1. New SNPs: Sift, Polyphen, etc. SIFT: predicting amino acid changes that affect protein function Pauline C. Ng and Steven Henikoff, Nucleic Acids Research, 2003, Vol. 3 1, No. 13 3812-3814

2. “Identification of BRCA1 missense substitutions that confer partial functional activity: potential moderate risk variants?” Lovelock, et al. 2007

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

4. 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)(Pub)

5. G6PD.. Malaria & HbS (food/drug induced hemolytic anemia) Thalassemia

6. G6PD

7. 7 Human Red Blood Cell ODE model GLC e GLC i G6P F6P FDP GA3P DHAP 1,3 DPG 2,3 DPG 3PG 2PG PEP PYR LAC i LAC e GL6PGO6PRU5P R5P X5P GA3P S7P F6P E4P GA3PF6P NADP NADPH NADP NADPH ADP ATP ADP ATP ADP ATP NADH NAD ADP ATP NADH NAD K+K+ Na + ADP ATP ADP ATP 2 GSHGSSG NADPHNADP ADO INO AMP IMP ADO e INO e ADE ADE e HYPX PRPP R1P R5P ATP AMP ATP ADP Cl - pH HCO 3 - ODE model Jamshidi et al. 2000 (Pub)(Pub)

8. 8 Sickle cell Average HbS  E6V Hb  E6W Modeling 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)(Pub) Pro3

9. 9 Red Blood Cell Function Transport O 2 from lungs to tissues – using hemoglobin to carry the O 2 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. 10 www.wadsworth.org/chemheme/heme/cytocritique Echinocytes (crenated); hyperosmotic medium Dacrocytes; pernicious anemia Acanthocytes; abetalipoproteinemia Macrocytes 9-12µm; megaloblastic anemia Spherocytes; enzyme deficiencies Sickle cell; HbS Normal RBC 6-8 µm; 4e12 per L 3D-structure to function to morphology to function

11. 11 Ab Initio Structure Prediction: CASP4 http://depts.washington.edu/bakerpg/ (2 pt for largely correct prediction, 1 point for a somewhat)

12. 12 Where do 3D structures come from? Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB)(RCSB PDB) HEADER COMPLEX (TRANSCRIPTION REGULATION/DNA) 23-NOV-93 1HCQ 1HCQ 2 COMPND 2 MOLECULE: HUMAN/CHICKEN ESTROGEN RECEPTOR; 1HCQ 4 REMARK 2 RESOLUTION. 2.4 ANGSTROMS 1HCQ 39 REMARK 3 PROGRAM 1 X-PLOR 1HCQ 42 REMARK 3 R VALUE 0.204 1HCQ 46 SEQRES 1 A 84 MET LYS GLU THR ARG TYR CYS ALA VAL CYS ASN ASP TYR 1HCQ 60 SEQRES 1 C 18 C C A G G T C A C A G T G 1HCQ 74 FORMUL 9 ZN 8(ZN1 2+) 1HCQ 107 FORMUL 10 HOH *158(H2 O1) 1HCQ 108 HELIX 1 1 GLU A 25 ILE A 35 1 1HCQ 109 ATOM 1 N MET A 1 50.465 24.781 79.460 1.00 60.88 1HCQ 133 ATOM 2 CA MET A 1 50.332 26.116 80.055 1.00 61.13 1HCQ 134 CONECT 2983 2747 2789 1HCQ4038 MASTER 22 3 8 9 8 0 0 6 3864 8 34 36 1HCQ4039 END 1HCQ4040

13. 13 NMR distance-constrained ensembles Crystallographic phases & electron density Ref1, 2, 2 C  trace

14. 14 Crystallographic refinement (ref)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.

15. 15 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/

16. 16 Measure Structure Quality R factor =  ||Fo|-|Fc|| /  |Fo| 0.4 crude Correlation Coefficient > 0.7 RMSD (root mean square deviation) = sqrt [  (X i1 - X i2 ) 2 ] compare models 1 & 2 i = 1 to n (#atoms) canonical peptide geometry

17. 17 20 Amino acids of 280 www.people.virginia.edu/~rjh9u/aminacid.html www-nbrf.georgetown.edu/pirwww/search/textresid.html T N CO R 19 L-amino acids: H toward you; CO R N clockwise.

18. 18 Favored peptide conformations fig 3(10)helix

19. 19 E = E b + E  + E  + E vdw + E electrostatic E b = 0.5 k b (r-r 0 ) 2 E   k   0  2 E  = k  [ 1 + cos( n  - l)] E vdw = A(r/r v0 ) -12 -B(r/r v0 ) -6 E electrostatic = qi qj / e r Molecular mechanics (Ref) F = m a -dE/dr i = F i = m i d 2 r i /dt 2 r = position (radius) dt ~= 1 fs (1e-15 sec) v i (t+dt/2) = v i (t-dt/2) + a i (t) dt update velocity & r r i (t+dt) = r i (t)= v(t+dt/2)dt b  

20. .