A novel peptide design aids in the expression and its simplified process of manufacturing of Insulin Glargine in Pichia pastoris.

Manufacturing of insulin and its analogues relied upon in vitro enzymatic cleavages of its precursor forms (single chain precursor, SCP) at both ends of a connecting peptide (C-peptide) that links the respective B-chain and A-chains to corresponding final forms. We have demonstrated a simplified approach of cleaving P. pastoris expressed SCP, distinctly at one site for conversion to insulin glargine. The design of the precursor was made in such a way that there is no C-peptide in the precursor which needs to be removed in the final product. Instead of traditional both side cleavage of the C-peptide and removing the C-peptide (by trypsin), followed by 2nd enzyme reaction (typically carboxipeptidase B), present work established only one side cleavage of the sequence by only trypsin converts the precursor to final insulin glargine product. The novel design of the precursor helped in producing insulin glargine in a single step with an application of single enzyme brought high degree of process efficiencies. Highly purified product was generated through two reversed phase high pressure chromatographic steps. Purified product was compared with the reference product Lantus®, for various physico-chemical and biological properties. Primary, secondary and tertiary structures as well as biological pharmaco-dynamic effects were found comparable. High cell density fermentation that gave a good yield of the SCP, a single step conversion to insulin glargine, enabled by a unique design of SCP and a distinct purification approach, has led to a simplified and economical manufacturing process of this important drug used to treat diabetes. KEY POINTS: • Novel concept for processing single chain precursor of insulin glargine • Simple and economic process for insulin glargine • Physicochemical characterization and animal Pharmacodynamics show similarity to Lantus.

Mass Spectrometry Based Mechanistic Insights into Formation of Tris Conjugates: Implications on Protein Biopharmaceutics.

We present here extensive mass spectrometric studies on the formation of a Tris conjugate with a therapeutic monoclonal antibody. The results not only demonstrate the reactive nature of the Tris molecule but also the sequence and reaction conditions that trigger this reactivity. The results corroborate the fact that proteins are, in general, prone to conjugation and/or adduct formation reactions and any modification due to this essentially leads to formation of impurities in a protein sample. Further, the results demonstrate that the conjugation reaction happens via a succinimide intermediate and has sequence specificity. Additionally, the data presented in this study also shows that the Tris formation is produced in-solution and is not an in-source phenomenon. We believe that the facts given here will open further avenues on exploration of Tris as a conjugating agent as well as ensure that the use of Tris or any ionic buffer in the process of producing a biopharmaceutical drug is monitored closely for the presence of such conjugate formation. Graphical Abstract ᅟ.

Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain Insulin glargine in Pichia pastoris.

Insulin glargine is a slow acting analog of insulin used in diabetes therapy. It is produced by recombinant DNA technology in different hosts namely E. coli and Pichia pastoris. In our previous study, we have described the secretion of fully folded two-chain Insulin glargine into the medium by over-expression of Kex2 protease. The enhanced levels of the Kex2 protease was responsible for the processing of the glargine precursor with in the host. Apart from the two-chain glargine product we observed a small proportion of arginine clipped species. This might be due to the clipping of arginine present at the C-terminus of the B-chain as it is exposed upon Kex2 cleavage. The carboxypeptidase precursor Kex1 is known to be responsible for clipping of C-terminal lysine or arginine of the proteins or peptides. In order to address this issue we created a Kex1 knock out in the host using Cre/loxP mechanism of targeted gene deletion. When two-chain glargine was expressed in the Kex1 knock out host of P. pastoris GS115 the C-terminal clipped species reduced by ∼80%. This modification further improved the process by reducing the levels of product related impurities.

Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.

Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.

Possible modification of Alzheimer's disease by statins in midlife: interactions with genetic and non-genetic risk factors.

The benefits of statins, commonly prescribed for hypercholesterolemia, in treating Alzheimer's disease (AD) have not yet been fully established. A recent randomized clinical trial did not show any therapeutic effects of two statins on cognitive function in AD. Interestingly, however, the results of the Rotterdam study, one of the largest prospective cohort studies, showed reduced risk of AD in statin users. Based on the current understanding of statin actions and AD pathogenesis, it is still worth exploring whether statins can prevent AD when administered decades before the onset of AD or from midlife. This review discusses the possible beneficial effects of statins, drawn from previous clinical observations, pathogenic mechanisms, which include ß-amyloid (Aß) and tau metabolism, genetic and non-genetic risk factors (apolipoprotein E, cholesterol, sex, hypertension, and diabetes), and other clinical features (vascular dysfunction and oxidative and inflammatory stress) of AD. These findings suggest that administration of statins in midlife might prevent AD in late life by modifying genetic and non-genetic risk factors for AD. It should be clarified whether statins inhibit Aß accumulation, tau pathological features, and brain atrophy in humans. To answer this question, a randomized controlled study using amyloid positron emission tomography (PET), tau-PET, and magnetic resonance imaging would be useful. This clinical evaluation could help us to overcome this devastating disease.

