WO2023095154A1 - Method for determining changes in a protein's structure - Google Patents

Abstract

The present invention includes a method of determining changes in a protein's structure based on silver nanoprism (AgNPR) detection platform (refereed as, Quality Check for Protein, QC prot) using only a basic UV-vis spectrophotometer. The method comprises measuring dipole resonance (D) and quadrupole resonance (Q) of protein of interest doped silver nanoprism and reference protein doped silver nanoprism; comparing Q/D ratio of the protein of interest doped silver nanoprism and reference protein doped silver nanoprism; and determining whether there is a difference in the structure of the protein of interest and reference protein if there is a difference in the comparing step.

Description

METHOD FOR DETERMINING CHANGES IN A PROTEIN’S STRUCTURE

REEATED APPLICATION

This application claims the benefit of Indian Patent Application No. 202141054362 filed on November 24, 2021, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention, in general, relates to nanobiotechnology, particularly to a method of determining changes in a protein’s structure, and more particularly to method of determining changes in a protein’s structure based on silver nanoprism (AgNPR) detection platform (referred to as, Quality Check for Protein, “QCprot”) using only a basic UV-vis spectrophotometer.

BACKGROUND OF THE INVENTION

Native structures of proteins are crucial for their functions. Many factors in real life such as disease conditions, laboratory conditions including storage, shipping, handling, batch to batch variation during laboratory synthesis, presence of contaminants can induce structural alterations or changes, which interfere with the normal functions of the proteins.

Therefore, it is extremely important to identify or determine even the minor structural alterations from the perspectives of quality control of protein as well as for developing a detection platform for disease conditions where the diagnosis is a direct consequence to the structural alterations or unfolding or mis-folding of the concerned protein. There have been methods, like kit-based techniques (Protein Thermal Shift™ Dye Kit, ProFoldin-Protein stability, and aggregation assay kit, PROTEOSTAT® Thermal shift stability assay kit) that majorly use fluorescence spectrophotometer; probe independent techniques; and spectroscopic methods like circular dichroism (CD) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy, to determine structural changes in a protein.

However, these methods have one or more drawbacks such as requiring high sample concentration and high-end instrumentation, complexity, high operation costs, low sensitivity, and low specificity. Further, most of these methods are useful only when there is a significant alteration in protein structure resulting in altered population distribution of secondary structures like a-helix, P-sheet, P-turns etc.

Thus, for determining changes in a protein structure, it is necessary to develop a method that overcome one or more of the problems of the prior art.

SUMMARY OF THE INVENTION

Accordingly, provided herein is a method for determining changes in a protein’s structure. The method comprises the steps of: selecting a protein of interest; doping the protein of interest within a silver nanoprism; measuring dipole resonance (D) and quadrupole resonance (Q) of the protein of interest doped silver nanoprism by ultraviolet (UV) visible spectrophotometer; selecting a reference protein; and doping the reference protein within a silver nanoprism; and measuring dipole (D) resonance and quadrupole (Q) resonance of the reference protein doped silver nanoprism by UV visible spectrophotometer.

In an aspect, the method further comprises: calculating Q/D ratio of the protein of interest doped silver nanoprism and reference protein doped silver nanoprism; and comparing the Q/D ratio of the protein of interest doped silver nanoprism and reference protein doped silver nanoprism; and determining whether there is a difference in the structure of the protein of interest and reference protein, and further determine if there is any difference in the comparing step(s).

In another aspect, the method of the present invention is simpler, economic, and more efficient. BRIEF DESCRIPTION OF THE FIGURES

The features of the present invention will become fully apparent from the following description taken in conjunction with the accompanying figures. With the understanding that the figures depict only several embodiments in accordance with the disclosure and are not to be considered limiting of its scope, the disclosure will be described further through use of the accompanying figures.

Figure 1 illustrates plot of perfectness factor, Pf (i.e., Q/D ratio) of GHb (Glycated hemoglobin) doped AgNPR (silver nanoprism) with respect to time (in days) at various glucose concentration.

Figure 2 illustrates plot of perfectness factor, Pf (i.e., Q/D ratio) of FHb (fructated hemoglobin) doped AgNPR with respect to time (in days) at various glucose concentration.

