with no application of the pump laser (‘laser off’). On–off pairs with increasing pump–probe time delays were measured in succession until all of the desired delay times were acquired, then the sequence was repeated as many as 50 times to improve the signal-to-noise ratio of the data. Note that the first measurement within each repeat is a control measurement, wherein the probe pulse arrives at the sample before the pump pulse (negative time delay).

NATURE CHEMISTRY | VOL 11 | NOVEMBER 2019 | 1058–1066 | www.nature.com/naturechemistry 1059

Thompson, M.C. et al., Nat. Chem. 11, 1058–1066 (2019)

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General Information and Inquiries

https://biology-lcls.slac.stanford.edu

Contact Mark Hunter (mhunter2@slac.stanford.edu)