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1.
Mol Pharm ; 20(6): 2951-2965, 2023 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-37146162

RESUMO

Therapeutic proteins can be challenging to develop due to their complexity and the requirement of an acceptable formulation to ensure patient safety and efficacy. To date, there is no universal formulation development strategy that can identify optimal formulation conditions for all types of proteins in a fast and reliable manner. In this work, high-throughput characterization, employing a toolbox of five techniques, was performed on 14 structurally different proteins formulated in 6 different buffer conditions and in the presence of 4 different excipients. Multivariate data analysis and chemometrics were used to analyze the data in an unbiased way. First, observed changes in stability were primarily determined by the individual protein. Second, pH and ionic strength are the two most important factors determining the physical stability of proteins, where there exists a significant statistical interaction between protein and pH/ionic strength. Additionally, we developed prediction methods by partial least-squares regression. Colloidal stability indicators are important for prediction of real-time stability, while conformational stability indicators are important for prediction of stability under accelerated stress conditions at 40 °C. In order to predict real-time storage stability, protein-protein repulsion and the initial monomer fraction are the most important properties to monitor.


Assuntos
Anticorpos Monoclonais , Quimiometria , Humanos , Estabilidade Proteica , Anticorpos Monoclonais/química , Desdobramento de Proteína , Conformação Proteica , Estabilidade de Medicamentos
2.
Mol Pharm ; 19(2): 508-519, 2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-34939811

RESUMO

Using light scattering (LS), small-angle X-ray scattering (SAXS), and coarse-grained Monte Carlo (MC) simulations, we studied the self-interactions of two monoclonal antibodies (mAbs), PPI03 and PPI13. With LS measurements, we obtained the osmotic second virial coefficient, B22, and the molecular weight, Mw, of the two mAbs, while with SAXS measurements, we studied the mAbs' self-interaction behavior in the high protein concentration regime up to 125 g/L. Through SAXS-derived coarse-grained representations of the mAbs, we performed MC simulations with either a one-protein or a two-protein model to predict B22. By comparing simulation and experimental results, we validated our models and obtained insights into the mAbs' self-interaction properties, highlighting the role of both ion binding and charged patches on the mAb surfaces. Our models provide useful information about mAbs' self-interaction properties and can assist the screening of conditions driving to colloidal stability.


Assuntos
Anticorpos Monoclonais , Anticorpos Monoclonais/química , Método de Monte Carlo , Espalhamento a Baixo Ângulo , Difração de Raios X , Raios X
3.
Mol Cell ; 51(5): 691-701, 2013 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-23973328

RESUMO

The Plk1-interacting checkpoint helicase (PICH) protein localizes to ultrafine anaphase bridges (UFBs) in mitosis alongside a complex of DNA repair proteins, including the Bloom's syndrome protein (BLM). However, very little is known about the function of PICH or how it is recruited to UFBs. Using a combination of microfluidics, fluorescence microscopy, and optical tweezers, we have defined the properties of PICH in an in vitro model of an anaphase bridge. We show that PICH binds with a remarkably high affinity to duplex DNA, resulting in ATP-dependent protein translocation and extension of the DNA. Most strikingly, the affinity of PICH for binding DNA increases with tension-induced DNA stretching, which mimics the effect of the mitotic spindle on a UFB. PICH binding also appears to diminish force-induced DNA melting. We propose a model in which PICH recognizes and stabilizes DNA under tension during anaphase, thereby facilitating the resolution of entangled sister chromatids.


Assuntos
Anáfase/genética , DNA Helicases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cromátides/metabolismo , DNA Helicases/química , DNA Helicases/genética , Humanos , Microscopia de Fluorescência/métodos , Ácidos Nucleicos Heteroduplexes/metabolismo , Nucleossomos/metabolismo , Transporte Proteico , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
4.
Mol Pharm ; 17(9): 3298-3313, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32609526

RESUMO

Therapeutic peptides and proteins show enormous potential in the pharmaceutical market, but high costs in discovery and development are limiting factors so far. Single or multiple point mutations are commonly introduced in protein drugs to increase their binding affinity or selectivity. They can also induce adverse properties, which might be overlooked in a functional screen, such as a decreased colloidal or thermal stability, leading to problems in later stages of the development. In this study, we address the effect of point mutations on the stability of the 4.4 kDa antimicrobial peptide plectasin, as a case study. We combined a systematic high-throughput biophysical screen of the peptide thermal and colloidal stability using dynamic light scattering and differential scanning calorimetry with structure-based methods including small-angle X-ray scattering, analytical ultracentrifugation, and nuclear magnetic resonance spectroscopy. Additionally, we applied molecular dynamics simulations to link obtained protein stability parameters to the protein's molecular structure. Despite their predicted structural similarities, all four plectasin variants showed substantially different behavior in solution. We observed an increasing propensity of plectasin to aggregate at a higher pH, and the introduced mutations influenced the type of aggregation. Our strategy for systematically assessing the stability and aggregation of protein drugs is generally applicable and is of particular relevance, given the increasing number of protein drugs in development.


