Thermal Stability of Heterotrimeric pMHC Proteins as Determined by Circular Dichroism Spectroscopy

Jia Li
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T cell receptor (TCR) recognition of foreign peptide fragments, presented by peptide major histocompatibility complex (pMHC), governs T-cell mediated protection against pathogens and cancer. Many factors govern T-cell sensitivity, including the affinity of the TCR-pMHC interaction and the stability of pMHC on the surface of antigen presenting cells. These factors are particularly relevant for the peptide vaccination field, in which more stable pMHC interactions could enable more effective protection against disease. Here, we discuss a method for the determination of pMHC stability that we have used to investigate HIV immune escape, T-cell sensitivity to cancer antigens and mechanisms leading to autoimmunity.

Keywords: Peptide-MHC stability(肽MHC稳定性), Circular dichroism(圆二色性), Thermal stability(热稳定性), T-cells(T细胞), Peptide vaccines(肽疫苗), Recombinant protein(重组蛋白), Protein folding(蛋白质折叠)


The ability of CD8+ T-cells to respond to foreign invaders or dysregulated self is dependent on stable pMHC class I (pMHCI) presentation at the cell surface. Structurally, MHCI molecules form a peptide binding groove formed of two parallel α helices with a floor of β sheets at the interface between the α1 and α2 domains (Latron et al., 1992). The peptide binding groove has primary peptide binding pockets (B and F) that tightly interact with specific amino acids towards the N- and C-terminals of bound peptides. Although these pockets can accommodate a range of amino acids, they exhibit preferences for certain side chains that have been characterized using structural and biochemical approaches (Parker et al., 1992). This information has been used to generate so called ‘heteroclitic’ peptides in which natural peptides that have poor MHC-anchors can be modified with amino acids that bind optimally to MHC for vaccination (Cole et al., 2010). Moreover, pMHC stability has been linked to HIV immune escape (Bronke et al., 2013) and the selection of autoreactive T-cell clones (Yin et al., 2011). Thus, understanding the mechanisms that control pMHC stability is important for therapeutic design and understanding complex human diseases. Here, we developed a protocol to accurately determine pMHC stability using circular dichroism spectroscopy. We have used this technique, together with structural, biophysical and cellular experiments, to provide new insight into the molecular factors that determined T-cell antigen recognition in the context of a range of human diseases (Kløverpris et al., 2015; Knight et al., 2015; Motozono et al., 2015; Cole et al., 2016; Jones et al., 2016; Cole et al., 2017).

Materials and Reagents

  1. Cellulose nitrate 0.45 µm filter papers (Sartorius, catalog number: 11306-47-N )
  2. 1.2 µm glass microfiber filters (GE Healthcare, catalog number: 1822-070 )
  3. 10 ml plastic syringes, Luer slip BD Plastipak (BD, catalog number: 302188 )
  4. 25 G needle (BD, catalog number: 300600 )
  5. 1 ml plastic syringes, Luer slip BD Plastipak (BD, catalog number: 303172 )
  6. 1.5 ml microcentrifuge tubes
  7. Amicon centrifugal concentrating tubes 4 ml MWCO 10 kDa (Merck, catalog number: UFC801096 )
  8. Phosphate buffered saline (PBS) made up from Dulbecco A tablets (137 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.3) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: BR0014G )
  9. Ultrapure water (> 18 MΩ cm) for buffer preparations
  10. Bolt® Bis-Tris 4-12% precast gels (Thermo Fisher Scientific, InvitrogenTM, catalog number: NW04120BOX )
  11. BlUeye prestained protein markers (Geneflow, catalog number: S6-0024 )
  12. Quick Coomassie Stain (Generon, catalog number: GEN-QC-STAIN-3L )
  13. Ethanol absolute (200 Proof)
  14. Nitric acid (HNO3), 70%


  1. Reusable bottle top filtration device (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: DS0320-5045 )
  2. Vacuum pump such as KNF Neuberger Vaccum Pump (KNF Neuberger, catalog number: 049268/018121 )
  3. 500 ml clear Duran bottles (Duran, catalog number: GL 45 )
  4. Liquid Chromatography system, with a 2 ml injection loop, and a fraction collector; we use the ÄKTA pure 25 L with an F9-R fraction collector (GE Healthcare, model: ÄKTA pure 25 L )
  5. Size exclusion chromatography column; we use a Superdex 200 Increase 10/300 GL column, bed volume 24 ml (GE Healthcare, catalog number: 28990944 )
  6. Benchtop refrigerated micro-centrifuge capable of 14,000 x g (e.g., Eppendorf, model: 5418 R )
  7. Far-UV spectrophotometer with a bandwidth < 1.8 nm and quartz cuvettes. We use a single-beam Beckman DU 800 instrument with microcells that allow measurements with volumes of 50 to 100 μl (Beckman Coulter, model: DU® 800 )
  8. A far-UV circular dichroism (CD) spectrometer with a temperature controlled cell holder. We use an AVIV Model 215 instrument (Aviv Biomedical, model: Aviv Model 215 ) with a single cell Peltier controlled cell holder, or make use of the Module B end-station spectrophotometer at the B23 Synchrotron Radiation CD (Diamond Light Source, model: B23 ) Beamline at the Diamond Light Source (Jávorfi et al., 2010; Cole et al., 2016). Alternative instruments are available from Applied Photophysics Ltd (Leatherhead, U.K.), JASCO Inc. (Easton, MD), and Olis Inc. (Bogart, GA)
  9. Strain free sealable quartz cuvettes of appropriate path length fitting the CD instrument’s cell holder. We use Teflon stoppered Hellma Suprasil cells of various thickness, mostly 0.1-cm
  10. A well-ventilated chemical fume hood should be accessible for cleaning cuvettes with HNO3