Figure 3 illustrates dose response bar diagram showing varied response from glycated and fructated Hb at different sugar concentration. Y axis denotes the slope of the linear plots presented in figure 1 and 2.

Figure 4 illustrates TEM (transmission electron microscopy) images of (a) native Hb (b) glycated Hb and (c) fructated Hb doped AgNPR.

Figure 5 illustrates XRD profile of AgNS (silver nano seeds) doped with Hb, GHb and FHb. The color-coded box table represents the deviation (green, red, and blue) or absence (Black) of different peaks compared to conventional AgNP.

Figure 6 illustrates plot of Q/D ratio of GHb doped AgNPR with respect to Glucose concentration

Figure 7 illustrates comparison of CD spectroscopy result (left panel) with QCprot (right panel) regarding the thermal melting experiment. Top half reports the actual spectra of protein asperginase and the bottom half describes the thermal denaturation curve indicating the Tm (temperature of melting) value.

Figure 8 illustrates comparison of CD spectroscopy result (left panel) with QCprot (right panel) regarding the thermal melting experiment. Top half reports the actual spectra of protein hemoglobin, and the bottom half describes the thermal denaturation curve indicating the Tm value.

Figure 9 illustrates comparison of CD spectroscopy result (left panel) with QCprot (right panel) regarding the thermal melting experiment. Top half reports the actual spectra of protein insulin, and the bottom half describes the thermal denaturation curve indicating the Tm value.

Figure 10 illustrates comparison of CD spectroscopy result (left panel) with QCprot (right panel) regarding the thermal melting experiment. Top half reports the actual spectra of protein streptokinase, and the bottom half describes the thermal denaturation curve indicating the Tm value.

Figure 11 illustrates pH induced aggregation profile of proteins such as bovine serum albumin (BSA), lysozyme (LYS), asperginase (ASP), trypsin (TYR), hemoglobin (Hb), insulin (INS) and streptokinase (STR).

Figure 12 illustrates (a) increase of hydrodynamic size of Ap42 (amyloid-beta42) peptide due to pH induced amyloidosis and (b) spectra of corresponding protein doped AgNPR.

Figure 13 illustrates constitution of AgNPR used in the present method along with the key dimension parameters like length (T), thickness (T) and internal angle (A) is on the upper panel and a characteristic spectrum (UV visible spectrum) in the lower panel.

Figure 14 illustrates two stage synthesis of AgNPR.

Figure 15 illustrates origin of AgNPR with different dimension features when doped with the native and unfolded state of the same protein.

DETAILED DESCRIPTION OF THE INVENTION

Before the methods of the present disclosure are described in greater detail, it is to be understood that the methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods.

Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, the term "comprises", "comprising", or “comprising of’ is generally used in the sense of include, that is to say permitting the presence of one or more features or components. As used herein, the term “invention” or “present invention” as used herein is a nonlimiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification and the claims.

Each embodiment is provided by way of explanation of the invention and not by way of limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made to the compounds, compositions and/or methods described herein without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment can be applied to another embodiment to yield a still further embodiment. Thus, it is intended that the present invention includes such modifications and variations and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are obvious from, the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only and is not to be construed as limiting the broader aspects of the present invention.

In an embodiment, the present invention provides a method for determining changes in a protein’s structure. The method employs a nanoprism (AgNPR) based detection platform (also referred to as Quality Check for Protein (QCprot)) using only a basic UV-vis spectrophotometer.

In certain embodiments, the present invention provides a method for determining changes in a protein’s structure, comprising the steps of: selecting a protein of interest; doping the protein of interest within a silver nanoprism; measuring dipole (D) resonance and quadrupole (Q) resonance of the protein of interest doped silver nanoprism by ultraviolet (UV) visible spectrophotometer; selecting a reference protein; doping the reference protein within a silver nanoprism; and measuring dipole (D) resonance and quadrupole (Q) resonance of the reference protein doped silver nanoprism by UV visible spectrophotometer.

In certain embodiments, the method further comprises the steps of: calculating Q/D ratio of the protein of interest doped silver nanoprism; and reference protein doped silver nanoprism; comparing the Q/D ratio of the protein of interest doped silver nanoprism; and reference protein doped silver nanoprism; and determining whether there is a difference in the structure of the protein of interest and reference protein if there is a difference in the comparing step.