Assuntos
Mutação Puntual/genética , Proteínas Citotóxicas Formadoras de Poros/química , Proteínas Citotóxicas Formadoras de Poros/genética , Biofísica/métodos , Varredura Diferencial de Calorimetria/métodos , Difusão Dinâmica da Luz/métodos , Concentração de Íons de Hidrogênio , Peptídeos/química , Peptídeos/genética , Agregados Proteicos/genética , Estabilidade Proteica/efeitos dos fármacos
5.
Mol Pharm ; 17(2): 426-440, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31790599

RESUMO

Therapeutic protein candidates should exhibit favorable properties that render them suitable to become drugs. Nevertheless, there are no well-established guidelines for the efficient selection of proteinaceous molecules with desired features during early stage development. Such guidelines can emerge only from a large body of published research that employs orthogonal techniques to characterize therapeutic proteins in different formulations. In this work, we share a study on a diverse group of proteins, including their primary sequences, purity data, and computational and biophysical characterization at different pH and ionic strength. We report weak linear correlations between many of the biophysical parameters. We suggest that a stability comparison of diverse therapeutic protein candidates should be based on a computational and biophysical characterization in multiple formulation conditions, as the latter can largely determine whether a protein is above or below a certain stability threshold. We use the presented data set to calculate several stability risk scores obtained with an increasing level of analytical effort and show how they correlate with protein aggregation during storage. Our work highlights the importance of developing combined risk scores that can be used for early stage developability assessment. We suggest that such scores can have high prediction accuracy only when they are based on protein stability characterization in different solution conditions.


Assuntos
Anticorpos Monoclonais/química , Descoberta de Drogas/métodos , Imunoglobulina G/química , Interferon alfa-2/química , Desdobramento de Proteína , Albumina Sérica Humana/química , Transferrina/química , Sequência de Aminoácidos , Armazenamento de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Agregados Proteicos , Estabilidade Proteica , Projetos de Pesquisa , Solubilidade
6.
Nucleic Acids Res ; 45(19): 11413-11424, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-28977671

RESUMO

PICH is a DNA translocase required for the maintenance of chromosome stability in human cells. Recent data indicate that PICH co-operates with topoisomerase IIα to suppress pathological chromosome missegregation through promoting the resolution of ultra-fine anaphase bridges (UFBs). Here, we identify the BEN domain-containing protein 3 (BEND3) as an interaction partner of PICH in human cells in mitosis. We have purified full length PICH and BEND3 and shown that they exhibit a functional biochemical interaction in vitro. We demonstrate that the PICH-BEND3 interaction occurs via a novel interface between a TPR domain in PICH and a BEN domain in BEND3, and have determined the crystal structure of this TPR-BEN complex at 2.2 Å resolution. Based on the structure, we identified amino acids important for the TPR-BEN domain interaction, and for the functional interaction of the full-length proteins. Our data reveal a proposed new function for BEND3 in association with PICH, and the first example of a specific protein-protein interaction mediated by a BEN domain.


Assuntos
Motivos de Aminoácidos , DNA Helicases/química , Domínios Proteicos , Proteínas Repressoras/química , Sequência de Aminoácidos , Sítios de Ligação/genética , Cristalografia por Raios X , DNA Helicases/genética , DNA Helicases/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitose/genética , Modelos Moleculares , Ligação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Homologia de Sequência de Aminoácidos
7.
Chembiochem ; 16(13): 1905-1918, 2015 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-26147795

RESUMO

De novo design and chemical synthesis of proteins and of other artificial structures that mimic them is a central strategy for understanding protein folding and for accessing proteins with new functions. We have previously described carbohydrates that act as templates for the assembly of artificial proteins, so-called carboproteins. The hypothesis is that the template preorganizes the secondary structure elements and directs the formation of a tertiary structure, thus achieving structural economy in the combination of peptide, linker, and template. We speculate that the structural information from the template could facilitate protein folding. Here we report the design and synthesis of three-helix-bundle carboproteins on deoxyhexopyranosides. The carboproteins were analyzed by CD, analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and NMR spectroscopy, and this revealed the formation of the first compact and folded monomeric carboprotein, distinctly different from a molten globule. En route to this carboprotein we observed a clear effect originating from the template on protein folding.