PMT1 gene plays a major role in O-mannosylation of insulin precursor in Pichia pastoris.

Protein mannosyltransferases (PMTs) catalyze the O-mannosylation of serine and threonine residues of proteins in the endoplasmic reticulum. The five PMT genes coding for protein mannosyltransferases, designated as PMT1, 2, 4, 5 and 6, were identified from Pichia pastoris genome based on the homology to PMT genes in Saccharomyces cerevisiae genome, which has seven PMT genes. The homologues of S. cerevisiae PMT 3 &7 genes are absent in P. pastoris genome. Approximately 5% of the recombinant insulin precursor expressed in P. pastoris is O-mannosylated. In this study, we attempted to prevent O-mannosylation of insulin precursor in vivo, through inactivation of the Pichia PMT genes. Since multiple PMTs are found to be expressed, it was important to understand which of these are involved in O-mannosylation of the insulin precursor. The genes encoding PMT1, 4, 5 and 6 were knocked out by insertional inactivation method. Inactivation of PMT genes 4, 5 and 6 showed ∼16-28% reductions in the O-mannosylation of insulin precursor. The PMT1 gene disrupted Pichia clone showed ∼60% decrease in O-mannosylated insulin precursor, establishing its role as an important enzyme for insulin precursor O-mannosylation.

Mass spectrometric distinction of in-source and in-solution pyroglutamate and succinimide in proteins: a case study on rhG-CSF.

Formation of cyclic intermediates involving water or ammonia loss is a common occurrence in any reaction involving terminal amines or hydroxyl group containing species. Proteins that have both these functional groups in abundance are no exception, and presence of amino acids such as asparagine, glutamines, aspartic acids, and glutamic acids aid in formation of such intermediates. In the biopharma scenario, such intermediates lead to product- or process-related impurities that might be immunogenic. Mass spectroscopy is a powerful technique that is used to decipher the presence and physicochemical characteristics of such impurities. However, such intermediates can also form in situ during mass spectrometric analysis. We present here the detection of in-source and in-solution formation of succinimide and pyroglutamate in the protein granulocyte colony stimulating factor. We also propose an approach for quick differentiation of such in-situ species from the tangible impurities. We believe that this will not only reduce the time spent in unambiguous identification of succinimide- and/or pyroglutamate-related impurity in bio-pharmaceutics but also provide a platform for similar studies on other impurities that may form due to stabilized intermediates.

Improvement of metabolic syndrome by irbesartan via the PPARγ/HGF pathway in apolipoprotein E knockout mice.

Irbesartan, a partial agonist of peroxisome proliferators activated receptor-γ (PPARγ), has been reported to improve insulin resistance and lipid profile in patients with diabetes mellitus or metabolic syndrome (MS). However, the down effectors of PPARγ have yet to be elucidated. Thus, in this study, we focused on the role of the hepatocyte growth factor (HGF) in the anti-metabolic effects of irbesartan, using apolipoprotein E (ApoE) knockout (KO) mice. ApoE KO mice placed on a high-fat diet (HFD) for 12 weeks were divided into four groups: i) the control (HFD only), ii) the HFD + irbesartan (5 mg/kg/day), iii) the HFD + irbesartan + GW9662, a PPARγ antagonist (0.5 mg/kg/day) and iv) the HFD + irbesartan + anti-HGF neutralizing antibody (200 µg/week). The liver and epididymal adipose tissues were evaluated histologically. Serum adiponectin and HGF levels were also measured by ELISA. Fatty liver (as detected by oil-red O staining) and macrophage infiltration were markedly reduced by irbesartan. Irbesartan treatment also reduced macrophage infiltration into epididymal adipose tissue and hypertrophy of adipocytes. However, these effects of irbesartan were attenuated by GW9662 as well as by anti-HGF neutralizing antibody. Serum and hepatic HGF levels were also markedly increased by irbesartan, whereas GW9662 decreased the HGF level. In conclusion, irbesartan, an angiotensin (Ang) receptor blocker (ARB) and partial agonist of PPARγ (metabosartan), demonstrated a reduction in fatty liver and chronic inflammation, such as macrophage infiltration, beyond its blood pressure-lowering effect. These favorable characteristics of irbesartan might be due to local HGF activation through its partial PPARγ agonistic action, in addition to Ang II blockade. Upregulation of local HGF by irbesartan might provide a novel advantage in a strategy for the prevention and treatment of cardiovascular diseases (CVDs).