In certain embodiments of the method, the protein of interest is selected from the group comprising: antibodies, regulatory factors, enzymes, and ligands. In further embodiments, the protein is a therapeutic protein or monoclonal antibody.

In certain embodiments, the protein is a globular protein or a fibrous protein. In further embodiments, the protein is a globular protein. In some instances, the globular protein is selected from the group comprising bovine serum albumin (BSA), lysozyme (LYS), asperginase (ASP), trypsin (TYR), hemoglobin (Hb), insulin (INS) and streptokinase (STR).

In certain embodiments, the protein is a fibrous protein with linear tubular shape. In certain embodiments, the fibrous protein is selected from the group comprising fibrinogen and collagen (including different types such as type I, type II, and type IV).

In certain embodiments, the reference protein is not exposed to the same handling, storage or shipping conditions, same processing, same batch, or the same manufacturing steps as the protein of interest.

In certain embodiments of the method, the protein (protein of interest or reference protein) is doped within the silver nanoprism structure at the time of synthesis.

In certain embodiments, the method as provided herein does not use any dye or high- end instrumentation. Silver nanoprism (AgNPR) displays characteristic quadrupole resonance peak (Q peak) about 400 nm wavelength and a highly sensitive dipole resonance peak (D peak) with a much larger dynamic range (about 450-1000 nm) in the UV visible spectrum. The highly sensitive nature of the D-peak depends on the thickness (T), length (L) and internal angle (A) of the plane (see figure 13). A minor change in any of these parameters leads to significant alteration or change in the spectroscopic property of the AgNPR. The present inventors found that this ultra-sensitive spectroscopic property can be used to determine or identify the presence of any structural alteration or change of a protein, when doped within the crystal planes of AgNPR.

AgNPR can be synthesized using any method known in the art. In certain embodiments, AgNPR is synthesised from the commercially available silver nitrate in a two- step pathway. In the first step, very small sized (~5 nm) spherical silver nanoparticles, designated as nanoseeds (AgNS) are prepared, which are subsequently aligned in a 2- dimensional fashion to form AgNPR (see figure 14). During the synthesis of AgNS, if any foreign agent (protein of interest in this case) is introduced (doped), that results in alteration/ differential arrangement of crystal planes of AgNS, which in turn results in quantitative alteration of one or more of the above parameters (L, T, A) of AgNPR and consequently in the alteration of intensity and location of D-peak. Due to incorporation of hundreds of seeds in the AgNPR, the overall extent of structural change gets amplified and enables convenient detection of any minor change in protein’s structure through simple UV-visible spectrometer and colorimetric changes. The relative absorbance of the Q-peak and D-peak, to be presented as QA/DA ratio, functions as a reasonable quantitative marker of the deformation of the crystal structure of the AgNPR.

Unfolding or denaturation of protein results in the alteration of its secondary and other higher order structure. This structural alteration occurs due to breaking of several bonds between different constituent amino acids, sometimes coupled with some new bond formation. [Di sulphide linkages are the dominant covalent bonds which are usually broken or formed during unfolding. Other interactions which get altered during unfolding are primarily non-covalent that includes electrostatic interaction, H-bonding, and van der Wall forces. Very strong ionic denaturants like guanidine hydrochloride can break 3 -dimensional structure and yield coiled structure. This phenomenon has two distinct effects on the overall property of the concerned protein: firstly, the loosening of the native structure, and secondly the altered surface charge distribution on the protein surface. As a result of this, when AgNS is doped with an unfolded protein, it distorts the crystal plane alignment differentially, compared to the native protein and results in the formation of differentially structured NS (PuF-AgNS, where PUF represents unfolded protein). Consequently, the alignment of PUF- AgNS and P-AgNS is different leading to formation of respective AgNPRs, having significantly different dimensions (L, T and A). As a result, the ratio of absorbances of Q peak to that of D peak in PuF-AgNPR will be different than that for P-AgNPR compared to AgNPR.