  1. An analysis software that can import the CD data files and allows curve fitting to a user defined set of equations is required. We use Origin version 7.5 and later (OriginLab Corp., Northampton, MA), but many other programs. e.g., Igor Pro, MATLAB, Micromath Scientist, SigmaPlot, etc. will work


  1. Purification and buffer exchange of pMHC protein
    HLA-A2 and human β2-microglobulin (β2m) sequences were generated as described and cloned into separate pGMT7 expression plasmids (Cole et al., 2007). Complexes of HLA-A2, β2m and peptides are refolded and purified as described (Bulek et al., 2012, MacLachlan et al., 2017) with the essential steps summarized below. For CD analysis, proteins should be of high purity and the preparation should lack the presence of denatured or incorrectly folded protein. In order to achieve this, the proteins are purified on the day of, or the day before analysis, and stored on ice to avoid any freeze thaw cycles.
    1. Prepare a 500 ml solution of PBS using 1 tablet per 100 ml of ultra-pure water. With the bottle top filtration device and vacuum pump, filter this solution through a cellulose nitrate 0.45 µm filter paper, with a 1.2 µm glass microfiber filter as a pre-filter, into a 500 ml Duran bottle. Prepare another 500 ml Duran bottle of ultra-pure water.
    2. Connect the gel filtration column to the ÄKTA FPLC system and wash with sterile ultra-pure water followed by equilibrating the system with PBS. Wash the 2 ml injection loop with ultra-pure water and equilibrate with PBS using a 10 ml syringe, taking special care to avoid the occurrence of any air gaps that can be transferred to the column.
    3. Using a 25 G needle transfer the protein solution, of no more than 1 ml, into the injection loop using a 1 ml syringe avoiding any air bubbles.
    4. Run the column at 0.5 ml/min and monitor elution by UV absorbance at 280 nm (Note 1). Collect 1-ml fractions (the number of fractions depends on the elution profile, but we try and take 2-4 fractions from the center of the peak) and analyse by SDS-PAGE under reducing conditions to check for purity of the pMHC sample. We use 4-12% gradient Bolt® Bis-Tris gels, BLUeye prestained protein ladder, NuPAGE MES running buffer, and Quick Coomassie Stain.
    5. Run an SDS-PAGE. Upon SDS-PAGE, pMHC dissociates into its constituent parts. Bands of HLA-A2 and β2m run at positions corresponding to ~32 and ~12 kDa, respectively. The peptide (~1 kDa) will run out of the gel (Figure 1).
    6. Combine only the purest two or three fractions and concentrate using an Amicon centrifugal filtration tube with an MWCO of 10 kDa. This provides protein of adequate purity and concentration for CD analysis.
    7. Directly before CD measurements, spin the samples at 14,000 x g for 20 min at 4 °C usually without resulting in any visible pellet.

      Figure 1. SDS-PAGE analysis of pMHCI. Approximately 20 µg of pMHCI (right lane) was loaded onto a 4-12% gradient Bolt® Bis-Tris gel with 3 µl BLUeye prestained protein ladder (left lane) and run at 200 V for 23 min in NuPAGE MES running buffer. The gel was stained for 5 min in Quick Coomassie Stain and destained with ddH2O. The pMHCI α chain runs as a separate band at ~32 kDa and β2m at ~12 kDa.

  2. Determination of parameters
    1. Record UV spectra of the supernatant (step A7) from 360 to 230 nm in a 1-cm quartz cuvette with the buffer baseline subtracted. Within the 360 to 320 nm range, the absorbance should be essentially zero; any slope is due to light scattering indicating the presence of large aggregates.
    2. Calculate the 280 nm absorption coefficient based on the amino acid composition (Pace et al., 1995) using the ProtParam website at http://web.expasy.org/protparam/.
      Due to the high amount of the aromatic residues Tyr (5.3%) and Trp (3.2%) within the HLA-A2 and β2-microglobulin sequences combined (see Supplements 1 and 2), a good estimate for (absorption coefficient) can be expected. As HLA-A2 and β2-microglobulin have two and one intra-chain disulphide bonds, respectively, the cystine contribution to the absorption is included (ProtParam option: ‘assuming all pairs of Cys residues form cystines’).
    3. Calculate protein concentration from the measured optical density at 280 nm as:

    4. Use ProtParam to calculate the molecular mass based on the average isotopic masses of amino acids. All three sequences of the HLA-A2/β2m/peptide complex are entered into the program input field together. To account for the two missing peptide bonds due to the three instead of single chain complex as seen by the program, two times the molecular mass of water (2 x 18.02) is added to the resulting mass Mr.
    5. Calculate the mean residue weight (MRW) required for the normalization of the CD data from this mass by dividing the number of peptide bonds:

      where, n is the number of amino acids within the trimer.

