Determining Biophysical Protein Stability in Lysates by a Fast Proteolysis Assay

The biophysical activity of a protein is an important parameter in studying their activities both in invitro and invivo conditions. Fast parallel proteolysis analyses the melting point of proteins in lysate medium. It combines the unfolding of proteins in a temperature gradient with proteolytic cleavage of this unfolded state of a protein. Thus it gives the stability of the single domains in presence of lysates. FASTpp can be done up to a protein molecule of size 10 kDa – 240 kDa. It can monitor folding, coupled folding and binding upon interaction with small-molecule ligands. Subtle stability in point mutations with high sensitivity can be detected in the range of minutes.

Activity and function depends on their structure and stability.The structure and stability of proteins is addected by various factors. Fators such as specific cellular environment or binding to a particualr ligands is the factor of interest in this technique. Specific metals, small molecules or a particular protein ligands presence is required for some proteins to perform their particular function. Intrinsically disordered proteins are such proteins which lacks the structure in isolation of binding proteins. Various ssays are used to analyse the probe structure and stability of these proteins. Spectroscopic methods like Circular Dichroism is used to study the secondary structure of the proteins. Tertiary analysis is done by intrinsic florescence and NMR is used is used for residue-specific information. Protein stability and interactions can be monitored by change in enthalphy and entropy in Differential Scanning Calorimetry (DSC), Isothermal Titration Calorimetry (ITC). Several techniques probe biophysical parameters both in invivo and exvivo conditions. Invivo folding sensors using florescent proteins, florescent small protein tags or exvivo pulse proteolysis are few of these methods. Proteolysis is a label free method. Thermostable protease Thermolysin (TL) cleaves the protein in hte hydrophobic residues Phe, Leu, Ile, Val. TL probe protein in unfolded states in lysates within seconds.  This is the principle of FASTpp.

Applications

FASTpp to assay protein stability

The unfolding temperature of a protein is an indicator for protein stability. Unfolding temperature can be affected by events that affect the stability. Protein structure shift caused by mutations changes the thermal unfolding to lower temperatures. Ligands that attach to the folded state not the unfolded state also increases the unfolding temperature. A thermostable protease that readily cuts the unfolded but not the folded fraction over a wide temperature range. FASTpp caan be used to find the biophysical protein stability. Protein of interest is exposed to a range of temperatures in presence of a thermostable protease. This becomes the underlying principle to use FASTpp in this application.  Temperature dependent changes of degradation of proteins can be obtained by fixing temperature ranges just below and above of the melting point of the protein.  The percision of this method depends on the precise control of the heating time (th), the period for the protein is exposed to a maximum period (melting time, tm) and the subsequent colling time (tc).

FASTpp assay consists of the followinfg steps

  1. Sample preparation of the protein of interest at 4oC.
  2. Addition of protease.
  3. Heating time (th) during which several aliquots of the same sample are heated up in parallel. Each aliquot reaches a specific maximal temperature; for instance the lowest sample 35oC and the highest 42oC.
  4. Melting time ™ during which aliquots are kept at defined maximum temperatures of the gradient for defined times.
  5. Cooling time (tc) of the protein samples down to 4oC.
  6. Stopping proteolysis by EDTA.
  7. Analysis of the reaction products by SDS-PAGE.

The steps 3–6 run in a thermal cycler with gradient control to ensure precision and reproducibility. Variation of th and tc influesnce the values determined by this assay.

All the values are instrument dependent.

Thermolysin is suitable for FASTpp

To valiadate a suitable protease enzyme for application. We will find the following factors/values;

  1. i) The cleavage rate of the enzyme over a range of temperature
  2. ii) Establish its specificity and test it on a range of protein folds.

Thermolysin (TL) is an enzyme due to the following characteristics  

(i) TL is thermostable up to 80oC.

(ii) TL preferentially cuts near exposed hydrophobic, bulky and aromatic amino acids, specifically Phe, Leu, Ala, Val and Ile. TL has a high specificity toward hydrophobic and aromatic residues. This makes TL applicable for FASTpp. Most of the protein molecules bury the amino acids inside the hydrophobic core. Thus it is neccessary to unfold these proteins and expose them for digestion with TL.

(iii) TL is stable over a wide pH range from 5.5 to 9, it remains active in the presence of high concentrations of chaotropic reagents such as 8 M urea and in the presence of EDTA-free protease inhibitors cocktails.

