Protein-DNA Interactions quilibria and free energies Estimate binding site occupancy Forces driving protein-DNA interactions Lambda repressor Methods to study protein-DNA interactions
Protein-DNA Interactions • Equilibria and free energies • Estimate binding site occupancy • Forces driving protein-DNA interactions • Lambda repressor • Methods to study protein-DNA interactions
Major groove Major groove H D CH DN H A N H D Minor groove Minor groove Adenine: Thymine Guanine: Cytosine
A A A A D D A A A A D M H 1
The 4 Major groove Patterns Are Distinct Maior Maior A-TA DA M G-C AADH TA MAD A C-G HDAA Minor Minor A-TA A G-C DA TAA A C-GA DA
A D A M Major A-T T-A M A D A Minor A-T T-A A A A A G-C A A D H C-G G-C C-G Major H D A A A D A A D A Minor The 4 Major Groove Patterns Are Distinct 2
Hyrdophobic groups on bases Major groove Major groove CH A H IN H H Minor groove Minor groove Adenine: Thymine Guanine: Cytosine
A A A A D D A A A A D M H 1 Hyrdophobic Groups on Bases
Helix-Turn-Helix motif 2 C 34A° N 4 2
3 Helix-Turn-Helix Motif 34 Aº
N-term
3 4 1 2 5 N -term 3’ 4
A REPRESSOR: A MODEL SYSTEM 13 Specificity Determinants? TABLE I Generic Interactions? Protein-DNA Intractions Major groove interactions dNa backbone interactions Hydrogen bonding groups P Adenine 2 O-IP... GIn-33 N N-6... Gln-44 OEI O-2P..Tyr-22 OH N-7...Gn-4NE2 O-2P,. Lys-26 NZ Guanine 4 N-7., Ser-45 OG OIP and O-2P... Lys-19 NZ Guanine 6 OIP.. GIn-33 NG2 N-7...Asn-55ND2 OIP.. Asn-52 ND2 O-6... Lys-4 NZ Guanine 8 O-IP.. Gly-43N N-7... Thr-2 OGl D Hydrophobic interactions O-IP and O-2P... Asn-61 ND2 Thymine l C-5 M.. GIn-44 CG O- IP and O-2P... Asn-58 ND2 Thymine 3 (in consensus half-site) C-5M... Ile-54 CDI OIP.. Ala-56 N Gly-48 main chain (in nonconsensus half-site) Ser-45 CB and main chain Thymine 5 C-5M... Gly-46 CA Ser-45 CA and CB
Specificity Determinants? Generic Interactions? 5
Table 13. 2 Thermodynamic Effects of Mutations in the a Repressor on Operator Binding AG Relative to Wild-Type, kcal mol Mutant Wild-Type Interaction Operator△ G Nonoperator△G yr-22- Phe Backbone phosphate 1.6 GIn-33- Ser Backbone phosphate Asn-52-Asp Backbone phosphate 6.2 >2.8 Gly-43- Glu Backbone phosphate 6.2 0.5 GIn-44-Ser Hydrogen Bond to A(2)N7,-NH 3.7 0.2 GIn-44- Leu Hydrogen Bond to A(2)N7,NH >6.2 0.2 Ser-45- Leu Hydrogen Bond to G(4)N7 >6.2 0.1 Asn-55- Lys Hydrogen Bond to G(6)N7, Lys-4 Lys-4-GIn Hydrogen Bond to G(6)06, Asn-55 5.2 0.5 Ala-49- Val Hydrophobic T(3)-CH 4.6 0.2 Ala-49-+Asp Hydrophobic T(3)CH >6.2 0.1 6
6 >104 >103
GIn3 3 networks with gin44 T N H一N N GIn-33CH2 CH2GIn-44 △△G CH 2 H 4.5 kcal △△G= 6.2 kcal er eu 7
Gln33 Networks with Gln44 Ser ∆ ∆G = 4.5 kcal Leu ∆ ∆G = >6.2 kcal 7
Conclusion dna binding specificity from major groove interactions Mutation in one Aa affects neighbors >Protein-DNa interfaces contain an intricate Network of hydrogen bonds
Conclusion • DNA binding specificity from major groove interactions • Mutation in one AA affects neighbors -->Protein-DNA interfaces contain an intricate Network of hydrogen bonds