During the doping process, when AgNS is synthesized in the presence of a protein, the extent of difference in the alignment of Ag fee (111) crystal planes depend on the structural difference between the two states of the proteins (native and unfolded). As the protein, specifically the globular one, gets unfolded, its size becomes slightly larger due to loss of a several cohesive forces like H-bonding, electrostatic interaction, and van der Waal forces, which hold the compact structure. A marginally larger size of dopant (here, unfolded protein) thus results in a slight increase of AgNS size and deviation from spherical structure. As a result of this, the thickness (T) of the nanoprism, which is determined by the diameter of the AgNS, increases marginally and becomes heterogenous based on the linking geometry between two adjacent AgNSs. An undoped nanoseed being spherical in shape; their linear alignment always results in a homogenous thickness of the resultant AgNPR. But due to doping of a protein, the shape of the P-AgNS deviates from being spherical. Consequently, when doped with a denatured protein, the shape of the PuF-AgNS also deviates from sphere but the deviation is much different from that of P-AgNS. Hence the thickness and dimensional features of both P-AgNPR and PuF-AgNPR become differentially heterogeneous resulting in differential spectral properties (see figure 15).

On the other hand, the linear alignment of hundreds of PuF-AgNS yields a multiplicative effect of the minute difference of size and shape between PuF-AgNS and P- AgNS. The slightly larger size of PuF-AgNS over P-AgNS leads to create more irregular alignment of the NSs, further compromising the perfectness of the AgNPR crystal. Hence, deviation from the perfect AgNPR structure is much higher for PuF-AgNPR compared to P- AgNPR. Hence, the D-peak absorbance decreases significantly for PuF-AgNPR compared to P-AgNPR. The inverse behaviour of Q-peak and D-peak with respect to protein unfolding, has thus paved a way to identify (QA)/(DA) ratio as a reporter of perfectness of the crystal and hence is called Perfectness Factor (Pf). Larger the unfolding stress, larger will be the size and shape variation of protein during unfolding. Consequently, larger will be the size and shape difference in the nanoseeds and finally, larger will be the imperfectness of the crystal, leading to a linear increase of the Pf.

Accordingly, in certain embodiments, the present invention also provides a method for determining quantification of deformation of structure (Ds) of a protein. The method comprises: measuring perfectness factor (Pf) (i.e. Q/D ratio) of the unfolded protein and native protein; and calculating deviation of perfectness factor (Pf) for the unfolded protein from that of the native protein structure, with respect to the complete unfolding range.

In certain embodiments of the method of quantification of Ds, the Pf of the unfolded protein and native protein can be calculated as described above; and the Ds can be calculated from the equation (1):

where, Pf(0) and Pf(ioo) are representative of the perfectness factor of the native state and the most extreme unfolded state that can be achieved experimentally for the corresponding unfolding route. The value ‘0’ and ‘100’ within the parentheses denote 0% and 100% unfolded state of the protein attained through thermal denaturation. The factor [Pflioo) - Pf₍o)] denotes the complete range of the unfolding.

In certain embodiments, the present invention further provides a method for quantifying protein modification during a process or due to aging. The protein modification may be quantified using the parameter relative distortion of structure (RDs) which is represented by equation 2:

Sign of the APf value depends on the nature of the unfolding. A positive value of APf - indicates that the structural distortion created by the sample protein between the fee (111) planes of Psample-AgNS is more than the distortion created by the reference protein between the fee (111) planes of the P_ref-AgNS and vis-a-vis. Theoretically, a single protein can show both positive and negative APf value if it undergoes different unfolding pathway under different unfolding stress.

In certain embodiments, the structural distortion of the protein is scaled between 0 to 100 representing the native and extremely unfolded state of the protein respectively.

In certain embodiments, the methods as provided herein are in vitro methods.

In certain embodiments, the methods as provided herein are useful to identify any minor alteration or change in protein’s structure and thus have important applications in protein’s quality control and in detection of diseases related to structural alterations or modifications, which otherwise requires elaborate methods for detection. The methods also useful in predicting aggregation of proteins using a common UV visible spectrophotometer.

In certain embodiments, the methods as provided herein are simple, require less time, low sample, and/or no high-end instrumentation. In further embodiments, the methods as provided herein are less or no complex, require low operation costs, provide high sensitivity and/or high specificity.