(iv) TL is instantly inhibited by addition of EDTA, which removes TL’s essential Ca2+ ion.

Validating the activity of TL in FASTpp conditions

Step 1: To test the temperature dependence of the proteolysis rate of TL. A flourogenic model substrate unfolded peptide ABZ-Ala-Gly-Leu-Ala-NBA is used. The florescence is proportional to the rate of cleavage. The floresence is thus used as a measure of reaction. Values are takenn from 20oC to 80oC for 3 to 6 nM. The values obtained are fitted to pseudo-first order kinetics. TL displayed a constant thermal activity from 33oC to 80oC.

 FASTpp reveals presence of the folded state

TL is further tested for specificity over unfolded protein chains. Cytochrome C is used to test TL for activity and specificity for unfolded proteins. Cytochrome C is obtained in two states in soluble state. Unfolded with heme and folded in presence of heme.TL cleaved unfolded protein at 4oC whereas folded protein is cleaved only at 60oC. TL possess both the activity (over a range of temperatures) and specificity (for folded and unfolded protein) thus suitable for FASTpp application.

FASTpp is insensitive to variation of TL concentration

Next step in FASTpp application is the refinement of experimental parameters. Maltose Binding Protein (MBP) is used as a substrate. MBP is structurally well characterized and folds both in presence and absence of a ligand. So it is used for this purpose.  We first probed the influence of TL concentration over four orders of magnitude on the apparent thermal melting temperature of MBP using a gradient of 50oC to 70oC and constant tm (6 s).  At the lowest TL concentration of 0.001 g/L, no detectable cleavage of MBP occurred. From 0.01 to 1 g/L TL (340 nM –34 mM).  A loss of thermal proteolysis resistance at 59oC. Assuming comparable cleavage kinetics of the model peptide substrate and unfolded protein, it is expected a minimal required cleavage time of approximately 6 s at 0.01 g/L TL to quench the unfolded fraction of protein under these conditions. TL titration results validated with the theoretical prediction. Interestingly, at 0.01 g/L TL,at a temperature of 61oC unfolding and concomitant cleavage of MBP was detected. An uncut MBP band however remained at temperatures from 63oC to 70oC. Kinetic competition between aggregation and cleavage at higher temperatures, which may protect MBP from complete cleavage because hydrophobic residues typically self-interact within aggregates may be the cause of this uncut band. TL  at a standard concentration of 0.1 g/L (3.4 mM) can be used for further experiments.

Kinetic protein stability can be probed by FASTpp at variable tm

The next step is to investigate how the apparent thermal unfolding transition in FASTpp is affected by tm. For this tm is varied from 6 s to 600 s. In parallel with a step-wise increase in tm, MBP digestion started at successively lower temperature. For instance at tm= 6 s, the unfolding occurred at 60oC while increasing tm to 600 s lowered the unfolding temperature to 49oC. Because all assay parameters are kept constant except for tm, we can monitor kinetic stability with this assay. Proteins are ‘‘kinetically-stable’’ under conditions where the unfolding is slow relative to the measurement time. For instance, MBP is kineticallystable at 40oC and kinetically-unstable at 60oC for all tm values analysed.

Ligand stabilisation can be revealed by FASTpp

This step is to illustrate and find the apacity of FASTpp to detect the effect of ligand binding on the biophysical stability of the protein. MBP is analysed over the ligand maltose. Using a temperature range from 50oC to 70oC at constant tm=6 s, apo MPB became susceptible to proteolysis at 58oC whereas maltose bound MBP resisted degradation up to 70oC. Intrinsic protein florescence data is used to study the protein stability. An onset of unfolding at 40oC for MBP-maltose and at 30oC for apo MBP, significantly lower absolute values compared to the FASTpp results.  Lower rate of temperature increase in the florescence experiment might be one of the reasons for this fact. Alternaivelyn discrepancies in the unfolding temperature is different for both the experiments. This could be due to the secondary and teritiary structures. Floresence can be sensitive to changes in the vicinity of tryptophans.  The stabilising effect of the maltose ligand on MBP, however, was approximately 10uC in both experiments. We therefore conclude, that FASTpp agrees qualitatively with fluorescence temperature dependence analysis about the stabilising effect of maltose on MBP. The FASTpp data confirmed a significantly stabilising effect of maltose on MBP.

