Mechanism of Antifreeze Proteins Studied by MQ Filtering ......MQ Pulse Sequence Time reversal process Even order MQCs are generated. 6 ... Liquid phase with the MQC labels Desorbed.

1

Mechanistic Study of AntifreezeProteins by MQ Filtering – Spin

Exchange NMR

Yong Ba

Department of Chemistry and Biochemistry,

California State University Los Angeles

Ba, Yong; Wongskhaluang; Jeff; Jiabo Li, Reversible Binding of the HPLC6 Isoform of Type I

Antifreeze Proteins to Ice Surfaces and the Antifreeze Mechanism Studied by Multiple

Quantum (MQ) Filtering - Spin Exchange NMR Experiment, (communication) J. Am.

Chem. Soc, 125(2), 330-331, 2003.

Ba, Yong; John A., Ripmeester, Multiple quantum filtering and spin exchange in solid state

nuclear magnetic resonance, J. Chem. Phys., 108, 20, 8589-8594, (1998).

2

Outline

Model system: molecular exchange betweenadamantane solid surface/adamantanebenzene-d6 solution

MQ Filtering – Spin Exchange NMR technique

Application to observe the reversibility ofHPLC6 isoform of type I AFPs binding to icesurfaces

Mechanism of binding of AFPs to ice surfaces

3

Proton NMR Spectrum ofAdamantane/Benzene-d6

4

MQ Transitions

M – magnetic quantum number

n – MQC order

5

MQ Pulse Sequence

Time reversal process

Even order MQCs are generated

6

MQ Filtering

Liquid phase

n=0

Solid phase

n=0, 2, 4, …

Nuclear spins arelabeled by MQCorders after the timereversal process.

7

MQ Filtering – Spin Exchange

The exchange mixing

time allows the spinscarried by the

adamantane molecules

exchange between thesolid surface and the

liquid phase.

Traveling of the spins

will be tracked by theMQC labels.

8

MQ Filtering – Spin Exchange

Molecules on solid

surfaces labeledwith MQCs

Liquid phase with

the MQC labels

Desorbed

9

1D Selective Detection of MQCs

10

Selective Detection

2, 6, 10, … MQC

selection

11

Spin Exchange Kinetics

12

HPLC6 Isoform of Type I AFPs

D TASDAAAAAAL TAANAKAAAEL TAANAAAAAAA TARAlanine rich

-helical

11-residue repeat unit

13

Function of AFPs

Inhibition for the growth ofseed ice crystals by bingingto specific ice surfaces

14

Binding of HPLC6 to IceSurfaces

Ice Ih latticeBipyramidal planeDirectional vector

)1202(

>< 2011

Davies,…,JBC, 273, 11714, 1998

15

Kelvin Effect Explanation

Local freezing-point depression

The binding of AFPs to ice surfaces causes thegrowing ice front to advance in spaces between the

AFP molecules

This causes local curvatures on ice surfaces

The physical change makes it energetically less

favorable for water molecules to join the ice lattice

Water

Ice

16

Irreversible Binding of KelvinEffect Explanation

Kelvin-effect explanation implies that AFPsessentially bind to ice surfaces in anirreversible manner

Because desorption of AFPs would allowsuper-cooled water to join the ice latticeinstantly

Water

Ice

17

Reversible binding of HPLC6 toIce Surfaces

Experimental result

of MQ filtering-spinexchange NMR for

HPLC6 peptides /

0.1 M ND4DCO3

deuterated

aqueous solution at–1.0 0C

18

Langmuir Model of Monolayer Adsorption

AFPsAdsorbedSitesBindingSurfaceIceAFPs +

A0Ak

k)1(AFP + +

B0Bk

k)1(AFP + +

( )0AAA 1)t(AFPk)t(k

dt

)t(dAFP= +

( )0BBB 1)t(AFPk)t(k

dt

)t(dAFP= +

E

00

E

0

A

A

A n

)S/n1(nk

V/n

S/nkk

+==

)]kt(Exp1[AFP)t(AFP EA =

19

Langmuir Model of Monolayer Adsorption

0.0 0.5 1.0 1.5 2.0 2.5 3.00.0

0.1

0.2

0.3

0.4

0.5

0.6

Fra

ctions o

f th

e H

PLC

6 p

etides

desorb

ed to the s

olu

tion

Exchange time (s)