In certain embodiments, the dose response of the (QA)/(DA) ratio follows strong correlation with the extent of unfolding stress within specific ranges of unfolding conditions like temperature difference of 5 °C, sugar concentration of 10 mg/dL and pH change of 1 unit.

In further embodiments, the methods as provided herein are more sensitive than the gold standard in the domain, i.e., CD (Circular Dichroism) spectroscopy. In some instances, the cost of the determination of alteration in the structure of protein using the methods provided herein is 5-10 times less than existing methods.

The efficiency of the present method is depicted in Tables 1 and 2 below. Table 1 provides a comparison of QCprot with the state-of-the-art technologies. Table 2 provides active dynamic range and resolution of QCprot.

Table 1: Comparison of QCprot with the state-of-the-art commercial technologies

Table 2: Active dynamic range and resolution of QCprot

The present invention is further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.

EXAMPLES

Example 1: Glycation of protein

Protein glycation is an essential post translational modification process which leads to the formation of a functional protein. The extent of glycation serves as an indicator of the available sugar in the bloodstream. Hence, glycated Hb (GHb) is measured as HbAlc test for determining the sugar control in the blood. The glycation study is envisaged in two stages. In stage 1, the study probed the effect of type of sugar and time of incubation on the whole process in a wide range of sugar concentration. In the stage 2, the method was tested in physiologically relevant range.

Inducing glycation:

Stage 1: Artificially created sugar induced stress to Hb for producing various degrees of GHb. Checked the kinetics of GHb formation as a function of time and checked the effect of sugar concentration as well as the nature of the sugar on the GHb formation by using ‘Ds’ as the quantifier. Two different sets of aqueous solution of Hb were prepared in mQ water of a concentration of Img/ml, using glucose and fructose respectively: keeping sugar concentration within the range of 0.1 to 0.5 mM. Buffering condition was avoided to account for the spontaneous misfolding of the protein. Aliquots from the unmodified (Hb) as well as glucose and fructose modified ones, i.e., GHb and FHb respectively, were collected at different time interval till 3 weeks and were studied under UV-visible spectroscopy before being used as a dopant during AgNPR synthesis. The study of glycation was performed with 100 uL of Hb.

Stage 2: In this stage, sugar (glucose) concentration ranging from 40mg/dL to 200mg/dL was taken to cover all three relevant stages of physiological glucose concentrations like hypoglycaemia (<80 mg/dL), normal (80-120 mg/dL) and hyperglycaemia (> 120 mg/dL).

Synthesis of AgNPR: a. Synthesis of nanoseed (AgNS): Into the mixture of aqueous trisodium citrate (5 mL, 2.5 mM), 10 pL protein solution was added followed by aqueous poly(sodium styrenesulphonate) of 1000 kDa (100 pL of 500 pg/mL) and 300 pL 10 mM freshly prepared aqueous NaBH4. After that, 5 mL of 0.5 mM aqueous AgNCF (was added to the solution at a rate of 2 mL/minute with continuous stirring). The protein modified AgNS was kept at 4°C for 10- 15min and then they were used for the next step. b. Alignment of AgNS to AgNPR: A reaction mixture was prepared with 5 mL distilled water, 75 mL of 10 mM aqueous ascorbic acid (75 pL, 10 mM), and seed solution of 300 pL. To this mixture, 3 mL of 0.5 mM aqueous AgNCL solution was added at a rate of 1 mL/minute with continuous stirring. So, the reaction took 3 min to complete. To the synthesized AgNPR, 500 pL of 2.5mM trisodium citrate was added to stabilize the nanoform for weeks.

Result:

Hb, GHb and FHb doped AgNPR solutions, mentioned as Hb@ AgNPR, GHb @ AgNPR and FHb @ AgNPR respectively, were prepared as described in the experimental section and were studied under UV-Vis spectrophotometer.