  3. Data acquisition
    1. Based on the concentration of the stock solution determined as described above, dilute an aliquot with buffer to result in ca. 600 μl of sample with c ~0.15 mg/ml (~3 μM), and record a further absorption spectrum to give the final protein concentration (Note 2).
    2. Following the usual start-up procedure of the CD instrument (30 min flushing with N2 followed by a further 30 min period for stabilizing the xenon arc lamp), record a spectrum of the buffer (step A1) in a clean 0.1-cm stoppered quartz cell from 260 to ~195 nm with the cell holder equilibrated to 4 °C. This spectrum is termed the baseline. We usually record spectra using a 1 nm bandwidth in 0.2 nm intervals with 3 or 4 sec measuring time per data point resulting in ca. 20 to 25 min per spectrum (Note 3).
    3. Record a further buffer baseline in the Kinetic Mode at a constant wavelength of 218 nm for 1 min in 2 sec intervals.
    4. Clean the cell with water by rinsing, followed by rinsing with absolute ethanol and dried under a stream of nitrogen.
    5. Fill the same cell completely with the protein solution (~550 μl), closed tightly with a Teflon stopper avoiding trapping of an air bubble, and place into the CD cell holder in the same orientation as used for recording the baseline.
    6. After ~2 min to allow for temperature equilibration, record the protein spectrum using the same instrument settings as used for the baseline. Data are reliable if the photomultiplier dynode voltage is < 500 V (Note 4).
    7. For temperature denaturation, set the wavelength to 218 nm (minimum in CD spectrum). Record changes in ellipticities in 0.5 °C intervals from 4 to ~80 °C using the following parameters: data collection 12 sec/point; temperature equilibration 12 sec; temperature dead band 0.3 °C; instrument heating rate 4 °C/min. During the intervals between data collection, the sample is shielded from the light beam by a closed slit. These settings result in an average heating rate of ~36 °C/h (Note 5).
    8. At elevated temperatures the HLA-A2/β2m/peptide complexes form large, visible aggregates (precipitation) that result in sharp changes of the melting profile and an increase of the dynode voltage indicating increased scattering. At this point, the measurement can be stopped.
    9. Clean protein precipitate containing cuvettes thoroughly rinsing with water followed by filling with 70% HNO3 in a fume hood, incubation for 2 h to overnight, rinsing with water, ethanol, and drying with a stream of N2.

Data analysis

  1. Import CD data files into the OriginLab software (Note 6).
  2. For spectra analysis, subtract the buffer baseline. Normalise Θ values recorded in mdeg to molar ellipticities [Θ]MRW in the classical units (deg cm2 dmol-1) according:

    with the concentration c in mg/ml and cell path length d in mm (Note 7).
  3. Subtract the mean of Θ recorded at 218 nm in the Kinetic Mode (step C3) from the thermal melt ellipticities, and normalize to [Θ]MRW218nm according to Eq. (3).
  4. Analysis of the thermal stability assumes a 2-state mechanism in which three unfolded polypeptide chains U combine to a native complex N:

    where, the equilibrium constant K is:

    with a total chain concentration c0 = cU +3CN and a degree of conversion to the native state F = 3 CN/c0 (see e.g., Engel et al., 1977; Marky and Breslauer, 1987). The standard Gibbs free energy can be written as:

    ΔG0 = -RT ln K = ΔH0 - TΔS0 (6)

    in which R is the gas constant (8.3145 J K-1 mol-1), ΔH0 the standard enthalpy, and ΔS0 the standard entropy. From Eqs. (5) and (6) it follows for the midpoint of transition, where F = 0.5 and T equals the melting temperature Tm, at which half of the proteins are in the folded and unfolded state, respectively, that

    This means that in contrast to a single chain polypeptide the melting temperature is concentration dependent. Tm and ΔH0 can be obtained by fitting the entire transition curve to the van’t Hoff equation:

    obtained by solving Eq. (7) for ΔS0 and substituting for ΔS0 in Eq. (6).
    The molar ellipticities for the native and unfolded state [Θ]n and [Θ]u show an additional temperature dependence which in a first approximation can be assumed as linear:

    [Θ]n = [Θ]n0 + bn T and [Θ]u = [Θ]u0 + bu T (9)

    where, [Θ]n0 and [Θ]u0 correspond to the [Θ] intercept at T = 0, and bn and bu describe the slopes of the native and unfolded states, respectively. The entire transition curve is thus described by:

    [Θ] = F ([Θ]n0 + bn T - [Θ]u0 - bu T) + [Θ]u0 + bu T (10)

    which is used for fitting with the Levenberg-Marquardt algorithm implemented in OriginLab. The essential parts of the fitting script are included as Supplement 4.
  5. As the HLA-A2/β2m/peptide complexes tend to precipitate within the transition region (step C8), we assume a common molar ellipticity [Θ]u0 = -4,500 deg cm2 dmol-1 and slope bu = 0. For many temperature denatured proteins and model peptides [Θ]u0 values in the -4,000 to -5,000 deg cm2 dmol-1 range are observed (Venyaminov et al., 1993) (Notes 8 and 9).
  6. Representative data
    The general procedure to analyse the stability of a trimeric pMHC complex is illustrated using HLA-A2/β2m/ILAKFLHWL as an example (Figure 2).This complex has been analysed in detail (Cole et al., 2017) and the structure has been solved in complex with a TCR (Protein Data Bank entry 5MEN).