8.
J Pept Sci ; 19(5): 283-92, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23505212

RESUMO

α-Helical coiled coil structures, which are noncovalently associated heptad repeat peptide sequences, are ubiquitous in nature. Similar amphipathic repeat sequences have also been found in helix-containing proteins and have played a central role in de novo design of proteins. In addition, they are promising tools for the construction of nanomaterials. Small-angle X-ray scattering (SAXS) has emerged as a new biophysical technique for elucidation of protein topology. Here, we describe a systematic study of the self-assembly of a small ensemble of coiled coil sequences using SAXS and analytical ultracentrifugation (AUC), which was correlated with molecular dynamics simulations. Our results show that even minor sequence changes have an effect on the folding topology and the self-assembly and that these differences can be observed by a combination of AUC, SAXS, and circular dichroism spectroscopy. A small difference in these methods was observed, as SAXS for one peptide and revealed the presence of a population of longer aggregates, which was not observed by AUC.


Assuntos
Peptídeos/química , Estrutura Secundária de Proteína , Sequências Repetitivas de Aminoácidos , Ultracentrifugação , Dicroísmo Circular , Modelos Moleculares , Peptídeos/síntese química , Dobramento de Proteína , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Espectroscopia por Absorção de Raios X , Raios X
9.
Biochemistry ; 51(36): 7189-201, 2012 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-22913742

RESUMO

S100B is a member of the S100 subfamily of EF-hand proteins that has been implicated in malignant melanoma and neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. Calcium-induced conformational changes expose a hydrophobic binding cleft, facilitating interactions with a wide variety of nuclear, cytoplasmic, and extracellular target proteins. Previously, peptides derived from CapZ, p53, NDR, HDM2, and HDM4 have been shown to interact with S100B in a calcium-dependent manner. However, the thermodynamic and kinetic basis of these interactions remains largely unknown. To gain further insight, we screened these peptides against the S100B protein using isothermal titration calorimetry and nuclear magnetic resonance. All peptides were found to have binding affinities in the low micromolar to nanomolar range. Binding-induced changes in the line shapes of S100B backbone (1)H and (15)N resonances were monitored to obtain the dissociation constants and the kinetic binding parameters. The large microscopic K(on) rate constants observed in this study (≥1 × 10(7) M(-1) s(-1)) suggest that S100B utilizes a "fly casting mechanism" in the recognition of these peptide targets.


Assuntos
Fatores de Crescimento Neural/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteínas S100/metabolismo , Sequência de Aminoácidos , Proteína de Capeamento de Actina CapZ/química , Proteína de Capeamento de Actina CapZ/metabolismo , Proteínas de Ciclo Celular , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Fatores de Crescimento Neural/química , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fragmentos de Peptídeos/química , Ligação Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/química , Termodinâmica , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo
10.
Proteins ; 78(6): 1376-81, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20112418

RESUMO

The thermodynamic properties of unfolding of the Trp-cage mini protein in the presence of various concentrations of urea have been characterized using temperature-induced unfolding monitored by far-UV circular dichroism spectroscopy. Analysis of the data using a two-state model allowed the calculation of the Gibbs energy of unfolding at 25 degrees C as a function of urea concentration. This in turn was analyzed by the linear extrapolation model that yielded the dependence of Gibbs energy on urea concentration, i.e. the m-value for Trp-cage unfolding. The m-value obtained from the experimental data, as well as the experimental heat capacity change upon unfolding, were correlated with the structural parameters derived from the three dimensional structure of Trp-cage. It is shown that the m-value can be predicted well using a transfer model, while the heat capacity changes are in very good agreement with the empirical models based on model compounds studies. These results provide direct evidence that Trp-cage, despite its small size, is an excellent model for studies of protein unfolding and provide thermodynamic data that can be used to compare with atomistic computer simulations.