FASTpp determines protein stability in lysate

In vitro stability of MBP to the ex vivo stability of E. coli lysate overexpressing MBP. MBP resists proteolysis in lysate up to 59 0C. From 61 0C to 70 0C, the apo MBP band intensity was nearly lost. In contrast, MBP’s proteolytic resistance persisted up to 70uC in presence of 5 mM maltose ligand. Both for purified protein and lysate samples, maltose addition increases the unfolding temperature by more than 10 0C. Interestingly, when compared with purified MBP, the apo MBP lysate displays a sudden unfolding transition between 59oC and 61oC while purified MBP has a much broader unfolding range between 50 0C and 58 0C. Lysate stabilized MBP without addition of maltose ligand. Since lysates are complex mixtures we assume that the balance of all (presumably mostly weak and transient) interactions determines the differences between biophysical stability of protein in lysates compared to experiments with purified proteins in more diluted solutions of isolated proteins. We conclude that FASTpp is suitable to monitor stability changes in whole cell lysates.

BSA thermostability is not affected by maltose

Excluding unspecific protein stabilization is important to increase the quality of FASTpp. TO demonstrate this process in FASTpp we use maltose as ligand and maltose non-binding protein BSA. FASTpp for BSA in presence and absence of maltose is done. Maltose did not change the thermal unfolding transition of BSA in a buffer with reducing redox potential between 4oC and 59oC. This corroborates our conclusion that FASTpp detects specific ligand stabilization effects.

Large proteins assemblies can be analysed with FASTpp

Tetrameric Pyruvate Kinase (PK), a 240 kDa is investigated using FASTpp on temperature range of 55oC and 65oC.  The protein starts to proteolyse from 59oC. However, 10% of PK ban intensity remains at higher temperature up to 65oC. This may be due to the fact thermal aggression competes with protein cleavage above this temperature.  Another cleavage resistant 34 kDa fragment appears above 60oC which may be either strong domain or an aggregating domain. This proves the applicability of FASTpp in large proteins.

FASTpp detects stability differences of point mutants

The ability of FASTpp to detect point mutantion is tested by comparing three evolved stated of Sortase A. Enhance transcriptase kinetics is studied for these molecule. The molecules are Sortase A triplemutant (36M), Sortase A tetramutant (46M), Sortase A pentamutant (56M). To achieve an accurate relative quantification, Coommassie dye enhanced with strong infrared florescence is used upon protein binding. Upon quantification, we obtained the following order of stability: 36M and 46M are equally stable with a transition starting above 40oC; the 56M variant displayed a less cooperative thermal unfolding transition consistent with an entropically broadened transition. Significantly more residual protein remained above 50oC for this protein variant.

Second, we used intrinsic fluorescence to probe stability differences. The variants 36M and 46M behaved very similar in this assay with non-linear fluorescence decay above a Tu of 40oC, while 56M appeared to be slightly more stable with linear decrease continuing up to a Tu of 43oC.

Qualitative validation of FASTpp data is done in comparison with floresecence due to the difference in two assays

  1. Heating times (hours in fluorescence, minutes in FASTpp)
  2. Fluorescence measures in equilibrium until unfolding and aggregation start while FASTpp constantly removes unfolded protein from the equilibrium – an effect that increases with tm. The difference qualitatively coherence with Sortace A variants. Thus FAASTpp is effective tool in detection of point mutation.

FASTpp is applicable to a wide range of protein folds

To reconcile our data in structural terms, we assessed the structure elements of the proteins analysed by FASTpp and compare these with our metapredictions of structural disorder using the PONDR-Fit algorithm in a simplified dichotomic representation discriminating well-structured/ordered and disordered regions. A broad range of folds compatible with the assay: all a-helical, a/b and mostly b-sheet. BSA is an example for a mostly a-helical protein containing multiple disulfide bonds. Also cytochrome C in the presence of heme as well as MBP contain a large a-helical fraction while cytochrome C in the absence of ligand was previously reported to be largely devoid of structure. Pyruvate kinase forms a 240 kDa complex with somewhat higher b-sheet content. The mostly b-sheet Sortase A protein was amenable to FASTpp analysis as well. This comparison of folds suggests that most folded domains without large internal disordered linkers may be amenable to analysis by FASTpp. Conversely, proteins containing large internal disordered regions are expected to be cleaved by default – unless they fold for instance by a coupled folding and binding mechanism in vivo. Accurate disorder predictions for watersoluble proteins such as PONDR-Fit might therefore be useful to preselect suitable candidate proteins for FASTpp assays and guide the data interpretation.

