KOSEN 한인과학기술자네트워크

>단백질 conformation의 미세한 변화를 감지하는데 쓰이는 기기(?)라고 알고 있습니다. > >논문에서 CD spectrum을 보니깐 x축은 파장, y축은 residue ellipticity이던데, 이걸 어떻게 해석하는건지요? ================================================ 첨부해 드린 파일은 “Theory for circular dichroism measurements“로서 측정법, 해석법등이 수록되어 있습니다. 참고하시기 바랍니다. CD의 해석법에 관련된 내용입니다. 저도 CD를 찍어봤는데 도무지 해석이 어떻게 되는지 몰랐습니다. 이 내용 읽고도 정확히 알지는 못하겠는데.. Circular dichroism circular dichroism, or CD, is defined as the differential absorption of left and right hand circularly polarized light. At a given wavelength: ΔA = (AL - AR) ΔA is the difference between absorbance of left circularly polarized and right circularly polarized light (this is what is usually measured). it can also be expressed as: ΔA = (εL - εR) C l where C is the molar concentration and l is path length and εL and εR are the molar extinction coefficients for RCP and LCP light then Δε = (εL - εR) - is the molar circular dichroism this is what is usually meant by the circular dichroism of the substance Although ΔA is usually measured, for historical reasons most measurements are reported in degrees of ellipticity. The molar ellipticity is: [θ] = 3298Δε In general, this phenomenon will be exhibited in absorption bands of any optically active molecule. As a consequence, circular dichroism is exhibited by biological molecules, because of the dextrorotary (e.g. some sugars) and levulorotary (e.g. some amino acids) molecules they contain. Noteworthy as well is that secondary structure will also impart a distinct CD to their respective molecules. Therefore, the alpha helix of proteins and the double helix of nucleic acids have CD spectral signatures representative of their structures. CD is closely related to the optical rotary dispersion (ORD) technique, and is generally considered to be more advanced. CD is measured in or near the absorption bands of the molecule of interest, while ORD can be measured far from these bands. In principle these two spectral measurements can be interconverted through an integral transform, if all the absorptions are included in the measurements. The ultraviolet CD spectrum of proteins can predict important characteristics of their secondary structure. CD spectra can be readily used to estimate the fraction of a molecule that is in the alpha-helix conformation, the beta-sheet conformation, the beta-turn conformation, or some other (random) conformation. These fractional assignments place important constraints on the possible secondary conformations that the protein can be in. CD can not, in general, say where the alpha helices that are detected are located within the molecule or even completely predict how many there are. Despite this, CD is a valuable tool, especially for showing changes in conformation. It can, for instance, be used to study how the secondary structure of a molecule changes as a function of temperature or of the concentration of denaturing agents. In this way it can reveal important thermodynamic information about the molecule that can not otherwise be easily obtained. Anyone attempting to study a protein will find CD a valuable tool for verifying that the protein is in its native conformation before undertaking extensive and/or expensive experiments with it. Also, there are a number of other uses for CD spectroscopy in protein chemistry not related to alpha-helix fraction estimation. CD spectroscopy is usually used to study proteins in solution, and thus it complements methods that study the solid state. This is also a limitation, in that many proteins are embedded in membranes in their native state, and solutions containing membrane structures are often strongly scattering. CD is sometimes measured in thin films. CD has also been studied in carbohydrates, but with limited success due to the experimental difficulties associated with measurement of CD spectra in the vacuum ultraviolet (VUV) region of the spectrum (100-200 nm), where the corresponding CD bands of unsubstituted carbohydrates lie. Substituted carbohydrates with bands above the VUV region have been successfully measured. Measurement of CD is also complicated by the fact that typical aqueous buffer systems often absorb in the range where structural features exhibit differential absorption of circularly polarized light. Phosphate, Sulfate, carbonate, and acetate buffers are generally incompatible with CD unless made extremely dilute. Borate and ammonium salts are often used to establish the appropriate pH range for CD experiments. Some experimenters have substituted fluoride for chloride ion because fluoride absorbs less in the far UV, and some have worked in pure water. Another, almost universal, technique is to minimize solvent absorption by using shorter path length cells when working in the far UV, 0.1 mm path lengths are not uncommon in this work. It may be of interest to note that the protein CD spectra used in secondary structure estimation are related to the π to π* orbital absorptions of the amide bonds linking the amino acids. These absorption bands lie partly in the so-called vacuum ultraviolet (wavelengths less than about 200 nm). The wavelength region of interest is actually inaccessible in air because of the strong absorption of light by oxygen at these wavelengths. In practice these spectra are measured not in vacuum but in an oxygen-free instrument (filled with pure nitrogen gas). Once oxygen has been eliminated, perhaps the second most important technical factor in working below 200 nm is to design rest of the optical system to have low losses in this region. Critical in this regard is the use of aluminized mirrors whose coatings have been optimized for low loss in this region of the spectrum. The usual light source in these instruments is a high pressure, short-arc xenon lamp. Ordinary xenon arc lamps are unsuitable for use in the low UV. Instead specially constructed lamps with envelopes made from high-purity synthetic fused silica must be used. At the quantum mechanical level, the information content of circular dichroism and optical rotation are identical.