Assuntos
Peptídeos/química , Peptídeos/metabolismo , Triptofano/química , Ureia/farmacologia , Simulação por Computador , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estrutura Secundária de Proteína , Termodinâmica
11.
Sci Rep ; 10(1): 10089, 2020 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-32572086

RESUMO

Fusion technology is widely used in protein-drug development to increase activity, stability, and bioavailability of protein therapeutics. Fusion proteins, like any other type of biopharmaceuticals, need to remain stable during production and storage. Due to the high complexity and additional intramolecular interactions, it is not possible to predict the behavior of fusion proteins based on the behavior the individual proteins. Therefore, understanding the stability of fusion proteins on the molecular level is crucial for the development of biopharmaceuticals. The current study on the albumin-neprilysin (HSA-NEP) fusion protein uses a combination of thermal and chemical unfolding with small angle X-ray scattering and molecular dynamics simulations to show a correlation between decreasing stability and increasing repulsive interactions, which is unusual for most biopharmaceuticals. It is also seen that HSA-NEP is not fully flexible: it is present in both compact and extended conformations. Additionally, the volume fraction of each conformation changes with pH. Finally, the presence of NaCl and arginine increases stability at pH 6.5, but decreases stability at pH 5.0.


Assuntos
Neprilisina/química , Engenharia de Proteínas/métodos , Albumina Sérica Humana/química , Albuminas/química , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Conformação Proteica , Estabilidade Proteica/efeitos dos fármacos , Espalhamento a Baixo Ângulo , Difração de Raios X/métodos
12.
Biochemistry ; 48(12): 2788-98, 2009 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-19275165

RESUMO

The S100 proteins make up a family of dimeric calcium binding proteins that function in response to changing calcium levels. Several S100 binding proteins have been identified; however, the exact biological functions of the S100 proteins are largely unknown as there are several factors which modulate their functions. To address these issues, the specificity of binding of representative members of the human S100 proteins to short N-terminal peptides of annexin I (AnI) and annexin II (AnII) was investigated under controlled experimental conditions. AnI and AnII have been shown previously to interact with S100A11 and S100A10, respectively. This provided a unique opportunity to determine their binding specificity with the other members of the human S100 protein family. It was found that AnI binds S100A6 or S100A11 while AnII binds S100A10 or S100A11. This is the first report of the interaction between S100A6 and AnI. The fact that AnI and AnII bind to selected members of the S100 protein family shows that these interactions are specific and that the mode of binding is different from that of calmodulin, as it was found not to bind AnI or AnII. From the analysis of the thermodynamics of interactions, the binding seems to be entropically driven. It was found that both AnI and AnII undergo a coil-to-helix transition upon binding to their respective binding partners. The observation that there is an overlap in functionality is not surprising due to considerable sequence homology between S100 protein family members. In fact, the functional overlap can explain previous failures of S100 knockout constructs to show any detectable changes in phenotype despite numerous implications of these proteins in important cellular processes.


Assuntos
Anexina A1/química , Anexina A2/química , Peptídeos/química , Proteínas S100/metabolismo , Termodinâmica , Sequência de Aminoácidos , Anexina A1/metabolismo , Anexina A2/metabolismo , Sítios de Ligação , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/metabolismo , Conformação Proteica , Proteínas S100/química , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por Substrato
13.
Methods Mol Biol ; 350: 105-13, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-16957320

RESUMO

Differential scanning calorimetry can measure the heat capacity of a protein/peptide solution over a range of temperatures at constant pressure, which is used to determine the enthalpy function of the system. There are several experimental factors that can have a significant impact on the determined enthalpy and subsequent derived thermodynamic parameters. These factors are discussed in terms of sample and instrument preparation, as well as data collection and analysis.


Assuntos
Varredura Diferencial de Calorimetria/métodos , Peptídeos/química , Conformação Proteica , Temperatura Alta , Termodinâmica
14.
Oncotarget ; 6(38): 40464-79, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26588054

RESUMO

DNA replication is a highly coordinated process that is initiated at multiple replication origins in eukaryotes. These origins are bound by the origin recognition complex (ORC), which subsequently recruits the Mcm2-7 replicative helicase in a Cdt1/Cdc6-dependent manner. In budding yeast, two essential replication factors, Sld2 and Mcm10, are then important for the activation of replication origins. In humans, the putative Sld2 homolog, RECQ4, interacts with MCM10. Here, we have identified two mutants of human RECQ4 that are deficient in binding to MCM10. We show that these RECQ4 variants are able to complement the lethality of an avian cell RECQ4 deletion mutant, indicating that the essential function of RECQ4 in vertebrates is unlikely to require binding to MCM10. Nevertheless, we show that the RECQ4-MCM10 interaction is important for efficient replication origin firing.


