Alignment of herg potassium channel with known structures of potassium channel Name: Prachi Sharma Superviser: Prof. Gert Vriend introduction

2. MATERIALS AND METHODS

2.1 KChannelDB: sequence-derived data One human sequence of potassium channel from every family was taken from the KCHANNELDB(30).

2.2 MRS BLAST

Human sequences obtained from KCHANNELDB were blasted against all database using MRS BLAST. Sequences with relevant e value (-4) were taken (31).

2.3 WHAT IF

Profiles were generated for each of the family with the sequences obtained from MRS BLAST.

Profile generation was done by WHAT IF software. Profiles were generated n the following way.

WALIGN option of WHATIF was run. Then following wahtif commands were given.

• 
  GETSEQ: asks for a seq one seq was given. This is the sequence against which blast was performed and results of blast are stored in the Fasta format.
  
• 
  FASTA3: it asks for a file having sequences in Fasta format. Blast results obtained above were entered.
  
• 
  NEWPRF: it asks for seq. First sequence was given. A profile was generated for the first seq
  
• 
  ALIPRF: It asks for a range. Whole range was given.
  
• 
  SRTPCT: it sorts sequence according to the similarity and identity.
  
• 
  MAKHSP: asks for range whole range was given. Then asks for file name.
  
• 
  MVIEW: Generates a file showing alignment of all sequences under profile
  
• 
  UPDPRF asks for sequence range sequences found more identical were then given eg 1 10 outof 77.
  
• 
  ORGBCK gives back original sequence. Asks for seq. Whole range was given.
  
• 
  Again ALIPRF, SRTPCT, MAKHSP and MVIEW command given.
  
• 
  MAKPRF command makes a profile file (32).
  

2.4 Profiles Corrected: Profiles were manually corrected. Gaps were avoided by increasing gap opening penalty at places where gaps were found and decreasing gap elongation penalty at some other places in alignment.
2.5 Prediction of Helices

Different Secondary structure predictors were used to find transmembrane helices. Following Secondary structure predictors were used

1. 
   TMpred - Prediction of Transmembrane Regions and Orientatio (33).
   
2. 
   NNPREDICT Protein Secondary Structure Prediction (34)
   

20

3. 
   PSIPRED Protein Structure Prediction Server (35).
   
4. 
   TMHMM Server v. 2.0 (36).
   

Final transmembrane helices were also determined from different literature. In the file obtained from MAKPRF option of WHAT IF, helices and Selectivity Filter were marked and profile was edited to remove gaps and insertions. Again new profile obtained with GETPRF command of WHAT IF.

2.6 Entropy/Variability values

To find entropy/variability values for all of the residue position WALVAR option of WHAT IF was used.
2.7 YASARA

YASARA software was used to visualize the structure of potassium channel. With YASARA we also colored potassium channel structures according to the entropy/variability value.

20

3. RESULT

Sequences for each family were aligned together and profiles for every family was made. These alignments, profiles and sequences of conserved structure of all potassium channel can be found on http://www.cmbi.ru.nl/~psharma/index.html.

Sequences for conserved structures of potassium channel like Helix5 which is the helix just before selectivity filter, sequences for selectivity filter and sequences for helix 6 which is the helix just after selectivity filter, from different families were aligned together. Sequence alignment of selectivity filter is given below

TM7 TMSTVGYG

TM6 VSMTTVGYG

TM4-1SILTTTGYG

TM4-2 SLSTIGLG

TM2 SQTTIGYG

Fig11: Sequence alignment of Selectivity filter of different families of potassium channel.TM4-1 is the first and TM4-2 is the second selectivity filter of the potassium channels having 4TM and twoP.

For helix 5 we made two types of alignment which are given below in (Fig12).

A.