Reversible binding

Monolayer adsorption

Interpeptide interaction does not exist

Independent binding of AFPs to ice surfaces

20

Experimental Facts

Specific ice surfaces possess affinities to AFPs

AFPs bind to ice surfaces in a reversible manner

AFPs bind to ice surfaces independently

21

Mechanism of Ice GrowthInhibition

There exists a concentration gradient of AFPs from an

ice surface to the solution

This results in a dense layer of AFPs in contact with theice surface

This dense layer of AFPs is able to depress the local

freezing point through colligative effect by reducing thechemical potential of the local liquid water

22

References1. Ewart, K.V., Q. Lin, and C.L. Hew, Structure, function and evolution of antifreeze proteins. Cell Mol Life Sci, 1999. 55(2): p. 271-83.2. Duman, J.G., D.W. Wu, T.M. Olsen, M. Urrutia, and D. Turaman, Thermal-Hysteresis Proteins. Adv. Low Temperature Biol.,1993. 2: p. 131.3. Duman, J.G., and A.L. DeVries, Isolation, characterization and physical properties of protein antifreeze from the winter flounder Pseudopleunectus americanus. Comp. Biochem. Physiol., 1976. B 54: p. 375-380.4. Harding, M.M., L.G. Ward, and A.D. Haymet, Type I 'antifreeze' proteins. Structure-activity studies and mechanisms of icegrowth inhibition. Eur J Biochem, 1999. 264(3): p. 653-65.5. Sicheri, F., and D.S. Yang, Ice-binding structure and mechanism of an antifreeze protein from winter flounder. Nature, 1995. 375(6530): p. 427-31.6. Knight, C.A., C.C. Cheng, and A.L. DeVries, Adsorption of alpha-helical antifreeze peptides on specific ice crystal surface planes. Biophys J, 1991. 59(2): p. 409-18.7. Houston, M.E., Jr., H. Chao, R.S. Hodges, B.D. Sykes, C.M. Kay, F.D. Sonnichsen, M.C. Loewen, and P.L. Davies, Binding of an oligopeptide to a specific plane of ice. J Biol Chem, 1998. 273(19): p. 11714-8.8. Devries, A.L., and Y. Lin, Structure of a peptide antifreeze and mechanism of adsorption to ice. Biochim Biophys Acta, 1977. 495(2): p. 388-92.9. Wen, D., and R.A. Laursen, Structure-function relationships in an antifreeze polypeptide. The role of neutral, polar amino acids. J Biol Chem, 1992. 267(20): p. 14102-8.10. Chao, H., M.E. Houston, Jr., R.S. Hodges, C.M. Kay, B.D. Sykes, M.C. Loewen, P.L. Davies, and F.D. Sönnichsen, A diminished role for hydrogen bonds in antifreeze protein binding to ice. Biochemistry, 1997. 36(48): p. 14652-60.11. Wilson, P.W., Explanation Thermal Hysteresis by the Kelvin Effect. Cryo-letters, 1993. 14: p. 31-36.12. Yeh, Y., and R.E. Feeney, Antifreeze proteins: Structures and Mechanisms of Function. Chemical Reviews, 1996. 96(2): p. 601-617.13. Ba, Y., and J.A. Ripmeester, Multiple quantum filtering and spin exchange in solid state nuclear magnetic resonance. J.Chem. Phys., 1998. 108(20): p. 8589-14.16. Wen, D., and R.A. Laursen, A model for binding of an antifreeze polypeptide to ice. Biophys J, 1992. 63(6): p. 1659-62.17. DeLuca, C.I., R. Comley, and P.L. Davies, Antifreeze proteins bind independently to ice. Biophys J, 1998. 74(3): p. 1502-8.18. Burcham, T.S., D.T. Osuga, Y. Yeh, and R.E. Feeney, A kinetic description of antifreeze glycoprotein activity. J Biol Chem, 1986. 261(14): p. 6390-7.19. Hew, C.L., and D.S. Yang, Protein interaction with ice. Eur J Biochem, 1992. 203(1-2): p. 33-42.

23

Acknowledgement

Cal State LA MBRS RISE Program

NIH MBRS-SCORE Grant 5S06 GM08101

Dr. Jiabo Li, Accelrys Inc., San Diego, CA

Jeff Wongskhaluang, Cal State LA Los Angeles