Inhibition of Lp(a)-induced functional impairment of endothelial cells and endothelial progenitor cells by hepatocyte growth factor.

BACKGROUND: Lipoprotein (a) (Lp(a)) is one of the risk factors for peripheral artery disease (PAD). Our previous report demonstrated that hepatocyte growth factor (HGF) gene therapy attenuated the impairment of collateral formation in Lp(a) transgenic mice. Since risk factors for atherosclerosis accelerate endothelial senescence and impair angiogenesis, we examined the role of Lp(a) in dysfunction and senescence of endothelial progenitor cells (EPC) and endothelial cells. METHODS: In vitro and in vivo incorporation assays were performed using ex-vivo expanded DiI-labeled human EPC. Senescence of cultured endothelial cells, production of oxidative stress and angiogenesis function were evaluated by SA-ß-galactosidase staining, dihydroethidium (DHE) staining and Matrigel assay, respectively. RESULTS: EPC transplantation significantly stimulated recovery of ischemic limb perfusion, while EPC pre-treated with Lp(a) did not increase ischemic limb perfusion. Impairment of angiogenesis by EPC with Lp(a) was associated with a significant decrease in CD31-positive capillaries and DiI-labeled EPC. Importantly, Lp(a) significantly accelerated the onset of senescence and production of reactive oxygen species (ROS) in human aortic endothelial cells, accompanied by a significant increase in the protein expression of p53 and p21. On the other hand, HGF significantly attenuated EPC dysfunction, senescence, ROS production, and p53 and p21 expression induced by Lp(a). CONCLUSION: Lp(a) might affect atherosclerosis via acceleration of senescence, ROS production, and functional impairment of the endothelial cell lineage. HGF might have inhibitory effects on these atherogenic actions of Lp(a).

A novel one-pot de-blocking and conjugation reaction step leads to process intensification in the manufacture of PEGylated insulin IN-105.

Bio-catalytic in vitro multistep reactions can be combined in a single step in one pot by optimizing multistep reactions under identical reaction condition. Using this analogy, the process of making PEGylated insulin, IN-105, was simplified. Instead of taking the purified active insulin bulk powder as the starting material for the conjugation step, an insulin process intermediate, partially purified insulin ester, was taken as starting material. Process intensification (PI) was established by performing a novel de-blocking (de-esterification) of the partially purified insulin ester and conjugation at B-29 Lys residue of B chain with a short-chain methoxy polyethylene glycol (mPEG) in a single-pot reactor. The chromatographic profile at the end of the reaction was found similar irrespective of whether both the reactions were performed sequentially or simultaneously. The conjugated product of interest, IN-105 (conjugation at LysB(29)), was purified from the heterogeneous mixture of conjugated products. The new manufacturing process was deduced to be more simplified and economical in making the insulin conjugates as several downstream purification steps could be circumvented. The physicochemical characteristics of IN-105 manufactured through this economic process was found to be indifferent from the product formed through the traditional process where the conjugation starting material was purified from bulk insulin.

Role of serotonin in angiogenesis: induction of angiogenesis by sarpogrelate via endothelial 5-HT1B/Akt/eNOS pathway in diabetic mice.

Serotonin (5-hydroxytryptamine, 5-HT) plays a crucial role in peripheral artery disease (PAD) and diabetes mellitus (DM). In these conditions, the balance between the 5-HT2A receptor in smooth muscle cells and the 5-HT1B receptor in endothelial cells (ECs) regulates vascular tonus. In the present study, we focused on the role of 5-HT in endothelial dysfunction using a selective 5-HT2A receptor blocker, sarpogrelate. In human EC, 5-HT markedly stimulated eNOS expression and the phosphorylation of eNOS, Akt and ERK1/2. In addition, a dose-dependent increase in tubule-formation on Matrigel was observed after 5-HT treatment. In contrast, high glucose significantly inhibited tubule formation and eNOS expression through inactivation of Akt, while 5-HT significantly attenuated these actions of high glucose (P<0.01). These results indicate that 5-HT stimulated angiogenesis through activation of Akt in ECs. However, in clinical situations, 5-HT seems to act as the "devil". To examine the role of 5-HT in diabetic PAD, a hindlimb ischemia model was created in diabetic mice. The blood flow ratio of the ischemic to non-ischemic limb was significantly lower in DM mice than in normal mice, while sarpogrelate significantly attenuated the decrease in the blood flow ratio compared to control (P<0.01). Consistently, the decrease in eNOS expression and Akt activity in DM mice was significantly attenuated by sarpogrelate. Overall, the present study demonstrated that selective inhibition of 5-HT2A by sarpogrelate significantly restored ischemic limb blood perfusion in a severe diabetic mouse model through stimulation of the eNOS/Akt pathway via the endothelial 5-HT1B receptor. Enhancement of vasodilation and angiogenesis by sarpogrelate might provide a unique treatment for PAD and DM patients.