TM7 K12 L11 V12 N12 L11 L22 S11 I11 F11 I11 S11 T22 W11

TM6 K11 A11 S11 M11 R11 E11 L11 G11 L11 L11 I11 F11

TM41 K23 P23 W23 A33 R23 Y23 L23 L23 L22 L22 M23 A22 H22

TM42 R12 W22 Q22 L23 A23 P23 A23 Q33 A23 A22 L23 L23 Q22

TM2 R11 Y11 K12 L12 L12 L12 F11 A12 T12 F11 A23 G12 T12

TM7 L11 T11 A11 A11 G11 F12 I11 H11 L11 V12 E11 N11 S11 G11 D11 P11 W22

TM6 F11 L11 F11 I11 G11 V11 I11 L11 F11 S11 S11 A11 V11 Y11 F11 A11 E11 A11 D11

TM41L23 L23 A12 M12 G23 L22 G11 A11 V23 V12 L23 Q23 A23 L22 E11 L23 P11 Y23 A12

TM42A22 A23 G23 L12 G22 L23 L23 V23 A23 C23 V23 F12 M22 L23 L23 P11 A12 L23 V12

TM2 W11 F11 L12 F11 G11 V12 V12 W12 Y11 L12 V12 A11 F23 A23 H11 G12 D11 L00

TM7

TM6 E11 R11

TM41 R23 H23 L23

TM42 L12 W23 G23 V12 Q12 G23 D22 C22 S22 L23 L11 E12 A12 I12

TM2 L22 E11 L12 G22 P23 P11 A12
B.
TM7 S11N22 S11 I11 K12 L11 V12 N12 L11 L22 S11 I11 F11 I11 S11 T22 W11

TM6 K11 A11 S11 M11 R11 E11 L11 G11 L11 L11 I11 F11

TM41 M GSL33 K23 P23 W23 A33 R23 Y23 L23 L23 L22 L22 M23 A22 H22 L23 L23

TM42 R12 W22 Q22 L23 A23 P23 A23 Q33 A23 A22 L23 L23 Q22

TM2 D11 M12 Q12 W11 R11 Y11 K12 L12 L12 L12 F11 A12 T12 F11 A23 G12 T12
20

TM7 L11 T11 A11 A11 G11 F12 I11 H11 L11 V12 E11 N11 S11 G11 D11 P11 W22

TM6 F11 L11 F11 I11 G11 V11 I11 L11 F11 S11 S11 A11 V11 Y11 F11 A11 E11 A11 D11

TM41A12 M12 G23 L22 G11 A11 V23 V12 L23 Q23 A23 L22 E11 L23 P11 Y23 A12 R23 H23

TM42A22 A23 G23 L12 G22 L23 L23 V23 A23 C23 V23 F12 M22 L23 L23 P11 A12 L23 V12

TM2 W11 F11 L12 F11 G11 V12 V12 W12 Y11 L12 V12 A11 F23 A23 H11 G12 D11 L00


TM7

TM6 E11 R11

TM41 L23

TM42 L12 W23 G23 V12 Q12 G23 D22 C22 S22 L23 L11 E12 A12 I12

TM2 L22 E11 L12 G22 P23 P11 A12

fig12:A. Shows alignment of helix5 in different families of potassium channel. With every residue its entropy/variability value is also shown. B. Shows new alignment according to the entropy/variability values.
Sequence alignment of Helix6 (helix before SF is given below)is given below.

TM7 : Y12 A12 K23 T11 T22 L11 G11 R11 L12 F11 M23 V11 F11 F11 I12 L11

TM6 : V11 P11 T11 T11 I11 G11 G11 K11 I11 V11 G11 S11 L11 C11 A11 I11

TM41: L22 S12 S23 G22 G11 K11 A22 F11 C12 V12 V23 Y11 A12 A12

TM42: A23 I12 Y23 H23 L22 G22 Q12 F23 A23 L12 L23 G23 Y22 L12 L23

TM2 :I12 S12 E11 E12 C11 P12 H23 A11 I11 F22 L11 L11 I12 A22 Q11 L12 V12 L12

TM7 :G22 G11 L11 A11 M11 F11 A11 S11 Y23 V12 P11 E11 I11 I22 E11 L12 I23 G11

TM6 :A11 G11 V11 L11 T11 I11 A11 L11 P11 V11 P11 V11 I11 V11 S11 N11 F11 N11

TM41:L12 G11 L12 P11 A23 S12 L12 A22 L12 V12 A12 A22 L23 R12 H23 C22 L12 L23

TM42:L22 G12 L12 L23 A12 M22 L22 L12 A22 V12 E22 T12 F12 S23 E23 L22 P23 Q23

TM2 :T12 T22 I12 L12 E12 I12 F11 I12 T12 G11 T12 F12 L12 A11 K11 I11 A12 R11

TM7 :N23 R12 K22 K11 Y11 G11 G11 S22

TM6 :Y11 F11 Y11 H11 R11 E11 T11 E11

TM41:P23 V23 F23 S23 R23 P23 G23

TM42:V23 R23 A23 M23 V23 K23

TM2 :P11 K11 K11 R11 A11 E12 T11

Fig13: Shows alignment of helix6 (Helix which is just before Selectivity Filter) in different families of potassium channel. With every residue its entropy/variability value is also shown.

20

Alignment of hERG with KCSA and TM6 profile

Fig14:Alignment above shows Anna's alignment of Kcsa and herg. In above alignment we have also aligned the profile of TM6 with the Kcsa and herg. from our alignment of TM6 profile with other potassium channel family profiles (see figures 11, 12, 13).

20