- The document summarizes Stanislav Andres' practical placement from July 1st to September 25th at the Latvian Institute of Organic Synthesis. - The goals were to obtain and analyze the BBA03 protein involved in Lyme disease infection, and to perform routine analysis of biological samples using NMR and LCMS techniques. - NMR experiments were performed to assign the backbone and sidechain structures of the BBA03 protein, with the longest experiments taking 9 days to complete.

1. Practical Placement
STANISLAV ANDRES

2. W
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e
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e
W
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Riga, Latvia
Latvian Institute of Organic
Synthesis (LOSI)
From July 1st till September 25th
Two parts:
- July 1st till August 30th
- September
- Obtainment and analysis of BBA03 protein
- Routine analysis of biological samples by NMR and LCMS

3. Kristaps JaudzemsZigmantas Toleikis
Direct mentor
and main
advisor in
BBA03 project
Head of high-
frequency
NMR
subgroup
Mentor at
LCMS group
Eduards Sevostjanovs
Edgars Suna
Official
coordinator
and mentor

4. NMR for biopolymers structure
Based on impulse technic
Requires multidimensional experiments
Heavy isotopes

5. BBA03
Borrelia burgdorferi infection apparatus protein
Takes part in Lyme disease infection. Potential target for antibodies
Small protein of 150 amino acids + 25 as initial domain
Structure of complex is mostly unknown

6. Sample growing and preparation
Culture of E.coli K12 was grown on LB medium
To transform M9, modified with 13
C-glucose, and 15
N-
ammonium, was used. To avoid contamination plasmid
contained kanamycin resistance gene, besides bba03.
Cells were sonicated for 25 minutes (5 sec in 20 sec cycle)
After short centrifugation - gel-filtration.
To check preliminary the efficiency of growth electrophoresis
was performed.
The solution was filtered through 10 kDa concentration tubes
and than till 500 ul through 3 kDa
For NMR 10% of D2O and 0,02% of Na-Azide

7. NMR technics for fast protein
assignment
For backbone assignment:
HNCA
CBCA(CO)NNH
HNCO
HN(CA)CO
For sidechains (just begun):
3D - NOESY (both N and C)
The longest takes 9 days to be performed, the volume of sample – 500ul, OD280= 0,5..0,9
Calibration is done against water shift on one of the spectra (normally HSQC)

8. HNCACBCA(CO)NNHHNCO HN(CA)CO
13C-NOESY-HSQC15N-NOESY-HSQC

10. For Glycine 94

11. Table of amino acids common shifts
ABCAmAc AmAc C CA CB
S Ser 174,66 58,75 63,79 -5,04 neg 0
below0T THR 174,59 62,27 69,71 -7,44 neg 0
D ASP 176,45 54,7 40,86 13,84 small 1
13-15
N ASN 175,31 53,56 38,69 14,87 small 1
L LEU 177,08 55,7 42,26 13,44 small 1
F PHE 174,49 58,14 39,93 18,21 lower 2
18-19Y TYR 175,47 58,19 39,27 18,92 lower 2
I ILE 175,93 61,68 38,58 23,1 mid 3
23-24
K LYS 176,73 56,99 32,77 24,22 mid 3
M MET 176,24 56,14 32,93 23,21 mid 3
R ARG 176,48 56,82 30,64 26,18 upper 4
25-28
C Cys 174,95 58,21 32,9 25,31 upper 4
E GLU 176,93 57,35 29,96 27,39 upper 4
Q GLN 176,38 56,62 29,15 27,47 upper 4
H HIS 175,27 56,52 30,22 26,3 upper 4
W TRP 176,23 57,74 29,97 27,77 upper 4
A Ala 177,82 53,19 18,96 34,23 big 5
29-34
P Pro 176,77 63,36 31,84 31,52 big 5
V VAL 175,71 62,57 32,7 29,87 big 5
G GLY 173,9 45,36 45,36

12. Thank you for attention