Assuntos
Neoplasias Ósseas/genética , Replicação do DNA , Proteínas de Manutenção de Minicromossomo/metabolismo , Osteossarcoma/genética , RecQ Helicases/metabolismo , Origem de Replicação/genética , Sequência de Aminoácidos , Animais , Apoptose , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proliferação de Células , Galinhas/genética , Cromatina/genética , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Componente 2 do Complexo de Manutenção de Minicromossomo/genética , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Dados de Sequência Molecular , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , RecQ Helicases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Células Tumorais Cultivadas
15.
Biophys Chem ; 151(3): 181-6, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20621410

RESUMO

It is well established that calcium binding leads to conformational changes in S100 proteins. These conformational changes are thought to activate the protein and render a protein conformation that is capable of binding other proteins. The basic quaternary structural motif of S100 proteins is a homodimer, however there is little information if higher order non-covalent oligomers are also formed and whether these oligomers are of functional relevance. To this end we performed equilibrium analytical ultracentrifugation experiments for 16 S100 proteins (S100A1, S100A2, S100A3, S100A4, S100A5, S100A6, S100A7, S100A8, S100A9, S100A10, S100A11, S100A12, S100A13, S100B, S100P, and S100Z) under reducing conditions in the absence and presence of calcium ions. We show that the addition of calcium promotes the formation of tetrameric structures which could be further enhanced under in vivo conditions where there is an additional effect of molecular crowding.


Assuntos
Cálcio/farmacologia , Estrutura Quaternária de Proteína/efeitos dos fármacos , Proteínas S100/química , Área Sob a Curva , Humanos , Modelos Moleculares , Multimerização Proteica/efeitos dos fármacos , Proteínas S100/metabolismo , Ultracentrifugação
16.
Biochemistry ; 46(10): 2876-80, 2007 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-17295518

RESUMO

Small proteins provide convenient models for computational studies of protein folding and stability, which are usually compared with experimental data. Until recently, the unfolding of Trp-cage was considered to be a two-state process. However, no direct experimental evidence for this has been presented, and in some cases, the contrary has been suggested. To elucidate a detailed unfolding mechanism, we studied the thermodynamics of unfolding of Trp-cage by differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. The observation that at low temperatures only approximately 90-95% of Trp-cage exists in the native conformation presented an analytical challenge. Nevertheless, it was found that the DSC and CD data can be fitted simultaneously to the same set of thermodynamic parameters. The major uncertainty in such a global fit is the heat capacity change upon unfolding, DeltaCp. This can be circumvented by obtaining DeltaCp directly from the difference between heat capacity functions of the native and unfolded states. Using such an analysis it is shown that Trp-cage unfolding can be represented by a two-state model with the following thermodynamic parameters: Tm = 43.9 +/- 0.8 degrees C, DeltaH(Tm) = 56 +/- 2 kJ/mol, DeltaCp = 0.3 +/- 0.1 kJ/(mol.K). Using these thermodynamic parameters it is estimated that Trp-cage is marginally stable at 25 degrees C, DeltaG(25 degrees C) = 3.2 +/- 0.2 kJ/mol, which is only 30% more than the thermal fluctuation energy at this temperature.


Assuntos
Varredura Diferencial de Calorimetria/métodos , Dicroísmo Circular/métodos , Peptídeos/química , Desnaturação Proteica , Dobramento de Proteína , Termodinâmica , Triptofano/química
17.
J Am Chem Soc ; 128(1): 30-1, 2006 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-16390106

RESUMO

beta-Sheets are a common secondary structural element found in proteins. The difficulty in studying beta-sheet folding and stability is that their formation is often dependent on the tertiary structural environment within the protein. However, the discovery of water-soluble beta-hairpin peptides has allowed them to be used as model systems because they represent the smallest units of beta-sheet structure independent of tertiary structural context. Trpzip4 has been used as a model beta-hairpin peptide to study beta-hairpin folding and stability because it is highly soluble in aqueous solutions, maintains its monomeric state, and shows reversible cooperative thermal unfolding. The previously determined thermodynamic parameters for trpzip4 thermal unfolding vary depending on the spectroscopic probe used, which questions the assumption that trpzip4 unfolds in a two-state manner. Here we provide direct calorimetric evidence that the unfolding of trpzip4 follows a two-state unfolding mode. Furthermore, the thermal unfolding of trpzip4 monitored using near- and far-UV-CD yielded thermodynamic parameters similar to those determined calorimetrically, providing additional evidence for a two-state unfolding mode.


Assuntos
Peptídeos/química , Dobramento de Proteína , Varredura Diferencial de Calorimetria/métodos , Dicroísmo Circular , Estrutura Secundária de Proteína , Termodinâmica , Triptofano/química
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