    Figure 2. Thermal stability of HLA A2/β2m/ILKA. A. Protein concentration is determined by UV absorbance spectroscopy. Wavelength scans from 360 to 230 nm are recorded in a 1-cm quartz cuvette using the same sample and buffer as used in subsequent CD measurements. Concentration is calculated assuming an absorbance coefficient = 2.257 as calculated based on the amino acid composition. B. A far-UV CD spectrum was recorded in a 0.1-cm quartz cuvette at 4 °C and buffer baselines measured in the same cell using the same instrument parameters were subtracted. The signals were normalized to mean-residue-weight (MRW) ellipticities [Θ]MRW acc. Eq. (3). C. Thermal stability was determined by following the ellipticity Θ at 218 nm upon increasing the temperature with measuring 12 sec per data point in ΔT = 0.5 °C intervals, an equilibration time of 12 sec between data points, a temperature dead band of ± 0.3 °C, and using slit closure between measurements resulting in a mean heating rate of 36 °C/h (red, left axis). The sample forms large, visible aggregates indicating precipitation in the 58 to 65 °C range (open circles). Data points below this temperature (filled circles) were fitted acc. Eq. (10) resulting in the dashed curve. Aggregate formation is accompanied by increasing light scattering resulting in a steep increase of the dynode voltage (blue, right axis) followed by a decrease when large particles sink to the bottom of the cuvette.

    1. Absorption coefficient and mean-residue-weight are calculated by putting all three sequences (Supplements 1 to 3) into the input frame of ProtParam (step B2) resulting in:

      Number of amino acids: 386
      Molecular weight: 44820.03
      Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
      Ext. coefficient   101675
      Abs 0.1% (=1 g/L)   2.269, assuming all pairs of Cys residues form cystines  

    2. The measured absorption of the protein solution (Figure 2A) shows = 0.283 resulting with Eq. (1) in a concentration of 0.283/2.269 = 0.125 mg/ml.
    3. MRW is calculated according to Eq. (2) as MRW = (44,820.03 + 2 x 18.02)/(386 - 3) = 117.12.
    4. A CD buffer baseline and spectra of both solutions are recorded according to steps C2 to C6 using a 0.1-cm path length cuvette. Baselines were subtracted, and the spectrum is normalised according to Eq. (3) (Figure 2B). At 195 nm, the dynode voltage was ~500 V. This reflects the high absorbance of the chloride ion included in PBS. The spectrum shows a minimum of [Θ]MRW = -8,950 deg cm2 dmol-1 at 218 nm.
    5. The melting curve is recorded as described in step C7 from 4 to 65 °C, the mean value of the baseline (step C3) is subtracted, and molar residue ellipticities calculated according to Eq. (3) (Figure 2C). Using the OriginLab script given as Supplement 4 fit the data according to Eq. (10). Values in the 59 to 65 °C range that show a pronounced deviation from a homogenous quasi-sigmoidal curve indicating protein aggregation (open dots in Figure 2C) were omitted from the fits. Fitting results in Tm = 54.5 ± 0.1 °C and ΔH0 = -500 ± 20 kJ/mol with a coefficient of determination r2 = 0.988.


  1. Peak fractionation of the pMHC will occur at an elution volume of ~12 ml; it is important to keep only the purest fractions and discard any that contain protein from more than one peak.
  2. To determine thermodynamic parameters, for HLA-A2/β2m/peptide complexes we aim for a concentration of ~0.15 mg/ml as for most peptides studied so far this can be relatively easily achieved and provides for a good signal-to-noise ratio to follow CD transition curves at 218 nm. Due to the concentration dependence of Tm for multi-chain proteins [Eq. (7)], it is important to settle for a narrow range to allow for comparison of data.
  3. A cuvette can be regarded as optically clean if the baseline using PBS or water runs about parallel to a spectrum recorded without a cell in the light beam (‘air baseline’); minima observed in the 200 to 225 nm range usually indicate that protein from previous measurements is absorbed to the quartz surface.
  4. On instruments of other manufacturers, the dynode voltage is called HT (high tension), and the reliable voltage range can differ depending on the type of photomultiplier and electronics.
  5. The heating parameters have been tested to result in the set equilibrium temperatures by measuring with a Pt100 Resistance Temperature Detector inside a 0.1-cm cuvette.
  6. The AVIV instrument software stores data in ASCII format. Some instruments from other manufacturers use propriety formats that require exporting into ASCII for analysis via other programs.
  7. [Θ]MRW can be converted into molar absorption units Δε = εL - εR = [Θ]MRW/3,298, where Δε is the absorption difference for left- and right-handed circularly polarized light.
  8. From the various MHC complexes studied in our lab, HLA-A*0101/β2m/peptide VTEHDTLLY exhibit high stability both with respect to Tm (68.9 °C) and enthalpy (-719 kJ mol-1); precipitation occurred only at T > 72 °C, [Θ]U0 > -5,200 deg cm2 dmol-1 (Jones et al., 2016). Using [Θ]U0 = -4,500 deg cm2 dmol-1 resulted in a r2 > 0.996.
  9. For strict thermodynamic analysis reversibility of the transition [Eq. (4)] is a prime requirement. It has been suggested, however, that even if reversibility is limited analysis is still of value as temperature induced cooperative unfolding is usually a much faster process than the aggregation of unfolded protein (Privalov, 2009). To allow for comparison of data, we use the same heating parameters for all samples (see step C7).