UV-Vis spectroscopy results show that for both GHb @ AgNPR and FHb @ AgNPR, the absorbance of D-peak is decreasing relative to the Q-peak for all sugar concentrations with increasing day of incubation, while insignificant change was observed for Hb@ AgNPR. To explore the extent of sugar induced deformation, the Pf of the samples, were plotted against the time of sugar incubation of the protein and was found to be a good linear fit with a positive slope for both glucose (see figure 1) and fructose (see figure 2). The positive sign of Pf is indicative of the fact that the addition of sugar molecules on the protein surface causes increasing hindrance towards forming the ideal AgNPR structure i.e., undoped AgNPR structure compared to their native counterpart. In addition, the goodness of linear fit over 0.95 for all the systems, indicate continuous as well as systematic structural deformation of the nanoplanar structure. Hence, sugar induced quantitative deformation of Hb structure is clearly observed by the Pf ratio trend. Importantly enough, this gives a clear account of the robustness of the Pf value, i.e., QA/DA ratio as the indicator of structural integrity of the protein.

For viewing the overall glycation and fructation kinetics as functions of structural deformation, the corresponding slope values from the Time vs. Pf plots for different sugar concentrations were presented in a bar diagram against sugar concentrations for both glucose and fructose (see figure 3). It is evident from the bar diagram that the slope values of various fructose concentrations are much higher than that of the corresponding glucose concentrations, which demonstrates for the fact that fructation of Hb results in the higher extent of denaturation compared to glycation.

The observation of the differential sugar response required some in-depth studies regarding the structure of the NPLs. To investigate the structural aspects of the phenomena, transmission electron microscopy (TEM) was performed. The TEM image, described in figure 4, showed the presence of various assembled structures of nanoforms in different sugar modified condition. The Hb@ AgNPR shows a regular hexagonal structure in figure 4a. The figure 4b, shows the assembly of ca. several GHb@AgNPR units [cone of Glc], and interestingly figure 4c shows a truncated assembly or disassembly of the planar unit for FHb@ AgNPR [cone of Fru] through some possible fault lines representing a population of broken NPLs. This highly degenerative impact of fructation, compared to glycation is reflective of the increasing slope of the sugar response curves presented in figure 1 to 3.

The highly differential spectroscopic properties between these AgNPRs are the cumulative manifestations of the AgNS structural difference. Hence, further investigated the core structural differences between the three variants of the AgNSs using XRD to clearly address these different planar structures viewed under TEM.

The XRD profiling is described in figure 5, where the crystal peaks for various NS species are compared when doped with Hb variants (i.e., normal Hb, GHb, FHb). The AgNS being too small (ca. 5nm), the peaks are very broad and of very low intensity. Within the figure, incorporated a table describing the deviation of crystal parameter (A a), calculated based on a

= 4.086 A (JCPDS file no. 04-0783). The deviation was measured in percentage for each crystal plane to describe the difference of differentially doped AgNS species from the undoped one.

The deviation is colour coded as described, Green represents <1%, Blue represents l%-2%, Red represents >2% and Black represents complete absence of that peak. This colour coded crystal plane difference matrix clearly shows how the crystal planes of Hb@AgNS, GHb @ AgNS and FHb @ AgNS are differently distributed and modified compared to the native or undoped AgNS crystal plane distribution. The significant difference between each of the sets are responsible to result in minute difference in the AgNS shapes. As modelled in figure 3 earlier, this shape difference is magnified many folds due to their collective alignment to form AgNPR, resulting in different dimensions (E, T and A), and finally in different intensity of the Q-peak and the D-peak.

Probing physiologically relevant range: The method was tested in physiologically relevant range of glucose to assess the potential of the present method in real life application. Hence, the range was chosen from highly hypoglycaemic (40 mg/dL) to significantly hyperglycaemic (200 mg/dL) range.

Figure 6 shows the linear response of QCprot to the glucose concentration. The normal glycaemic range of 80-120 mg/dL was highlighted (in green) for a better understanding for the glycation response curve. The appreciable linearity (R² = 0.95), of the response signifies that QCprot has significant potential to be used as a substitute technique for conventional HbAic method.

Example 2: Thermal folding Measurement of thermal melting has been a standard method for determining a protein’s folding state. The gradual loss of structural integrity of protein with increasing temperature was monitored by various methods. The temperature where 50% loss of foldedness was observed, was defined as the melting temperature (Tm) of a protein. The thermal melting on asperginase, hemoglobin, insulin and streptokinase were studied and calculated their Tm by using both Circular Dichroism (CD) spectroscopy and our AgNPR based spectroscopic method. The present method was compared with CD spectroscopy, which is still considered as gold standard in secondary structure assessment.