Discussion

FASTpp as a biophysical tool to monitor structural protein stability both in individual form and in lysate. FASTpp is applicable for i) Detection of  protese activity in high temperature range and thermostable applications, ii) Interaction of  a folded protein with a ligand in either presence or absence of cellular lysate.

Applicability of FASTpp to determine the absolute melting point of the proteins need some prerequisties. The determination requires equilibrium conditions, which can be achieved by calorimetric methods. The unfolding temperature values depend on the experimental conditions such as temperature range, heating rates, protein concentration and protease susceptibility of the protein of interest. These prohibit the determination of melting point by FASTpp but the experiment provides the relative stability. Relative stability analysis for proteins with diferent case scenario of ligand binding, point mutation and differenet environment conditions can be precisely determine byFASTpp for different applications.

Fluorescence is widely used due to its high sensitivity and in many cases sufficient intrinsic label concentrations of either naturally occurring tryptophanes or genetically engineered fluorescent tags. FASTpp is a useful complementation to fluorescence-based assays in cases where intrinsic labels are below detection levels or genetic manipulation is not possible.

FASTpp has the ability to analyse protein stability at low concentrations and in complex solutions, such as lysates and primary patient samples. Specific antibodies allow stability analysis by FASTpp of cell or tissue-derived samples without the need for tagging or purification.  To investigate tumor mutations possible links between biophysical and pathological mechanisms can be analyzed. Putative stability changes in disease-related proteins such as kinases and tumor suppressors from patient tissues are used for analyses.  FASTpp experiments can be done in laboratories equipped with standard biochemistry instruments and do not require advanced biophysical equipment.

FASTpp is also an alternative for Pulse Proteolysis. In this ex vivo assay, equilibrium unfolding at room temperature in urea precedes a short proteolysis pulse to probe unfolding. Several features of FASTpp differ significantly from Pulse Proteolysis:

  1. The rapid temperature increase in FASTpp significantly increases the denaturation rate of kinetically-stable proteins compared to urea titrations at room temperature, e.g. for ligand-bound maltose binding protein.
  2. High temperature (up to 80oC) has little effect on the intrinsic proteolysis rate; high urea concentrations however inhibit the enzyme.
  3. Temperature gradients reveal quickly self-aggregating unfolded species while urea may dissolve aggregates.

Taken together, both approaches have complementary benefits: FASTpp gives insight into thermal stability, Pulse Proteolysis into equilibrium unfolding. FASTpp, however, requires less experimental time. Considering the broad range of folds that can be analysed by FASTpp and the specificity, robustness and speed of the method, we anticipate a broad range of future applications.

Minimal sample preparation requirements and use of standard molecular biological techniques allow applications in protein engineering, cell biology and biomedical research.

Methods

Thermal Proteolysis

  1. A 5 g/L stock solutions of TL as described earlier.
  2. The proteolysis assay buffer contained 10 mM CaCl2, 20 mM sodium phosphate buffer at pH 7.2 and 150 mM NaCl for purified proteins and 5 mM DTT for cytosolic proteins.
  3. Protein concentrations were between 0.15–1 g/L.
  4. Digestion was performed in a C1000 thermal cycler.
  5. Protein amounts were quantified by coommassie fluorescence in an Odyssey scanner; specific fluorescence enhancement of coommassie upon binding to protein was measured and the integrated fluorescence intensity per protein band was compared to the corresponding two-fold dilution series of undigested proteins of known concentration to fit the parameters of a second-order polynom describing the dependence of fluorescence on protein concentration.

Determination of the temperature dependence of the intrinsic proteolysis rate of TL

We determined temperature dependence of TL activity analogous to a previous approach for monitoring urea dependence of TL activity. Briefly, we used 6 nM and 3 nM TL to cleave a fluorigenic model substrate (ABZ-Ala-Gly-Leu-Ala-NBA) to monitor the reaction by fluorescence dequenching of this substrate at various temperatures. For quantification we used a pseudo-firstorder kinetic model that assumes a constant concentration of the catalyst (TL) over the course of the experiment and full accessibility of the substrate. As fluorescence increases relative to the extent of dequench, we fitted the intrinsic rate by using the formula:

F=F0(Fmax-F0)(1-e(-kt))

F is fluorescence,

F0 is the initial fluorescence,

Fmax is the fluorescence after complete cleavage

k is the intrinsic rate of proteolysis at the specific enzyme concentration used,

t is the observation time.