Single-molecule analytical characterization: a window into

biomolecular heterogeneity

Jose Casas-Finet, Ph.D., D.Sc.Analytical Biochemistry Dept.CMC Forum – WCBP 2010 – Jan. 24, 2010

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Outline

Rationale for Structural Analysis in Protein TherapeuticsMolecular Electron Tomography: a new structural toolCase Examples: Electron Tomography of anti-RSV MAbDevelopment of Quantitative Electron TomographyLow-voltage EM: the leading edgeConclusions

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Structural Analysis in Protein Therapeutics

Protein function depends on both its three-dimensional shape and dynamical properties

Substantial effort is devoted by academic and commercial laboratories to determine protein structure at (or near) atomic resolution

Knowledge of structure-function relationship can be applied to molecular engineering or securing IP

Recent technological developments permit to image proteins and interrogate their function with single molecule sensitivity in the industrial analytical laboratory setting

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Trends in Structural Analysis

XX--ray Crystallographyray CrystallographyNMRNMRCryoCryo--Electron MicroscopyElectron MicroscopyCircular Circular DichroismDichroismMass SpectroscopyMass SpectroscopyDynamic Light ScatteringDynamic Light Scattering

COMPLEXITY RESOLUTION

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New Trends in Structural Analysis

COMPLEXITY RESOLUTION

Free Electron Laser ScatteringFree Electron Laser ScatteringMolecular Molecular Electron TomographyElectron TomographySingle Molecule Fluorescence Single Molecule Fluorescence SpectroscopySpectroscopyIn situIn situ Electron TomographyElectron TomographySingle Cell SensingSingle Cell Sensing

NON-AVERAGED PROPERTIES

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Electron Tomography in Structural Biology

Advantages of Electron Tomography:Requires small amount of material

Provides molecular shapes (down to 20 Å) without crystallization

Visualizes individual molecules (including membrane proteins)

Types of Electron Tomography:In situ: studies of proteins on intact cells, decorated with a primary antibody to which a secondary, gold-label antibody attaches

In vitro: studies on purified protein in physiological or formulation buffer

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Electron Tomography: sample preparation

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Electron Tomography: measurement

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Electron Tomography: data collection

Tilt series+/- 60 degr.

Sample volume

COMET is a maximum-entropy image refinement algorithm developed by Dr. Ulf Skoglund, a founding member of Sidec (Stockholm, Sweden)

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Electron Tomography: data analysis

Protein tomography directly visualizes the polymorphism of a biological structure; there is no image averaging (unlike in cryo-EM)

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Electron Tomography of Antibodies

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The Electron Tomography Advantage

““It is believed by many people that eventually, by one It is believed by many people that eventually, by one strategy or another, one may extend the singlestrategy or another, one may extend the single--particle electron microscopy approach to comparable particle electron microscopy approach to comparable resolutions [to xresolutions [to x--ray crystallography]ray crystallography]””

Roger KornbergRoger KornbergProfessor of Structural Biology, Stanford UniversityProfessor of Structural Biology, Stanford University2006 Chemistry Nobel Laureate2006 Chemistry Nobel Laureate

in in ““The bigger picture: Dramatic progress in imaging The bigger picture: Dramatic progress in imaging and microscopy enables scientists to see even greater and microscopy enables scientists to see even greater detaildetail”” ((EMBO Rep.EMBO Rep. 66 (12), December 2005: 1113(12), December 2005: 1113––16)16)

Protein tomography directly visualizes the polymorphism of a biological structure; there is no image averaging of large numbers of conformers.

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Electron Tomography of Anti-RSV MAbAnti-RSV antibodies (far left; Fab: blue, Fc: gray) show high flexibility and correlated domain motions (left)Rigid-body docking (below) fits a full-length IgG1 structure to the anti-RSV MAb tomogram (lower left)

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Anti-RSV MAb Bound to RSV F-protein F-protein shows the computer-predicted ‘lollipop’ shapeAnti-RSV MAb binds to the ‘stalk’ region of F-proteinAnti-RSV MAb dimer retains F-protein binding ability

Anti-RSV MAb Anti-RSV MAb/F-protein complex

F-protein

Fab Fab

Fc

Fab Fab

Fc stalkhead stalk head

10 nm

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Electron Tomography of Anti-RSV MAb Dimer

Electron tomography is one of the few techniques capable of imaging antibody oligomersAnti-RSV MAbdimers form through Fab-Fab interactionsTwo hydrophobic contact regions were identified

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Dynamics of Anti-RSV MAb

Molecular Dynamics calculations (Dr. Raul Cachau, Advanced Biomedical Computing Center) show that non-canonical MAbconformations should be common

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Coarse Grain Molecular Dynamics of Anti-RSV MAb (microseconds trajectory)

Structures from coarse grain molecular dynamics (movie) can be sorted energetically (50 ns/trajectory, ΔGa = 7.3 kcal mol-1)

MD1

MD2

MD3

MD4

MD5

MD6

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Quantitative Electron TomographyIf the frequency of occurrence of conformers can be tallied, sample quality could be assessed in the development labAutomated image analysis software will provide aggregation and polymorphism statistics from raw electron tomograms

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Low-voltage EM: the next wave

All imaging modes in a single instrument with similar resolutionand increased scale range (nano to sub-mili)Dramatically higher image contrast does not require specimen fixing and staining, or noise-filtering algorithms Imaging in liquid without freezing can monitor biological eventsLower cost instrumentation facilitates the quantitation of protein aggregation and polymorphism characterization

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Low-voltage EM: the next wave

Multi-length scaleHigh resolutionHigh contrastElement-specific imagingChemical/electron statesNon-destructive testingLower cost instrumentation

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Atomic Force Microscopy (AFM) imaging

AFM is a type of scanning probe microscopy in which a cantileverwith an ultra-sharp tip senses specimen height in a raster modeSingle antibody molecules can be imaged in liquidAntibody recognition imaging with a derivatized tip acts as identity test at the single-molecule level

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Anti-RSV MAb Force Spectroscopy by AFMForce measurements (binding affinity) of single molecules are possible (e.g., immobilized Anti-RSV MAb to tethered F-peptide)Anti-RSV MAb grabs one F-peptide with 74 pN force, on average. The Gaussian force distribution shows molecule-to-molecule variation (16 pN width). Divalent binding is also observed.From the rupture force as a function of pulling rate, the off-rate of Anti-RSV MAb can be derived (~ 1 s-1)

0 50 100 150 2000

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40

60

80

100MPF 74±4W idth 16Height 97

0.3 Hz

Monovalent binding

Divalent bindingForce (pN)

104

105

70

80

90

100

110

120

130

Loading rate, pN/sR

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Conclusions

New technologies complement or replace established techniques are emerging that:

Provide information about protein conformationAssess the relationship between macromolecular conformation and molecular processes Enhance our understanding of protein functionDetermine analytical properties on single molecules Assess product qualityStrengthen IP position or create value in licensing deal