DKC is a Wellcome Trust Research Career Development Fellow (WT095767). AKS is a Wellcome Trust Senior Investigator. This and original work using the described procedures was supported by the UK Biotechnology and Biological Sciences Research Council (BBSRC) (Grant BB/H001085/1). Purchase of the CD instrument was partially funded by BBSRC grant 75/REI18433.


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  20. Yin, Y., Li, Y., Kerzic, M. C., Martin, R. and Mariuzza, R. A. (2011). Structure of a TCR with high affinity for self-antigen reveals basis for escape from negative selection. EMBO J 30(6): 1137-1148.


由肽主要组织相容性复合物(pMHC)提供的外源肽片段的T细胞受体(TCR)识别控制T细胞介导的针对病原体和癌症的保护。 许多因素控制T细胞敏感性,包括TCR-pMHC相互作用的亲和力和pMHC在抗原呈递细胞表面的稳定性。 这些因素对于肽疫苗接种领域尤其重要,其中更稳定的pMHC相互作用可以实现更有效的防止疾病的保护。 在这里,我们讨论一种测定pMHC稳定性的方法,我们已经用来调查HIV免疫逃逸,T细胞对癌症抗原的敏感性和导致自身免疫的机制。
【背景】CD8 + T细胞对外来入侵者或失调自身的反应的能力取决于细胞表面上稳定的pMHC I类(pMHCI)表达。在结构上,MHCI分子在α1和α2结构域之间的界面处形成由两个平行的α螺旋形成的肽结合槽,其具有β片的底部(Latron等人,1992)。肽结合槽具有与结合肽的N-和C-末端紧密相互作用的特异性氨基酸的主要肽结合口袋(B和F)。虽然这些口袋可以适应一系列氨基酸,但它们表现出对使用结构和生物化学方法表征的某些侧链的偏好(Parker等人,1992)。该信息已被用于产生所谓的“异型”肽,其中具有差的MHC锚的天然肽可以用与MHC最佳结合的氨基酸进行修饰(Cole等人,2010 )。此外,pMHC稳定性与HIV免疫逃逸(Bronke等人,2013)有关,并且选择了自身反应性T细胞克隆(Yin等人,2011) 。因此,了解控制pMHC稳定性的机制对于治疗设计和了解复杂的人类疾病是重要的。在这里,我们开发了一个使用圆二色光谱法准确测定pMHC稳定性的方案。我们已经将这种技术与结构,生物物理学和细胞学实验结合起来,为在一系列人类疾病的背景下确定T细胞抗原识别的分子因子提供了新的见解(Kløverpris等人 2015年; Knight等人,2015年; Motozono等人,2015年; Cole等人,2016; Jones et al。,2016; Cole等人,2017)。

关键字:肽MHC稳定性, 圆二色性, 热稳定性, T细胞, 肽疫苗, 重组蛋白, 蛋白质折叠


  1. 硝酸纤维素0.45μm滤纸(Sartorius,目录号:11306-47-N)
  2. 1.2微米玻璃微纤维过滤器(GE Healthcare,目录号:1822-070)
  3. 10 ml塑料注射器,Luer滑BD Plastipak(BD,目录号:302188)
  4. 25 G针(BD,目录号:300600)
  5. 1 ml塑料注射器,Luer打印BD Plastipak(BD,目录号:303172)
  6. 1.5 ml微量离心管
  7. Amicon离心浓缩管4ml MWCO 10kDa(Merck,目录号:UFC801096)
  8. 由Dulbecco A片剂(137mM NaCl,3mM KCl,8mM Na 2 HPO 4,1.5mM KH 2)组成的磷酸盐缓冲盐水(PBS) / sub> PO 4,pH 7.3)(Thermo Fisher Scientific,Thermo Scientific TM,目录号:BR0014G)
  9. 用于缓冲液制剂的超纯水(>18MΩcm)
  10. 螺栓 Bis-Tris 4-12%预制凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:NW04120BOX)
  11. BlUeye预染色蛋白标记(Geneflow,目录号:S6-0024)
  12. 快速考马斯染色(Generon,目录号:GEN-QC-STAIN-3L)
  13. 乙醇绝对(200证明)
  14. 硝酸(HNO 3),70%


  1. 可重复使用的瓶顶过滤装置(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:DS0320-5045)
  2. 真空泵如KNF Neuberger Vaccum Pump(KNF Neuberger,目录号:049268/018121)
  3. 500毫升透明杜兰瓶(杜兰,目录号:GL 45)
  4. 液相色谱系统,2ml注射回路和馏分收集器;我们使用ÄKTA纯25 L与F9-R级分收集器(GE Healthcare,型号:ÄKTA纯25 L)
  5. 尺寸排阻色谱柱;我们使用Superdex 200增加10/300 GL柱,床体积24毫升(GE Healthcare,目录号:28990944)
  6. 具有14,000 x g(例如Eppendorf型号:5418 R)的台式冷冻微型离心机
  7. 带有紫外线分光光度计的远紫外分光光度计1.8 nm和石英比色皿。我们使用单光束Beckman DU 800仪器与微电池,允许测量体积为50至100μl(Beckman Coulter,型号:DU 800)
  8. 具有温度控制的电池座的远紫外圆形二色性(CD)光谱仪。我们使用一个单细胞珀耳帖控制电池座的AVIV 215型仪器(Aviv Biomedical,型号:Aviv Model 215),或者使用B23同步辐射CD(Diamond Light Source,Diamond Light Source)型号的模块B终端分光光度计B23)金刚石光源的光束(Jávorfi等人,2010; Cole等人,2016)。替代工具可从Applied Photophysics Ltd(Leatherhead,U.K.),JASCO Inc.(Easton,MD)和Olis Inc.(Bogart,GA)获得
  9. 具有适合路径长度的无菌密封石英比色皿,可安装CD仪器的电池座。我们使用特氟隆阻塞的各种厚度的Hellma Suprasil细胞,大多是0.1厘米的
  10. 应使用通风良好的化学通风柜进行清洗,使用HNO 3


