PMID- 22769726
OWN - NLM
STAT- MEDLINE
DCOM- 20121126
LR  - 20240321
IS  - 1520-4995 (Electronic)
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 51
IP  - 30
DP  - 2012 Jul 31
TI  - Specific fluorine labeling of the HyHEL10 antibody affects antigen binding and 
      dynamics.
PG  - 6017-27
AB  - To more fully understand the molecular mechanisms responsible for variations in 
      binding affinity with antibody maturation, we explored the use of site specific 
      fluorine labeling and (19)F nuclear magnetic resonance (NMR). Several 
      single-chain (scFv) antibodies, derived from an affinity-matured series of 
      anti-hen egg white lysozyme (HEL) mouse IgG1, were constructed with either 
      complete or individual replacement of tryptophan residues with 5-fluorotryptophan 
      ((5F)W). An array of biophysical techniques was used to gain insight into the 
      impact of fluorine substitution on the overall protein structure and antigen 
      binding. SPR measurements indicated that (5F)W incorporation lowered binding 
      affinity for the HEL antigen. The degree of analogue impact was 
      residue-dependent, and the greatest decrease in affinity was observed when (5F)W 
      was substituted for residues near the binding interface. In contrast, 
      corresponding crystal structures in complex with HEL were essentially 
      indistinguishable from the unsubstituted antibody. (19)F NMR analysis showed 
      severe overlap of signals in the free fluorinated protein that was resolved upon 
      binding to antigen, suggesting very distinct chemical environments for each (5F)W 
      in the complex. Preliminary relaxation analysis suggested the presence of 
      chemical exchange in the antibody-antigen complex that could not be observed by 
      X-ray crystallography. These data demonstrate that fluorine NMR can be an 
      extremely useful tool for discerning structural changes in scFv antibody-antigen 
      complexes with altered function that may not be discernible by other biophysical 
      techniques.
FAU - Acchione, Mauro
AU  - Acchione M
AD  - Structural Biophysics Laboratory, Center for Cancer Research, Frederick National 
      Laboratory for Cancer Research, Frederick, MD 21702, USA.
FAU - Lee, Yi-Chien
AU  - Lee YC
FAU - DeSantis, Morgan E
AU  - DeSantis ME
FAU - Lipschultz, Claudia A
AU  - Lipschultz CA
FAU - Wlodawer, Alexander
AU  - Wlodawer A
FAU - Li, Mi
AU  - Li M
FAU - Shanmuganathan, Aranganathan
AU  - Shanmuganathan A
FAU - Walter, Richard L
AU  - Walter RL
FAU - Smith-Gill, Sandra
AU  - Smith-Gill S
FAU - Barchi, Joseph J Jr
AU  - Barchi JJ Jr
LA  - eng
GR  - HHSN261200800001C/RC/CCR NIH HHS/United States
GR  - HHSN261200800001E/CA/NCI NIH HHS/United States
GR  - Z01 BC010739-03/Intramural NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
PT  - Research Support, N.I.H., Intramural
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
DEP - 20120716
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Antibodies, Monoclonal)
RN  - 0 (Antigens)
RN  - 0 (Immunoglobulin G)
RN  - 284SYP0193 (Fluorine)
RN  - EC 3.2.1.- (hen egg lysozyme)
RN  - EC 3.2.1.17 (Muramidase)
SB  - IM
MH  - Animals
MH  - Antibodies, Monoclonal/*chemistry/metabolism
MH  - Antigens/chemistry/*metabolism
MH  - Binding Sites, Antibody
MH  - Crystallography, X-Ray/methods
MH  - Fluorine/*metabolism
MH  - Immunoglobulin G/chemistry/metabolism
MH  - Isotope Labeling/methods
MH  - Mice
MH  - Molecular Dynamics Simulation
MH  - Muramidase/*chemistry/immunology/metabolism
MH  - Nuclear Magnetic Resonance, Biomolecular/methods
MH  - Protein Binding/immunology
MH  - Protein Structure, Secondary
MH  - Protein Structure, Tertiary
PMC - PMC3508667
MID - NIHMS421456
COIS- The authors declare no competing financial interest.
EDAT- 2012/07/10 06:00
MHDA- 2012/12/10 06:00
PMCR- 2012/11/28
CRDT- 2012/07/10 06:00
PHST- 2012/07/10 06:00 [entrez]
PHST- 2012/07/10 06:00 [pubmed]
PHST- 2012/12/10 06:00 [medline]
PHST- 2012/11/28 00:00 [pmc-release]
AID - 10.1021/bi300455t [doi]
PST - ppublish
SO  - Biochemistry. 2012 Jul 31;51(30):6017-27. doi: 10.1021/bi300455t. Epub 2012 Jul 
      16.