간단하게만 설명하겠습니다. 시료에 선편광된 빛을 입사하면 원편광 이색성(circualr dichriosm)을 갖는 물질로 인해 빛의 편광이 타원형으로 되는 경우가 있습니다. ellipticity라고 하는 것은 원형에서 얼마나 찌그러져있는가를 평가하는 지수입니다. ellipsometer에서도 같은 의미로 사용하고 있습니다.

>단백질 conformation의 미세한 변화를 감지하는데 쓰이는 기기(?)라고 알고 있습니다. > >논문에서 CD spectrum을 보니깐 x축은 파장, y축은 residue ellipticity이던데, 이걸 어떻게 해석하는건지요? CD가 생물학에도 쓰이는군요. 물리학적 관점에서 보면 CD 는 레이저에서 나오는 linearly plane polarized light (선형편광)을 시료의 표면에 입사시켜 반사되어 나오는 빛의 편광 상태로 부터 얻을 수 있는 물리양입니다. Linearly plane polarized light은 공기나 진공중에서 Right-circularly polarized (RCP) light 과 Left-circularly polarized (LCP) light 으로 decompose 할 수 있으며, 이때 RCP와 LCP의 위상과 진폭 (또는 세기) 는 동일합니다. 이 둘은 진행해 오면서 회전하는 방향이 서로 반대입니다. 이 둘을 Vector Sum 하면 Linearly plane polarized light이 됨을 쉽게 알 수 있읍니다. 이들 RCP 와 LCP 는 이등방성 (anisotropy) 성질이 있는 시료에 입사되면 흡수율이 서로 다르게 되고 위상에도 차이가 발생할 수 있읍니다. 즉 어느 한쪽 원형편광의 크기가 (반지름) 작아집니다. 따라서 반사되어 나올때, 이들 두개의 vector sum은 더 이상 Linearly plane polarized light 이 아니고, elliptically polarized light이 됩니다. 위상차는 타원편광의 장축의 회전으로 나타나고, 흡수율의 차이는 타원편광의 단축과 장축의 ratio로 표시됩니다 (원형편과의 경우 이 ratio는 1 이 됩니다). 즉, RCP 와 LCP가 시료안의 전자분포와 상호작용으로 인하여 각각의 위상이나 세기성분이 달라져 elliptically polarized light (EPL) 이 됩니다. 서로 달라진 위상으로 인하여 EPL의 장축이 회전하게 되며, 서로 달라진 세기 성분으로 인하여 찌그러진 elliptical 형태로 변합니다. CD는 얼마나 RCP와 LCP의 세기성분이 시료의 이등방성에 의해 달라진가를 나타내는 척도가 됩니다. 이런 현상은 빛과 물질안의 전자들과의 상호작용에 의해 발생하는데 이는 전자가 자기적, 전기적 특성에 민감하게 반응하여 대칭성이 깨지기 때문이거난 자체적으로 이등방성 성질이 있기 때문입니다. 이를 통하여 시료의 내부 전자구조적 특성을파악할 수 있읍니다. 레이저 대신에 흰광원 (모든 파장 포함한 빛) 을 사용하고 편광자를 사용하면, 적외선, 가시광선, 자외선 영역을 스캔하여, x축을 파장 y축을 ellipticity (단축과 장축의 비) 로 표현할 수 있읍니다.

저도 매우 궁금해 하는 질문입니다. 물리화학적 background가 부족하여 CD spec.의 원리와 개념, 용어들이 익숙하지 않아서요... 전문가님이 나타나셔서 시원해 해결해 주시길 기대할께요...