Molecular biology, isotopic labeling, and

refolding of membrane proteins in phospholipid

bilayers.

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Preparation of membrane proteins for NMR experiments.

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• Established bacterial overexpression systems.

• Over forty different constructs of various membrane proteins.

• Improved purification and refolding protocols.

• Efficient detergent removal and sample quality control by lipid analysis.

• Implemented various lipid bilayer systems.

• Proteoliposomes.

• Nanodiscs and macrodiscs.

• Bicelles.

• Implemented various isotopic labeling schemes.

• Several types of sparse labeling.

• Complementary selective labeling.

• Perdeuteration.

Preparation of membrane proteins for NMR experiments plays an important role in our BTRC program together with

the development in NMR probes, pulse programs, and structure calculations.

General protocol for sample preparation of membrane proteins.

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Before reconstitution After reconstitution

retention (ml)

Lipid analysis by Evaporative Light Scattering Detection (ELSD)

Vpu TM

CXCR1

Vpu TM CXCR1

The protocol can be applied to a wide range of

membrane proteins from a small single

transmembrane protein to a large seven

transmembrane helical protein.

Expression, purification, and refolding of human GPCRs.

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Function

& related diseases

Fusion

protein

Expression vector

& host cells

Reconstitution

lipids

Yield (mg/L culture)

CXCR1

Chemokine receptor

Inflammatory responsesGST

pGEX-2T

BL21

DMPC

DMPC/POPC

8

AVPR2

Arginine vasopression receptor

Nephrogenic diabetes insipidusKSI pET-31b(+)

C43(DE3)

DMPC

DMPC/POPC

2

b2ARAdrenergic receptor

Asthma, obesity, type 2

diabetes

KSI pET-31b(+)

BL21(DE3)plysS

DPPC/CHS 3

CB2*Cannabinoid receptor

Immune system,

Neurodegenerative disorders

GST pGEX-2T

BL21 codon plus

DMPC ~1

*Collaboration with Sean Xie’s group at the University of Pittsburgh.

The refolding and reconstitution protocol originally developed for CXCR1 was successfully applied to other GPCRs

with minor modifications, and the multi-milligram quantities of the functional receptors were obtained.

Refolded GPCRs have biological activities.

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CXCR1

b2ARCB2 in DMPC binding H3CP55940

-12 -10 -8 -6 -4

0

50

100

log[SR144528] M

%sp

ecif

ic b

ind

ing

Xie and coworkers

CB2

Ki = 4.74 nM

EC50 = 1 nM

G-protein activation assay NMR binding experiments

Fluorescent antagonist binding Competitive ligand displacement assay

unbound

CXCR1 bound

3HCP55,940

H shift (ppm)1

Two-dimensional 13C/13C correlation MAS solid-state NMR

spectra of three constructs of Vpu from HIV-1.

6~ 2 mg of uniformly 13C/15N-labeled protein, lipid/protein = 5−10 (w/w)

Vpu Full Vpu Cyto Vpu TM

Vpu Full QPIQIAIVALVVAIIIAIVVWSIVIIEYRKILRQRKIDRLIDRLIERAEDSGNESEGEISALVELGVELGHHAPWDVDDL

Vpu Cyto EYRKILRQRKIDRLIDRLIERAEDSGNESEGEISALVELGVELGHHAPWDVDDL

Vpu TM QPIQIAIVALVVAIIIAIVVWSIVIIEGRGGKKKK

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Membrane environment affects the structure of p7 from

hepatitis C virus.

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N C

C

P7 ALENLVVLNAASVAGAHGILSFLVFFSAAWYIKGRLAPGAAYAFYGVWPLLLLLLALPPRAYA63

DHPC detergent micelles DMPC lipid bilayers

N

In general, membrane proteins are more stable in lipid bilayers than in

detergent micelles, suggesting that the p7 structure in lipid bilayers is more

relevant to the native structure in cell membranes.

Terminal truncation affects the structure

of the bacterial mercury transporter MerF.

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* *

A19

A52

* *

A

MerF MerFt

N C

N

C

* * 81

MerF KDPKTLLRVSIIGTTLVALSSFTPVLVILLGVVGLSALTGYLDYVLLPALAIFIGLTIYAIQRKRQADASSTPKFNGVKKS

MerFt IGTTLVALSSFTPVLVILLGVVGLSALTGYLDYVLLPALAIFIGLTIYAIQRKRQADASS

It is important to study the full-length membrane proteins in lipid bilayers in order to obtain accurate structure-

function relationships of membrane proteins.

Mobile N-terminal region of CXCR1 is immobilized

upon interaction with its ligand interleukin-8.

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15N-1TM-CXCR1

unlabeled IL-8

Unbound 1TM-CXCR1 IL-8 bound 1TM-CXCR1

15N shift (ppm) 15N shift (ppm)

15N-1TM-CXCR1

We have determined the structure of the unmodified full-length chemokine receptor CXCR1 in lipid bilayers. And now we continue working on the

structure of the complex of the receptor, ligand and G-proteins in order to understand the activation mechanism of CXCR1.

Cell-free expression of membrane proteins.

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• Quick production of membrane proteins.

• Selective amino acid labeling.

• Unnatural amino acid incorporation.

1. Supernatant in the presence of nanodiscs

2. Precipitate in the absence of nanodiscs

3. 1TM-CXCR1 and GST in the presence of detergents

Nanodisc

lipids

MSP

Cell-free Cell-based

1 2 3

1TM-CXCR1

1TM-CXCR1

GST

1TM-CXCR1

MSP21.5

31.0

36.5

14.4

6.0

3.52.5

MembraneMax expression kit: http://www.lifetechnologies.com

Unnatural amino acid incorporation into membrane proteins.

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• HQA1: (2-Amino-3-(8-hydroxyquinolin-3-yl)propanoic acid.

• Forms stable complexes with metal ions and lanthanides.

• Apply to distance measurement by Paramagnetic Relaxation Enhancement.

1. Lee HS, Spraggon G, Schultz PG, Wang F J Am Chem Soc (2009).

2. Yong TS, Ahmand I, Yin JA, Schultz PG J Mol Biol (2010).

pEVOL-aaRS2

pEVOL

6124 bp

aaRS

aaRS araC

CmR

araBADglnS'

proK promp15A

rrnB

proK termtRNA

SalI (3453)

BglII (2526)

Nde I (3874)

PstI (4801)

Xho I (5336)

ApaLI (5112)

glnS T

Segmental labeling of membrane proteins using Sortase A.

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• Sortase A: Staphylococcus aureus transpeptidase.

• Reduce NMR spectral complexity.

• Generate post-translationally modified proteins.

Sortase A

CXCR1 Gai

Nanodiscs

Various CXCR1 constructs

Segmental labeling is used to specifically label a protein segment with NMR active nuclei, and as a result it reduces NMR spectral complexity,

providing more flexibility in sample preparations and facilitate structural studies of multi-domain membrane proteins.