• 생명과학회지
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• 제16권7호
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• pp.1225-1228
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• 2006

cAMP와 복합체를 형성한 cAMP 수용성 단백질은 DNA와 결합하여 ${\sim}90$도 정도의 예리한 DNA bending을 유도한다. 그러나 이전의 논문[5]에 의하면 돌연변이 CRP:cGMP 복합체는 돌연변이 CRP:cAMP 복합체보다 아크릴아미드 겔에서 상대적으로 빠른 이동속도를 보였다. CRP와 cyclic nucleotide 존재하에서 DNA의 구조 변화를 알아보기 위하여 6가지 준비된 DNA조각들을 사용하여 몰 고리화 인자(molar cyclization factor)[13]를 측정하였다. 이들 자료를 사용하여 nonlinear regression analysis를 통하여 cGMP 존재하에서 돌연변이 CRP는 DNA bending을 형성하지 않으나 CAMP 존재하에서 나선 꼬임과 같은 DNA 구조 변화없이 DNA bending을 형성한다.

• 생명과학회지
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• 제13권5호
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• pp.751-755
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• 2003

Cyclic AMP receptor proteins(CRP) activate many genes in Escherichia coli by binding of cAMP with not fully known mechanism. CRP existed as apo-CRP in the absence of cAMP, $CRP;(cAMP)_2$$_2$ at low(micromolar) cAMP concentration, or $CRP;(cAMP)_4$ at high(millimolar) concentration of cAMP. This study is designed to measure the thermal stability of S83G CRP, which substituted glycine for serine at amino acid 83 position, with CD spectrapolarimeter at 222nm by the constant elevation of temperature from $20^{\circ]C\; to\; 90^{\circ}C\; at\; 1^{\circ}C/min$. The non-linear regression analysis showed that melting temperatures were 68.4, 72.0, and $82.3^{\circ}C$ for no cAMP, 0.1mM cAMP, and 5mM cAMP, respectively. Result showed the strong thermal stability of CRP by binding of additional cAMP molecules to region between the hinge region and helix-turn-helix(HTH) motif at 5mM cAMP concentration.

• 생명과학회지
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• 제13권5호
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• pp.746-750
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• 2003

Lactose operon contains two CRP binding sites at promoter(CRP1 site) and operator(CRP2 site) regions at lac operon. CRP protein can bind to both sites with the different binding affinity. CRP1 site, major CRP binding site, acts the transcription activation with the fully unknown mechanism by binding of CRP. In this study, the binding affinities of CRP1 site and CRP2 site were measured with the fluorescein-labeled oligomers, which contain CRP1 site and the three different spacing sequences between GTGA and TCAC at CRP2 site. Results showed that CRP:cAMP complex bound to CRP1 site 3 times more strongly than CRP2 site and the base spacing between GTGA and TCAC was not the only factor to affect the binding affinity of CRP to CRP2 site.

• 통합자연과학논문집
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• 제4권3호
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• pp.222-226
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• 2011

Cyclic AMP receptor protein(CRP) is involved in the activation of many genes corresponding to catabolite enzymes in Escherichia coli. In this study, mutant CRP(S128A) was used to elucidate the effect of Ser 128 on the cAMP-induced structural change. Based on the protease digestion and thermal analysis, serine 128 in CRP affects the cAMP binding capability and then structural change of CRP protein. In addition, CD spectra in near UV region revealed that S128A CRP retained the sensitive conformation to thermal effect relative to that of wild-type CRP, in spite of identical Tm values in the absence of cAMP.

• 생명과학회지
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• 제14권2호
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• pp.309-314
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• 2004

cAMP수용성 단백질(CRP)은 E. coli의 100가지 이상의 유전자 전자조절에 관계된다. CRP는 dimer로 존재하며 cAMP의 결합으로 활성인 형태로 전환된다. 이중체인 CRP 단백질의 열 안정성과 구조 전이의 연구에 효과적인 온도 기울기 전기영동장치를 이용하여 확인하였다. 본 연구에서 야생형과 S83C CRP 단백질의 melting temperature (Tm)는 산성인 완충용액[89.8 mM Glycine, 24mM Boric acid (pH 5.8)]에서 57$\pm$1(야생형 CRP)과 55$\pm$1$^{\circ}C$ (S83G CRP)였다. 그리고 온도에 따른 CRP 단백질의 구조변화도 protease digestion과 CD spectropolarimeter을 이용하여 확인하였다.