Inducing thermal melting:

A constant temperature water bath was used to attain temperature from 30°C to 70°C. Aqueous protein solution of Img/mL concentration was used as stock. The temperature was increased by 10°C each time and kept at a specific temperature for 15 minutes after attaining the desired temperature. Then l OpL of the protein aliquote was taken from the water bath setup to add to the reaction cocktail for AgNS synthesis. The protein modified AgNS was then used to synthesize AgNPR by the process described earlier. The as formed AgNPR was then studied by a UV-vis spectrophotometer to calculate the structural parameters to obtain Ds value. Similarly, CD spectroscopy was performed on all the proteins at those temperature points. Plot of Temperature vs. Ds was compared to the plot of Temperature vs. Ellipticity (mdeg) obtained from CD results. Model globular proteins asperginase (ASP), hemoglobin (Hb), insulin (INS) and streptokinase (STR) having similar melting temperature of (64-65°C) were used.

Synthesis of AgNPR:

AgNPR was prepared using the similar protocol as depicted in Example 1.

Result:

Figures 7-10 show the snapshots of thermal denaturation of ASP, HB, INS and STR respectively, probed by CD and QCprot separately. The left panel of each of the figure shows the probing by CD spectroscopy and the right panel shows the probing using QCprot. Upper half of each panel shows the whole spectra of the protein at different temperature, whereas the lower half shows the kinetics of the melting process. Table 3 summarizes the accuracy of QCprot to determine the Tm, compared to the CD spectroscopy. The accuracy values were calculated using the equation 1, where the absolute value for the difference between reported Tm and measured Tm with respect to the reported Tm was calculated to obtain the accuracy.

* Accuracy (%) = 100 100 Eq. i

In all four cases, QCprot has demonstrated significantly higher degree of accuracy compared to CD spectroscopy. The remarkably high accuracy for ASP and STR is highly noteworthy. Accuracy for INS is little less (89.3%) compared to others. This may be due to the temperature mediated fibrillization of INS, which falls under the borderline space for applying QCprot, which works perfectly for the globular protein but behaves a bit erratically in the presence of fibrous proteins. Fibrous protein with linear tubular shape may facilitate the formation of AgNPR compared to the other globular protein. Table 3: Comparison of the Tm value detection between CD and QCprot

Example 3: pH induced Aggregation pH induced aggregation was studied because different proteins are stable under different pH based on their isoelectric point. Proteins like BSA, Hb, lysozyme, trypsin, insulin, streptokinase, asperginase were used in the experiments. The proteins were incubated in a large range of pH from 4 to 10 and probed them by the present method.

Inducing Aggregation:

Three types of buffers were prepared to create a pH range of 4-10 for the study. Citrate buffer (lOOmM) was used for the pH range 4-5, while phosphate buffer (lOOmM) for pH 6-8 and Tris buffer (lOOmM) for pH 9-10. All the proteins were incubated separately in all the buffered solutions at a concentration of Img/mL. From those samples, 10 pl aliquots of each were used to synthesize the corresponding protein doped AgNPR. Upon synthesis, the stability profile of the proteins was constructed by plotting the Q/D values of AgNPRs at various pH.

Synthesis of AgNPR:

AgNPR was prepared using the similar protocol as depicted in Example 1.

Result:

Figure 11 demonstrates the pH induced unfolding profile for the proteins as labelled in the image. The vertically aligned panels show the plot of Q/D vs. pH for the proteins BSA, HB, LYS, TRY, INS, ASP, STR. These proteins have demonstrated remarkable transition behaviour based on critical pH values. Proteins like BSA, HB, TRY and ASP were shown transition from higher Q/D to lower Q/D state with increasing pH, denoting transition from an acid induced unfolded or aggregated state to the base induced refolded or de-aggregated state. On the contrary, LYS, INS and STR demonstrated the reverse trend where transition from the base induced unfolded or aggregated state to the acid induced refolded or deaggregated state was observed.

Example 4: Amyloidosis Amyloidosis is another well-studied phenomenon having huge clinical significance. The alteration of the oligomeric assembly for the amyloids is very important to form amyloid plaques, which are the key diagnostic markers for various amyloid related diseases like Alzheimer’s Disease. pH mediated aggregation of amyloids was studied and calculated the RDs value to measure the relative structural distortion for the process.