  1. 需要一个可以导入CD数据文件并允许曲线拟合到用户定义的方程组的分析软件。我们使用Origin版本7.5及更高版本(OriginLab Corp.,Northampton,MA),但还有许多其他程序。例如,Igor Pro,MATLAB,Micromath Scientist,SigmaPlot,等。将工作


  1. pMHC蛋白的纯化和缓冲液交换
    如所述产生HLA-A2和人β2-微球蛋白(β2m)序列并克隆到单独的pGMT7表达质粒中(Cole等人,2007)。 HLA-A2,β2m和肽的复合物如(Bulek等人,2012,MacLachlan等人,2017)所述重新折叠和纯化,其基本步骤如下。对于CD分析,蛋白质应该具有高纯度,并且制剂应该缺乏变性或错误折叠的蛋白质的存在。为了达到此目的,蛋白质在分析前一天或分析前一天进行纯化,并储存在冰上以避免任何冻融循环。
    1. 使用1ml / 100ml超纯水制备500ml PBS溶液。使用瓶顶过滤装置和真空泵,通过硝酸纤维素0.45μm滤纸将该溶液过滤,将1.2μm玻璃微纤维过滤器作为预过滤器,倒入500ml Duran瓶中。准备另外500毫升杜兰瓶超纯水。
    2. 将凝胶过滤柱连接到ÄKTAFPLC系统,用无菌超纯水洗涤,然后用PBS平衡系统。用超纯水清洗2ml注射回路,并使用10ml注射器与PBS平衡,特别注意避免发生可转移到柱中的任何气隙。
    3. 使用25 G针将不超过1毫升的蛋白质溶液使用1毫升注射器将注射液转移到注射回路中,以避免任何气泡。
    4. 以0.5ml / min运行柱,并通过280nm处的UV吸光度监测洗脱(注1)。收集1ml级分(分数取决于洗脱曲线,但是我们尝试从峰的中心取2-4个级分),并通过SDS-PAGE在还原条件下分析以检查pMHC样品的纯度。我们使用4-12%梯度Bolt Bis-Tris凝胶,BLUeye预染蛋白梯,NuPAGE MES运行缓冲液和Quick Coomassie Stain。
    5. 运行SDS-PAGE。在SDS-PAGE上,pMHC解离成其组成部分。 HLA-A2和β2m的带分别在对应于〜32和〜12kDa的位置运行。肽(〜1 kDa)将从凝胶中流出(图1)
    6. 仅使用最纯净的两个或三个级分组合并使用具有10kDa的MWCO的Amicon离心过滤管进行浓缩。这为CD分析提供了足够的纯度和浓度的蛋白质
    7. 在CD测量之前,通常在4℃下将样品以14,000 x g旋转20分钟,通常不会产生任何可见的颗粒物。

      图1. pMHCI的SDS-PAGE分析将约20μg的pMHCI(右泳道)加载到4-12%梯度Bolt的Bis-Tris凝胶上,其中3 μlBLUeye预染蛋白梯(左泳道),并在NuPAGE MES运行缓冲液中在200V下运行23分钟。将凝胶在Quick Coomassie Stain中染色5分钟,并用ddH 2 O进行脱色。 pMHCIα链在〜32 kDa和β2m处以〜12 kDa的单独条带运行。

  2. 参数的确定
    1. 在1厘米石英比色皿中记录上清液(步骤A7)的紫外光谱从360至230nm,减去缓冲液基线。在360至320 nm范围内,吸光度应基本为零;任何斜率都是由于光散射,表明存在大的聚集体
    2. 使用ProtParam网站 http://web.expasy.org/protparam/
      由于HLA-A2和β2-微球蛋白序列组合中的芳香残基Tyr(5.3%)和Trp(3.2%)含量高(参见补充1和2 ),对于(吸收系数)。由于HLA-A2和β2-微球蛋白分别具有两个和一个链内二硫键,因此包括胱氨酸对吸收的贡献(ProtParam选项:“假设所有成对的Cys残基形成胱氨酸”)。
    3. 从280nm处测量的光密度计算蛋白质浓度 as:

    4. 使用ProtParam计算基于氨基酸的平均同位素质量的分子质量。 HLA-A2 /β2m/肽复合物的所有三个序列一起输入到程序输入区。为了解释由于程序所见的三个而不是单链复合物的两个缺失的肽键,将两倍于水的分子量(2×18.02)加入到所得质量M r / 。
    5. 计算通过除以肽键的数量从该质量标准化CD数据所需的平均残留量(MRW):


