New SNPs: Sift, Polyphen, etc.

SIFT: predicting amino acid changes that affect protein functionPauline C. Ng and Steven Henikoff, Nucleic Acids Research, 2003, Vol. 3 1, No. 13 3812-3814

“Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?”

Lovelock, et al. 2007

From SNPs to form &function:100% sequence identity, but different fold & function

4

Gene-Gene Interactions

"Model...erythrocytes of [glutathione peroxidase] GPX1*2 heterozygotes should be more efficient in sheltering the cell

membrane from irreversible oxidation and binding of hemoglobin caused by the oxidant stress exerted by Plasmodium

falciparum... we observed a clear trend toward a dissociation between the HBB*A/*S and GPX1*2/*1 genotypes in the

overall data." Destro-Bisol et al. Hum Biol 1999; 71:315-32. (Pub)

G6PD ..

Malaria & HbS

(food/drug inducedhemolytic anemia)

Thalassemia

G6PD

7

Human Red Blood CellODE model

GLCe GLCi

G6P

F6P

FDP

GA3P

DHAP

1,3 DPG

2,3 DPG

3PG

2PG

PEP

PYR

LACi LACe

GL6P GO6P RU5PR5P

X5P

GA3P

S7P

F6P

E4P

GA3P F6P

NADPNADPH

NADPNADPH

ADPATP

ADPATP

ADP ATPNADHNAD

ADPATP

NADHNAD

K+

Na+

ADP

ATPADP

ATP

2 GSH GSSGNADPH NADP

ADO

INO

AMP

IMPADOe

INOe

ADE

ADEeHYPX

PRPP

PRPP

R1P R5PATP

AMPATP

ADP

Cl-

pH

HCO3-

ODE model

Jamshidi et al.

2000 (Pub)

8

Sickle cell

Average HbSE6V HbE6WModeling SNP to 3D to

cell morphology & pathology

Harrington et al. Crystal structure of deoxy-human hemoglobin 6 Glu -->

Trp. Implications for the structure and formation

of the sickle cell fiber. J Biol Chem. 1998,

273:32690-6. (Pub)

Pro3

9

Red Blood Cell Function• Transport O2 from lungs to tissues – using

hemoglobin to carry the O2

• Hemoglobin is maintained in its functional state (reduced) by the metabolic machinery

• Cell membrane separates the internal environment from the external environment– subject to physicochemical constraints

• Electroneutrality, Osmotic balance

– Cause of the imbalance:• impermeable polyions inside the cell

– hemoglobin, organic phosphates

10

www.wadsworth.org/chemheme/heme/cytocritique

Echinocytes (crenated);

hyperosmotic medium

Dacrocytes;

pernicious anemiaAcanthocytes;

abetalipoproteinemia

Macrocytes 9-12µm;

megaloblastic anemia

Spherocytes;

enzyme deficiencies

Sickle cell;

HbS

Normal RBC 6-8 µm;4e12 per L3D-structure to function

to morphology to function

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Ab Initio Structure Prediction: CASP4

http://depts.washington.edu/bakerpg/

(2 pt for largelycorrect prediction, 1

point for a somewhat)

12

Where do 3D structures come from?

Research Collaboratory for Structural BioinformaticsProtein Data Bank (RCSB PDB)

HEADER COMPLEX (TRANSCRIPTION REGULATION/DNA) 23-NOV-93 1HCQ 1HCQ 2COMPND 2 MOLECULE: HUMAN/CHICKEN ESTROGEN RECEPTOR; 1HCQ 4REMARK 2 RESOLUTION. 2.4 ANGSTROMS 1HCQ 39REMARK 3 PROGRAM 1 X-PLOR 1HCQ 42REMARK 3 R VALUE 0.204 1HCQ 46SEQRES 1 A 84 MET LYS GLU THR ARG TYR CYS ALA VAL CYS ASN ASP TYR 1HCQ 60SEQRES 1 C 18 C C A G G T C A C A G T G 1HCQ 74FORMUL 9 ZN 8(ZN1 2+) 1HCQ 107FORMUL 10 HOH *158(H2 O1) 1HCQ 108HELIX 1 1 GLU A 25 ILE A 35 1 1HCQ 109ATOM 1 N MET A 1 50.465 24.781 79.460 1.00 60.88 1HCQ 133ATOM 2 CA MET A 1 50.332 26.116 80.055 1.00 61.13 1HCQ 134CONECT 2983 2747 2789 1HCQ4038MASTER 22 3 8 9 8 0 0 6 3864 8 34 36 1HCQ4039END 1HCQ4040

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NMR distance-constrained ensembles Crystallographic phases & electron density

Ref1, 2

C trace

14

Crystallographic refinement

(ref)

Minimize Fo-Fc.Linearize with a

first order Taylor expansion; parameters p (e.g. = x,y,z)

Fourier transform relates scattered X-rays, F, to electron density, k is the scattering vector.

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Crystallography & NMR System(CNS)X-plor

Heavy atom searching, experimental phasing (MAD & MIR), density modification, crystallographic refinement with

maximum likelihood targets.

NMR structure calculation using NOEs, J-coupling, chemical shift, & dipolar coupling data.

http://cns.csb.yale.edu/v1.0/

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Measure Structure Quality

R factor = ||Fo|-|Fc|| / |Fo| < 0.25 good > 0.4 crude

Correlation Coefficient > 0.7

RMSD (root mean square deviation) = sqrt[ (Xi1 - Xi2)2 ] compare models 1 & 2 i = 1 to n (#atoms)

canonical peptide geometry

17

20 Amino acids of 280

www.people.virginia.edu/~rjh9u/aminacid.htmlwww-nbrf.georgetown.edu/pirwww/search/textresid.html

T

N CO

R

19 L-amino acids:H toward you; CO R N

clockwise.

18

Favored peptide conformations

fig

3(10)helix

19

E = Eb + E + E+ Evdw + Eelectrostatic

Eb = 0.5 kb(r-r0)2

Ek02

E = k [ 1 + cos( n - l)]

Evdw = A(r/rv0)-12 -B(r/rv0)-6 Eelectrostatic = qi qj / e r

Molecular mechanics

(Ref)

F = m a-dE/dri = Fi = mi d

2ri/dt2 r = position (radius)

dt ~= 1 fs (1e-15 sec)vi(t+dt/2) = vi(t-dt/2) + ai(t) dt update velocity & r

ri(t+dt) = ri(t)= v(t+dt/2)dt

b

.