SARS coronavirus unique domain: three-domain molecular architecture in solution and RNA binding.

Nonstructural protein 3 of the severe acute respiratory syndrome (SARS) coronavirus includes a "SARS-unique domain" (SUD) consisting of three globular domains separated by short linker peptide segments. This work reports NMR structure determinations of the C-terminal domain (SUD-C) and a two-domain construct (SUD-MC) containing the middle domain (SUD-M) and the C-terminal domain, and NMR data on the conformational states of the N-terminal domain (SUD-N) and the SUD-NM two-domain construct. Both SUD-N and SUD-NM are monomeric and globular in solution; in SUD-NM, there is high mobility in the two-residue interdomain linking sequence, with no preferred relative orientation of the two domains. SUD-C adopts a frataxin like fold and has structural similarity to DNA-binding domains of DNA-modifying enzymes. The structures of both SUD-M (previously determined) and SUD-C (from the present study) are maintained in SUD-MC, where the two domains are flexibly linked. Gel-shift experiments showed that both SUD-C and SUD-MC bind to single-stranded RNA and recognize purine bases more strongly than pyrimidine bases, whereby SUD-MC binds to a more restricted set of purine-containing RNA sequences than SUD-M. NMR chemical shift perturbation experiments with observations of (15)N-labeled proteins further resulted in delineation of RNA binding sites (i.e., in SUD-M, a positively charged surface area with a pronounced cavity, and in SUD-C, several residues of an anti-parallel beta-sheet). Overall, the present data provide evidence for molecular mechanisms involving the concerted actions of SUD-M and SUD-C, which result in specific RNA binding that might be unique to the SUD and, thus, to the SARS coronavirus.

Nuclear magnetic resonance structure of the nucleic acid-binding domain of severe acute respiratory syndrome coronavirus nonstructural protein 3.

The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand beta-sheet holding two alpha-helices of three and four turns that are oriented antiparallel to the beta-strands. Two antiparallel two-strand beta-sheets and two 3(10)-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.

Nuclear magnetic resonance structure shows that the severe acute respiratory syndrome coronavirus-unique domain contains a macrodomain fold.

The nuclear magnetic resonance (NMR) structure of a central segment of the previously annotated severe acute respiratory syndrome (SARS)-unique domain (SUD-M, for "middle of the SARS-unique domain") in SARS coronavirus (SARS-CoV) nonstructural protein 3 (nsp3) has been determined. SUD-M(513-651) exhibits a macrodomain fold containing the nsp3 residues 528 to 648, and there is a flexibly extended N-terminal tail with the residues 513 to 527 and a C-terminal flexible tail of residues 649 to 651. As a follow-up to this initial result, we also solved the structure of a construct representing only the globular domain of residues 527 to 651 [SUD-M(527-651)]. NMR chemical shift perturbation experiments showed that SUD-M(527-651) binds single-stranded poly(A) and identified the contact area with this RNA on the protein surface, and electrophoretic mobility shift assays then confirmed that SUD-M has higher affinity for purine bases than for pyrimidine bases. In a further search for clues to the function, we found that SUD-M(527-651) has the closest three-dimensional structure homology with another domain of nsp3, the ADP-ribose-1"-phosphatase nsp3b, although the two proteins share only 5% sequence identity in the homologous sequence regions. SUD-M(527-651) also shows three-dimensional structure homology with several helicases and nucleoside triphosphate-binding proteins, but it does not contain the motifs of catalytic residues found in these structural homologues. The combined results from NMR screening of potential substrates and the structure-based homology studies now form a basis for more focused investigations on the role of the SARS-unique domain in viral infection.

Proteomics analysis unravels the functional repertoire of coronavirus nonstructural protein 3.

Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructural protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.

NMR comparison of the native energy landscapes of DLC8 dimer and monomer.