  3. 数据采集
    1. 基于如上所述确定的储备溶液的浓度,用缓冲液稀释等分试样以导致ca。 600μlc〜0.15 mg / ml(〜3μM)的样品,并记录进一步的吸收光谱,得到最终的蛋白质浓度(注2)。
    2. 按照CD仪器的常规启动程序(30分钟用N 2冲洗,接着再进行30分钟时间以稳定氙弧灯),记录缓冲液的光谱(步骤A1)在260至195nm的干净的0.1-cm塞电石英池中,电池座平衡至4℃。该谱称为基线。我们通常使用1 nm带宽以0.2 nm的间隔记录光谱,每个数据点的测量时间为3或4秒,导致ca 。每光谱20至25分钟(注3)
    3. 在动态模式下,以恒定波长的218 nm,以2秒的间隔记录1分钟的进一步缓冲基线
    4. 用水冲洗清洁细胞,然后用无水乙醇冲洗,并在氮气流下干燥
    5. 用蛋白质溶液(〜550μl)完全填充相同的细胞,用Teflon塞子紧密封闭,避免捕获气泡,并以与记录基线相同的方向放入CD细胞座。
    6. 在〜2分钟后允许温度平衡,使用与基线相同的仪器设置记录蛋白质谱。如果光电倍增管的电压为< 500 V(注4)。
    7. 对于温度变性,将波长设置为218 nm(CD光谱最小)。使用以下参数在0.5°C间隔内记录椭圆度的变化,从4到〜80°C:数据采集12秒/点;温度平衡12秒;温度死区0.3°C;仪器加热速率为4°C / min。在数据采集间隔期间,样品通过闭合的狭缝与光束屏蔽。这些设置导致平均加热速度为〜36°C / h(注5)
    8. 在升高的温度下,HLA-A2 /β2m/肽复合物形成大的可见聚集体(沉淀),导致熔化曲线的急剧变化,并且增加了表示增加的散射的倍增极电压。此时可以停止测量。
    9. 清洁含有比色皿的蛋白质沉淀物,用水彻底冲洗,然后在通风橱中填充70%HNO 3,孵育2小时至过夜,用水,乙醇冲洗,并用N sub> 2 。


  1. 将CD数据文件导入到OriginLab软件中(注6)。
  2. 对于光谱分析,减去缓冲液基线。将经典单位(deg cm 2 dmol)中记录的mdeg至摩尔椭圆度Θ[Θ] 归一化Θ -1 )根据:

    浓度(mg / ml),细胞路径长度(mm)(mm)(注7)。
  3. 在动力学模式(步骤C3)中从热熔体椭圆度减去在218nm处记录的Θ的平均值,并归一化为[Θ] > 218nm 根据等式(3)
  4. 热稳定性的分析假设其中三个未折叠的多肽链U结合到天然复合物N的2-状态机制:


    具有总链集合sub> +3 CN ,并且转换为本机状态的程度 = 3 > 0 (参见 eg ,Engel等人,1977; Marky和Breslauer,1987)。标准吉布斯自由能可写为:

    (3)ΔG 0 = -RT ln K =ΔH 0 - TΔS 0 (6)
    其中R是气体常数(8.3145 JK -1 mol -1 ),ΔH标准焓和标准熵的标准焓值,和/或标准熵。从等式(5)和(6)之后,对于转变的中点,其中,f'= 0.5和等于解链温度T m > ,其中一半的蛋白质分别处于折叠和展开状态,即

    这意味着与单链多肽相反,熔融温度是浓度依赖性的。 和 通过将整个过渡曲线拟合为van't Hoff方程:

    通过求解方程(7)对于ΔS 而代之以/ sup> (6)。
    用于原始和解折叠状态[Θ] 和[Θ] 的摩尔椭圆度表示另外的温度依赖性,其在第一近似中可以假定为线性: />
    [Θ] n = [Θ] n 0 + b T和[Θ] = [Θ] u 0 + b u T(9)

    其中,[Θ] n 0 和[Θ] 0 对应于T处的[Θ]截距= 0,并且b sub分别描述天然和未折叠状态的斜率。整个过渡曲线由以下描述:

    [Θ] = F([Θ] n 0 + b T - [Θ] T(10)


  5. 由于HLA-A2 /β2m/肽复合物倾向于在过渡区域内沉淀(步骤C8),所以我们假设共同的摩尔椭圆率[Θ] 对于许多温度变性蛋白质和模型肽[Θ] = 0,观察到在-4,000至-5,000 deg cm 2 dmol -1 范围内的值(Venyaminov et al。 / em>。,1993)(注8和9)
  6. 代表数据
    使用HLA-A2 /β2m/ ILAKFLHWL作为实例说明分析三聚体pMHC复合物的稳定性的一般程序(图2)。该复合物已经被详细分析(Cole等人, 2017),结构已经与TCR(蛋白质数据库条目5MEN)复杂化。