Inducing amyloidosis:

To validate the ability of AgNPR to detect amyloidosis, amyloid Ap42 peptide was selected, which is known to exist as small oligomers at physiological pH while forms higher order of aggregates under acidic condition (~pH 5). An aqueous solution of Ap42 of a concentration of Img/mL was prepared. The pH of the solution was adjusted using citrate buffer to prepare two sets of solution having pH 5.0 and 7.0. The size analysis was performed using dynamic light scattering (DLS) to measure the hydrodynamic radius of the amyloid oligomers. Now, from each of the stock, lOuL was taken for the synthesis of AgNPR by the method described earlier.

Synthesis of AgNPR:

AgNPR was prepared using the similar protocol as depicted in Example 1.

Result:

Aggregation was induced by lowering the pH of an aqueous solution of Ap42 peptide. The aggregation behaviour was further verified by hydronamic size measurement using Dynamic light scattering instrument, which showed the predominant species at pH 7 has a hydrodynamic diameter of 19 nm, and that at pH 5 has a hydrodynamic diameter of 170 nm. AgNPRs were synthesised using the Ap42 peptide solutions at pH 7 and pH 5. As demonstrated in Figure 12, AgNPR synthesised using Ap42 peptide solution at pH 5 showed markedly different colour and spectral signatures. To assess the overall structural distortion, the RDs was calculated as per equation 2, and was found to be about 16%. Hence, the relative structural distortion can also be reported by using Pf.

Claims

The Claims:

1. A method for determining changes in a protein’s structure, comprising: selecting a protein of interest; doping the protein of interest within a silver nanoprism; measuring dipole (D) resonance and quadrupole (Q) resonance of the protein of interest doped silver nanoprism by ultraviolet (UV) visible spectrophotometer; selecting a reference protein; doping the reference protein within a silver nanoprism; and measuring dipole (D) resonance and quadrupole (Q) resonance of the reference protein doped silver nanoprism by UV visible spectrophotometer.

2. The method as claimed in claim 1, further comprises: calculating Q/D ratio of the protein of interest doped silver nanoprism; and reference protein doped silver nanoprism; comparing the Q/D ratio of the protein of interest doped silver nanoprism; and reference protein doped silver nanoprism; and determining whether there is a difference in the structure of the protein of interest and reference protein if there is a difference in the comparing step.

3. The method as claimed in claim 1, wherein the protein of interest is selected from the group comprising: antibodies, regulatory factors, enzymes, and ligands.

4. The method as claimed in claim 1, wherein the protein is a globular protein or a fibrous protein.

5. The method as claimed in claim 1, wherein the protein is a globular protein.

6. The method as claimed in claim 1, wherein the protein is a fibrous protein with linear tubular shape.

7. The method as claimed in claim 5, wherein the globular protein is selected from the group comprising bovine serum albumin (BSA), lysozyme (LYS), asperginase (ASP), trypsin (TYR), hemoglobin (Hb), insulin (INS) and streptokinase (STR).

8. The method as claimed in claim 1, wherein the reference protein is not exposed to the same handling, storage or shipping conditions, same processing, same batch, or the same manufacturing steps as the protein of interest.

9. The method as claimed in claim 1, wherein the protein of interest is doped within the silver nanoprism structure at the time of synthesis.

10. A method for determining quantification of deformation of structure (Ds) of a protein, comprising: measuring perfectness factor (Pf) (i.e. Q/D ratio) of the unfolded protein and native protein; and calculating deviation of perfectness factor (Pf) for the unfolded protein from that of the native protein structure, with respect to the complete unfolding range.

11. The method as claimed in claim 11, wherein Ds is calculated from the equation:

where, Pf(0) and Pfqoo) are representative of the perfectness factor of the native state and the most extreme unfolded state that can be achieved experimentally for the corresponding unfolding route.

12. A method for quantifying protein modification during a process or due to aging, wherein the protein modification is quantified using the parameter relative distortion of structure (RDs) which is represented by equation 2:

13. The method as claimed in any one of the claims 1 to 12, wherein the method is an in vitro method.

14. The method as claimed in any one of the claims 1 to 13, wherein the method does not use any dye or high-end instrumentation.