• Molecules and Cells
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• 제26권1호
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• pp.61-66
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• 2008

Cyclic AMP receptor protein (CRP) is allosterically activated by cAMP and functions as a global transcription regulator in enteric bacteria. Structural information on CRP in the absence of cAMP (apo-CRP) is essential to fully understand its allosteric behavior. In this study we demonstrated interdomain interactions in apo-CRP, using a comparative thermodynamic approach to the intact protein and its isolated domains, which were prepared either by limited proteolysis or using recombinant DNA. Thermal denaturation of the intact apo-CRP, monitored by differential scanning calorimetry, revealed an apparently single cooperative transition with a slight asymmetry. Combined with circular dichroism and fluorescence analysis, the thermal denaturation of apo-CRP could be interpreted as a coupled process involving two individual transitions, each attributable to a structural domain. When isolated individually, both of the domains exhibited significantly altered thermal behavior, thus pointing to the existence of non-covalent interdomain interactions in the intact apo-CRP. These observations suggest that the allosteric conformational change of CRP upon binding to cAMP is achieved by perturbing or modifying pre-existing interdomain interactions. They also underline the effectiveness of a comparative approach using calorimetric and structural probes for studying the thermodynamics of a protein.

• 생명과학회지
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• 제10권3호
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• pp.279-285
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• 2000

cAMP 수용성 단백질인 CRP는 Escherichia coli에서 대사와 관련된 유전자의 전사를 조절한다. 본 연구는 야생형과 돌연변이 CRP 단백질의 열적 안정성과 온도에 따른 단백질의 구조변화를 관찰하기 위 하여 proteolytic digestion, UV spectrophotometer, CD spectrapolarimeter 등의 방법을 사용하였다. cAMP가 없을 때에는 야생형, S83G, S128A CRP가 열적 안정성에서 큰 차이를 보이지 않았지만, cAMP가 존재할 때 야생형 CRP가 다른 돌연변이 CRP보다 열적으로 더욱 안정함을 보였다. 그리고 protease digestion 실험을 통하여 높은 온도에서 cAMP의 존재와 무관하게 돌연변이 CRP에서 단백질 의 변성으로 인한 절단된 단백질띠를 관찰할 수 있었다. 그리고 55$^{\circ}C$에서 측정한 CD 스펙트럼에서 단백 질의 2차 구조인 $\alpha$-helix 구조가 부분적으로 파괴되었음이 관찰되었다.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1392-1396
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• 2005

Proteolytic digestion and CD measurement of wild-type and mutant cyclic AMP receptor proteins (CRPs) were performed either in the presence or absence of cyclic nucleotide. Results indicated that transition of a structural change to the hinge region by the binding of cAMP to the anti site was required for the binding of cAMP to the syn site near the hinge region and, although the occupancy of cAMP in the anti site increased the protein stability, CRP adopted more a stable conformation by the binding of cAMP to the syn site.

• 한국생물물리학회:학술대회논문집
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• 한국생물물리학회 1996년도 정기총회 및 학술발표회
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• pp.19-19
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• 1996

Cyclic AMP receptor protein (CRP) from E. Coli plays a key role in regulation of the expression of more than 20 genes of the bacterium. CRP binds in the presence of cAMP to a specific target site near the promoter of each gene under its regulation. CRP is a dimer (Mr～47,000) of two identical subunits. There are two binding domains in the CRP monomer, one for the binding of the cAMP and the other for the binding of specific DNA sequences. (omitted)

• 한국자기공명학회논문지
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• 제1권1호
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• pp.31-44
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• 1997

The effect of the binding of CRP to the symmetrically synthetic 22, 28, and 30 bp lac promoter was investigated by 1H NMR. The binding of cAMP*CRP to the 22 bp DNA did not bring about any changes in the chemical shift values, but did cause selective line broadening of imino proton resonances of specific base pairs. However, The binding of cAMP*CRP to the 28 and 30 bp DNA brought about large changes on the imino proton resonances that seems to be induced by DNA bending. We studied also the role of cAMP as an activator of DNA/CRP complex formation by gel mobility shift assay. Gel mobility shift assay revealed that the cAMP*CRP complex was not able to bind to the 22 bp DNA fragment, but was able to bind to the 28 bp DNA fragment of lac promoter region.