PMID- 8784201
OWN - NLM
STAT- MEDLINE
DCOM- 19961011
LR  - 20131121
IS  - 0006-2960 (Print)
IS  - 0006-2960 (Linking)
VI  - 35
IP  - 35
DP  - 1996 Sep 3
TI  - Induction of alpha-helix in the beta-sheet protein tumor necrosis factor-alpha: 
      acid-induced denaturation.
PG  - 11454-60
AB  - Acid-induced unfolding of proteins often results in an intermediate structure, 
      called the molten globule structure or "A" state, which retains at least partial 
      secondary structure but lacks a rigid tertiary structure. Acid-induced unfolding 
      has been studied extensively for alpha-helical proteins, while few studies have 
      been done on proteins containing only beta-strands. Tumor necrosis factor-alpha 
      (TNF-alpha) is a trimer in which the individual subunits consist of antiparallel 
      beta-sheet, organized into a jellyroll beta-sandwich. We have found previously 
      [Narhi et al. (1996) Biochemistry 35, 11447-11453] that thermal denaturation of 
      TNF-alpha results in an aggregate which contains a substantial amount of 
      alpha-helix and that the addition of trifluoroethanol induces alpha-helix in both 
      murine and human TNF-alpha. Here we show that acid also can induce alpha-helix in 
      these proteins. At acidic pH (below 4), both human and murine TNF-alpha convert 
      to a monomeric form, as determined by sedimentation and diffusion constants 
      obtained from sedimentation velocity experiments. The sedimentation coefficient 
      indicated that this monomer was only slightly expanded relative to the native 
      state. Near-UV circular dichroic (CD) analysis showed a loss of tertiary 
      structure. These structural features coincide with the notion that the 
      acid-induced structure of TNF-alpha is a molten globule. What is unique in this 
      protein is that TNF-alpha acquires alpha-helical structure, which is not present 
      in the native structure as determined by both CD and Fourier transform infrared 
      spectroscopy. Even more surprising is that TNF-alpha at pH 3.3 undergoes a very 
      gradual noncooperative change in secondary structure upon heating, which results 
      in an increase in alpha-helical content. At pH 2.2 in the absence of salt, 
      TNF-alpha shows considerable alpha-helix, although heating does not change the 
      spectrum. At pH 2.2, physiological salt decreases the amount of alpha-helix at 
      ambient temperature, and upon heating, we see the noncooperative increase in 
      alpha-helix as observed at pH 3.3 with low salt. The addition of salt at low pH 
      induces reassociation but to a range of oligomers rather than a unique trimer 
      structure. This acid-induced formation of an alpha-helical monomer of TNF-alpha 
      may be related to its known interaction with lipid bilayers.
FAU - Narhi, L O
AU  - Narhi LO
AD  - Amgen Inc., Amgen Center, Thousand Oaks, California 91320-1789, USA.
FAU - Philo, J S
AU  - Philo JS
FAU - Li, T
AU  - Li T
FAU - Zhang, M
AU  - Zhang M
FAU - Samal, B
AU  - Samal B
FAU - Arakawa, T
AU  - Arakawa T
LA  - eng
PT  - Journal Article
PL  - United States
TA  - Biochemistry
JT  - Biochemistry
JID - 0370623
RN  - 0 (Phosphoric Acids)
RN  - 0 (Tumor Necrosis Factor-alpha)
RN  - 660YQ98I10 (Potassium Chloride)
RN  - E4GA8884NN (phosphoric acid)
RN  - QTT17582CB (Hydrochloric Acid)
SB  - IM
MH  - Animals
MH  - Circular Dichroism
MH  - Diffusion
MH  - Humans
MH  - Hydrochloric Acid/pharmacology
MH  - Hydrogen-Ion Concentration
MH  - Mice
MH  - Phosphoric Acids/pharmacology
MH  - Potassium Chloride/pharmacology
MH  - Protein Conformation
MH  - *Protein Denaturation
MH  - *Protein Structure, Secondary
MH  - Protein Structure, Tertiary
MH  - Spectroscopy, Fourier Transform Infrared
MH  - Tumor Necrosis Factor-alpha/*chemistry
MH  - Ultracentrifugation
EDAT- 1996/09/03 00:00
MHDA- 1996/09/03 00:01
CRDT- 1996/09/03 00:00
PHST- 1996/09/03 00:00 [pubmed]
PHST- 1996/09/03 00:01 [medline]
PHST- 1996/09/03 00:00 [entrez]
AID - bi952767n [pii]
AID - 10.1021/bi952767n [doi]
PST - ppublish
SO  - Biochemistry. 1996 Sep 3;35(35):11454-60. doi: 10.1021/bi952767n.