Practical PlacementSTANISLAV ANDRES

Where

When

What

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

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

NMR for biopolymers structure Based on impulse technic

Requires multidimensional experiments

Heavy isotopes

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

Sample growing and preparation Culture of E.coli K12 was grown on LB medium

To transform M9, modified with -glucose, and -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 O and 0,02% of Na-Azide

NMR technics for fast protein assignment

For backbone assignment:HNCA CBCA(CO)NNHHNCO 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, = 0,5..0,9Calibration is done against water shift on one of the spectra (normally HSQC)

HNCA CBCA(CO)NNHHNCO HN(CA)CO

13C-NOESY-HSQC15N-NOESY-HSQC

For Glycine 94

Table of amino acids common shifts

ABCAmAc AmAc C CA CBS Ser 174,66 58,75 63,79 -5,04 neg 0

below0T THR 174,59 62,27 69,71 -7,44 neg 0D ASP 176,45 54,7 40,86 13,84 small 1

13-15N ASN 175,31 53,56 38,69 14,87 small 1L LEU 177,08 55,7 42,26 13,44 small 1F PHE 174,49 58,14 39,93 18,21 lower 2

18-19Y TYR 175,47 58,19 39,27 18,92 lower 2I ILE 175,93 61,68 38,58 23,1 mid 3

23-24K LYS 176,73 56,99 32,77 24,22 mid 3M MET 176,24 56,14 32,93 23,21 mid 3R ARG 176,48 56,82 30,64 26,18 upper 4

25-28

C Cys 174,95 58,21 32,9 25,31 upper 4E GLU 176,93 57,35 29,96 27,39 upper 4Q GLN 176,38 56,62 29,15 27,47 upper 4H HIS 175,27 56,52 30,22 26,3 upper 4W TRP 176,23 57,74 29,97 27,77 upper 4A Ala 177,82 53,19 18,96 34,23 big 5

29-34P Pro 176,77 63,36 31,84 31,52 big 5V VAL 175,71 62,57 32,7 29,87 big 5G GLY 173,9 45,36 45,36

Thank you for attention