    图2. HLA A2 /β2m/ ILKA的热稳定性。 A.蛋白浓度通过紫外吸收光谱法测定。使用与随后的CD测量中使用的相同的样品和缓冲液,将记录在1cm的石英比色杯中的从360nm到230nm的波长扫描。基于氨基酸组成计算出的吸光度系数 = 2.257计算浓度。 B.在4℃下在0.1cm石英比色杯中记录远紫外光谱,减去使用相同仪器参数在相同电池中测量的缓冲液基线。信号被归一化为平均残差权重(MRW)椭圆率[Θ] MRW 式。 (3)。 C.通过以ΔT= 0.5℃的间隔以每个数据点测量12秒,在数据点之间的平衡时间为12秒,温度死区为±0.3,在增加温度时,以218nm的椭圆率Θ测定热稳定性°C,并且在测量之间使用狭缝封闭,导致36℃/ h的平均加热速率(红色,左轴)。样品形成大的可见聚集体,表明在58至65℃范围内的沉淀(空心圆圈)。低于该温度(实心圆)的数据点符合式。 (10)导致虚线。聚集形成伴随增加的光散射,导致倍增电压(蓝色,右轴)急剧增加,随后大颗粒沉入比色皿底部时减少。

    1. 通过将所有三个序列(补充1到3 )进入ProtParam的输入框(步骤B2),导致:

      消光系数以M为单位 - 1 cm - 1 style =“font-family:Courier New;”>,以280nm测量水中。
      Ext。系数&NBSP;&NBSP; 101675
      Abs 0.1%(= 1 g / L)&nbsp;&nbsp;假设所有成对的Cys残基都形成胱氨酸 &nbsp;

    2. 测量的蛋白质溶液的吸收(图2A)显示了等式1的 = 0.283。 (1)浓度为0.283 / 2.269 = 0.125mg / ml
    3. MRW根据公式(2)为MRW =(44,820.03 + 2×18.02)/(386-3)= 117.12。
    4. 根据步骤C2至C6使用0.1cm路径长度的比色皿记录两个溶液的CD缓冲液基线和光谱。减去基线,根据方程式对光谱进行归一化。 (3)(图2B)。在195nm处,倍增极电压为〜500V。这反映了PBS中包含的氯离子的高吸光度。光谱在218nm处显示出最小的 [Θ] = -8,950 deg cm 2 dmol -1 。
    5. 如步骤C7中所述从4℃至65℃记录熔解曲线,减去基线的平均值(步骤C3),并根据方程式计算摩尔残差椭圆度。 (3)(图2C)。使用“补充4 < / a>根据公式拟合数据(10)。从拟合中省略了显示与表示蛋白质聚集的均匀准S形曲线(图2C中的露点)显着偏差的59至65℃范围内的值。拟合结果 = 54.5±0.1°C和 = -500±20kJ / mol,测定系数r = 0.988。


  1. pMHC的峰分离将以约12ml的洗脱体积进行;重要的是保持最纯净的部分,并丢弃任何含有超过一个峰值的蛋白质。
  2. 为了确定热力学参数,对于HLA-A2 /β2m/肽复合物,我们的目的是浓度为〜0.15mg / ml,因为迄今为止研究的大多数肽可以相对容易地实现,并提供良好的信噪比遵循218 nm的CD转换曲线。由于多链蛋白质的浓度依赖性[等式[ (7)],重要的是定义一个狭窄的范围以允许数据比较。
  3. 如果使用PBS或水的基线平行于光束中记录的光谱(“空气基线”),则可以将比色杯视为光学清洁。在200至225nm范围内观测到的最小值通常表示来自先前测量的蛋白质被吸收到石英表面
  4. 在其他制造商的仪器上,倍增电压称为HT(高压),可靠的电压范围可能因光电倍增器和电子设备的类型而不同。
  5. 已经测试了加热参数,通过在0.1厘米比色杯内使用Pt100电阻温度检测器进行测量,得出设定的平衡温度。
  6. AVIV仪器软件以ASCII格式存储数据。其他制造商的一些仪器使用必要的格式,需要通过其他程序导出为ASCII进行分析
  7. 可以将MRW 转换成摩尔吸收单位Δε=ε L / sub> = [Θ] MRW / 3,298,其中Δε是左旋和右旋圆偏振光的吸收差。
  8. 从我们实验室研究的各种MHC复合物中,HLA-A * 0101 /β2m/肽VTEHDTLLY在相对于Tm(68.9℃)和焓(-719kJ mol / -1 );降水仅在T> 72℃,[Θ] 0 (Jones等人,2016)。使用[Θ] U 2>>>>>>>>>>>>>>>>>>>>>>>>>>> &gt; 0.996。
  9. 对于严格的热力学分析,转变的可逆性[等式(4)]是主要要求。然而,有人建议,即使可逆性有限,分析仍然是有价值的,因为温度诱导的协同展开通常比未折叠蛋白质的聚集快得多(Privalov,2009)。为了允许比较数据,我们对所有样品使用相同的加热参数(参见步骤C7)。


DKC是威康信托研究职业发展研究员(WT095767)。 AKS是Wellcome Trust高级研究员。英国生物技术与生物科学研究理事会(BBSRC)(Grant BB / H001085 / 1)支持了使用所述程序的原始作品。购买CD文件部分由BBSRC 75 / REI18433资助。


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引用:Fuller, A., Wall, A., Crowther, M. D., Lloyd, A., Zhurov, A., Sewell, A. K., Cole, D. K. and Beck, K. (2017). Thermal Stability of Heterotrimeric pMHC Proteins as Determined by Circular Dichroism Spectroscopy. Bio-protocol 7(13): e2366. DOI: 10.21769/BioProtoc.2366.

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