CONFORMATIONAL STABILITY OF HEME-PROTEINS IN VACUO

被引:41
作者
KONISHI, Y
FENG, R
机构
[1] National Research Council, Biotechnology Research Institute, Montreal, Quebec, H4P 2R2
[2] American Cyanamid Co., Princeton, NJ 08540
关键词
D O I
10.1021/bi00198a041
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The function and conformational stability of myoglobin were studied in vacuo by using an electrospray mass spectrometer. The electrospray technique gently transfers protein molecules from the solution phase to the gas phase, and solvent-free protein ions are produced in the mass spectrometer. Horse myoglobin was dissolved at neutral pH, Fe3+ in the heme was reduced to Fe2+ to produce the biologically active oxymyoglobin in solution, and then the protein was isolated in vacuo. A molecular ion (17 601 Da) corresponding to the molecular mass of oxymyoglobin (17 600.0 Da) was observed in the mass spectrum. This demonstrates that the protein retains a heme and an oxygen molecule in the gas phase. Since the biological function of myoglobin is to carry an oxygen molecule, this is the first observation that a protein is functional in the absence of solvent. Gas-phase ''unfolding'' of myoglobin was also studied. Collisions of accelerated protein ions with nitrogen curtain gas at a quadrupole guidance lens or argon gas introduced at a second quadrupole increase the ''molecular temperature'' of myoglobin, resulting in release of the heme from myoglobin. Apomyoglobin produced at the quadrupole guidance lens showed a larger collisional cross section than that of myoglobin, revealing conformational disordering of the protein. The gas-phase unfolding of horse and whale myoglobins and the alpha-chain of human hemoglobin induced at the second quadrupole were studied as a function of the argon gas thickness. Horse and whale myoglobins showed the same gas-phase stability, whereas the alpha-chain of human hemoglobin was less stable. Consequently, the amino acid sequence by itself has sufficient information to stabilize the gas-phase conformation of the heme proteins.
引用
收藏
页码:9706 / 9711
页数:6
相关论文
共 33 条
[1]   DENATURATION OF RIBONUCLEASE-A BY COMBINATIONS OF UREA AND SALT DENATURANTS [J].
AHMAD, F ;
BIGELOW, CC .
JOURNAL OF MOLECULAR BIOLOGY, 1979, 131 (03) :607-617
[2]   FAST ATOM BOMBARDMENT OF SOLIDS (FAB) - A NEW ION-SOURCE FOR MASS-SPECTROMETRY [J].
BARBER, M ;
BORDOLI, RS ;
SEDGWICK, RD ;
TYLER, AN .
JOURNAL OF THE CHEMICAL SOCIETY-CHEMICAL COMMUNICATIONS, 1981, (07) :325-327
[3]   UNFOLDING PATHWAY OF MYOGLOBIN - EVIDENCE FOR A MULTISTATE PROCESS [J].
BISMUTO, E ;
COLONNA, G ;
IRACE, G .
BIOCHEMISTRY, 1983, 22 (18) :4165-4170
[4]   ION SPRAY INTERFACE FOR COMBINED LIQUID CHROMATOGRAPHY/ATMOSPHERIC PRESSURE IONIZATION MASS-SPECTROMETRY [J].
BRUINS, AP ;
COVEY, TR ;
HENION, JD .
ANALYTICAL CHEMISTRY, 1987, 59 (22) :2642-2646
[5]   PROBING CONFORMATIONAL-CHANGES IN PROTEINS BY MASS-SPECTROMETRY [J].
CHOWDHURY, SK ;
KATTA, V ;
CHAIT, BT .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 1990, 112 (24) :9012-9013
[6]   FREE-ENERGY CHANGES IN ALPHA-LACTALBUMIN DENATURATION [J].
CONTAXIS, CC ;
BIGELOW, CC .
BIOCHEMISTRY, 1981, 20 (06) :1618-1622
[7]  
Covey T R, 1988, Rapid Commun Mass Spectrom, V2, P249, DOI 10.1002/rcm.1290021111
[8]  
COVEY TR, 1993, J AM SOC MASS SPECTR, V4, P613
[9]   FOLDING OF RIBONUCLEASE-A FROM A PARTIALLY DISORDERED CONFORMATION - KINETIC-STUDY UNDER FOLDING CONDITIONS [J].
DENTON, JB ;
KONISHI, Y ;
SCHERAGA, HA .
BIOCHEMISTRY, 1982, 21 (21) :5155-5163
[10]   MOLECULAR BEAMS OF MACROIONS [J].
DOLE, M ;
MACK, LL ;
HINES, RL .
JOURNAL OF CHEMICAL PHYSICS, 1968, 49 (05) :2240-&