Characterization of the low energy excited states on the energy landscape of a protein is one of the exciting and challenging problems in structural biology today. In this context, we present here residue level NMR description of the low energy excited states representing locally different alternative conformations in the dynein light chain protein, in its dimeric as well as monomeric forms. Important differences have been observed between the two cases and these are not necessarily restricted to the dimer interface. Simulations indicate that the low energy excited states are within a free energy of 2-3 kcal/mol above the native state. In both the monomer and the dimer the energy landscape is very sensitive to small pH perturbations. Nearly 25% of the residues (total of residues at pH 3.0 and 3.5 for the monomer, and at pH 7.0 and 6.0 for the dimer) access alternative conformations. The observations have been rationalized on the basis of protonation-deprotonation equilibria in the side chains; histidines in the case of the dimer and aspartates/glutamates in the case of the monomer. The possible relationship of the observed ruggedness of the native energy landscape with the protein structure, and its implications to protein adaptability and unfolding have been discussed.

NMR assignment of the nonstructural protein nsp3(1066-1181) from SARS-CoV.

Sequence-specific NMR assignments of the globular core comprising the residues 1066-1181 within the non-structural protein nsp3e from the SARS coronavirus have been obtained using triple-resonance NMR experiments with the uniformly [(13)C, (15)N]-labeled protein. The backbone and side chain assignments are nearly complete, providing the basis for the ongoing NMR structure determination. A preliminary identification of regular secondary structures has been derived from the (13)C chemical shifts.

NMR assignment of the domain 513-651 from the SARS-CoV nonstructural protein nsp3.

Sequence-specific NMR assignments of an internal domain of the protein nsp3, nsp3(513-651), which is a part of the SARS coronavirus (SARS-CoV) replicase polyprotein, have been determined, using triple-resonance NMR experiments with the uniformly [(13)C,(15)N]-labeled protein. The complete assignments (>99%) provide the basis for the ongoing three-dimensional structure determination.

Equilibrium unfolding of DLC8 monomer by urea and guanidine hydrochloride: Distinctive global and residue level features.

We present circular dichroism (CD), steady state fluorescence and multidimensional NMR investigations on the equilibrium unfolding of monomeric dynein light chain protein (DLC8) by urea and guanidine hydrochloride (GdnHCl). Quantitative analysis of the CD and fluorescence denaturation curves reveals that urea unfolding is a two-state process, whereas guanidine unfolding is more complex. NMR investigations in the native state and in the near native states created by low denaturant concentrations enabled residue level characterization of the early structural and dynamic perturbations by the two denaturants. Firstly, (15)N transverse relaxation rates in the native state indicate that the regions around N10, Q27, the loop between beta2 and beta4 strands, and K87 at the C-terminal are potential unfolding initiation sites in the protein. Amide and (15)N chemical shift perturbations indicate different accessibilities of the residues along the chain and help identify locations of the early perturbations by the two denaturants. Guanidine and urea are seen to interact at several sites some of which are different in the two cases. Notable among the common interaction site is that around K87 which is in close proximity to W54 on the protein structure, but the interaction modes of the two denaturants are different. The secondary chemical shifts indicate that the structural perturbation by 1M urea is small, compared to that by guanidine which is more encompassing over the length of the chain. The probable (phi, psi) changes at the individual residues have been calculated using the TALOS algorithm. It appears that the helices in the protein are significantly perturbed by guanidine. Further, comparison of the spectral density functions of the native and the two near native states in the two denaturants implicate greater loosening of the structure by guanidine as compared to that by urea, even though the structures are still in the native state ensemble. These differences in the early perturbations of the native state structure and dynamics by the two denaturants might direct the protein along different pathways, as the unfolding progresses on further increasing the denaturant concentration.

Following autolysis in proteases by NMR: insights into multiple unfolding pathways and mutational plasticities.

Biophysical studies in proteases are severely hampered due to the auto-cleavage property of these enzymes. In this context, we develop here a kinetic model and an NMR-based strategy to use this very autolytic property to derive useful insights into multiple unfolding pathways and mutational plasticities in these proteins. The basic idea lies in the interpretation of the auto-cleavage-driven decay of the folded protein peaks in the HSQC spectra as a function of time. The different peaks are seen to decay at different rates. As unfolding is the rate-determining step in the auto-cleavage reaction, the NMR spectral changes reflect on local unfolding processes at the residue level. A formalism is presented to gain insights into unfolding free energies and evaluate local perturbations due to single point mutations. The model is applied to HIV-1 protease-tethered dimer as an example, considering mutations at a particular site. Significant perturbations are seen even at very remote areas from the site of the mutation.