Exposure Of Hydrophobic Surfaces Research Articles

A structurally derived universal mechanism for the catalytic cycle of the tail-anchored targeting factor Get3.

Tail-anchored (TA) membrane proteins, accounting for ∼2% of proteomes, are primarily targeted post-translationally to the ER membrane by the Guided Entry of TA proteins (GET) pathway. For this complicated process, it remains unknown how the central targeting factor Get3 uses nucleotide to facilitate large conformational changes to recognize then bind clients while also preventing exposure of hydrophobic surfaces. We have identified the GET pathway in Giardia intestinalis. Using x-ray crystallography and cryo-EM, I will present a series of structures including the first structure of the Get3/client complex in the critical post-nucleotide-hydrolysis state, demonstrating that Get3 reorganizes the client-binding domain (CBD) to accommodate and shield the client transmembrane helix. Four additional structures of GiGet3, spanning the nucleotide-free (apo) open to closed transition and the ATP-bound state, reveal the details of nucleotide stabilization and occluded CBD. This work provides the first direct data of the GET pathway outside of opishthokonts and resolves key conundrums, allowing for a complete model of the dramatic conformational landscape of Get3. I will further discuss an exciting Get3-like protein found associated with photosynthesis.

Heparin-Induced Changes of Vascular Endothelial Growth Factor (VEGF165) Structure.

Vascular endothelial growth factor-A (VEGF-A), a secreted homodimeric glycoprotein, is a critical regulator of angiogenesis in normal and pathological states. The binding of heparin (HE) to VEGF165 (the major form of VEGF-A) modulates the angiogenesis-related cascade, but the mechanism of the observed changes at the structural level is still insufficiently explored. In the present study, we examined the effect of HE on the structural and physicochemical properties of recombinant human VEGF165 (rhVEGF165). The HE binding results in an increase of hydrophobic surface exposure in rhVEGF165 without changes in its secondary structure. Differential scanning calorimetry measurements for intact and HE-bound rhVEGF165 reveals the absence of any pronounced thermally induced transitions in the protein in the temperature range from 20 to 100 °C. The apolar area increase during the heparin binding explains the pronounced HE-induced oligomerization/aggregation of rhVEGF165, as studied by chemical glutaraldehyde cross-linking and dynamic light scattering. Molecular modeling and docking techniques were used to model the full structure of dimeric VEGF165 and to reveal putative molecular mechanisms underlying the function of the VEGF165/HE system. In general, the results obtained can be a basis for explaining the modulating effect of HE on the biological activity of VEGF-A.

Conformational stability of SARS-CoV-2 glycoprotein spike variants

The severe acute respiratory syndrome spread worldwide, causing a pandemic. SARS-CoV-2 mutations have arisen in the spike, a glycoprotein at the viral envelope and an antigenic candidate for vaccines against COVID-19. Here, we present comparative data of the glycosylated full-length ancestral and D614G spike together with three other transmissible strains classified by the World Health Organization as variants of concern: beta, gamma, and delta. By showing that D614G has less hydrophobic surface exposure and trimer persistence, we place D614G with features that support a model of temporary fitness advantage for virus spillover. Furthermore, during the SARS-CoV-2 adaptation, the spike accumulates alterations leading to less structural stability for some variants. The decreased trimer stability of the ancestral and gamma and the presence of D614G uncoupled conformations mean higher ACE-2 affinities compared to the beta and delta strains. Mapping the energetics and flexibility of variants is necessary to improve vaccine development.

A finely tuned interplay between calcium binding, ionic strength and pH modulates conformational and oligomerization equilibria in the Respiratory Syncytial Virus Matrix (M) protein

As in most enveloped RNA viruses, the Respiratory Syncytial Virus Matrix (RSV-M) protein plays key roles in viral assembly and uncoating. It also plays non-structural roles related to transcription modulation through nucleo-cytoplasmic shuttling and nucleic acid binding ability. We dissected the structural and conformational changes underlying the switch between multiple functionalities, identifying Ca2+ binding as a key factor. To this end, we tackled the analysis of M's conformational stability and equilibria. While in silico calculations predict two potential calcium binding sites per protomer, purified RSV-M dimer contains only one strongly bound calcium ion per protomer. Incubation of RSV-M in the presence of excess Ca2+ leads to an increase in the thermal stability, confirming additional Ca2+ binding sites. Moreover, mild denaturant concentrations trigger the formation of higher order oligomers which are otherwise prevented under Ca2+ saturation conditions, in line with the stabilizing effect of the additional low affinity binding site. On the other hand, Ca2+ removal by chelation at pH 7.0 causes a substantial decrease in the thermal stability leading to the formation of amorphous, spherical-like aggregates, as assessed by TEM. Even though the Ca2+ content modulates RSV-M oligomerization propensity, it does affect its weak RNA binding ability. RSV-M undergoes a substantial conformational change at pHs 4.0 to 5.0 that results in the exposure of hydrophobic surfaces, an increase beta sheet content but burial of tryptophan residues. While low ionic strength promotes dimer dissociation at pH 4.0, physiological concentrations of NaCl lead to the formation of soluble oligomers smaller than 400 kDa at pH 4.0 or insoluble aggregates with tubular morphology at pH 5.0, supporting a fine tuning by pH. Furthermore, the dissociation constants estimated for the low- and high affinity calcium binding sites are 13 μM and 58 nM, respectively, suggesting an intracellular calcium sensing mechanism of RSV-M upon infection.We uncover a finely tuned interplay between calcium binding, ionic strength, and pH changes compatible with the different cellular compartments where M plays key roles, revealing diverse conformational equilibria, oligomerization, and high order structures, required to stabilize the virion particle by a layer of molecules positioned between the membrane and the nucleocapsid.

Molecular organization of the early stages of nucleosome phase separation visualized by cryo-electron tomography

It has been proposed that the intrinsic property of nucleosome arrays to undergo liquid-liquid phase separation (LLPS) invitro is responsible for chromatin domain organization invivo. However, understanding nucleosomal LLPS has been hindered by the challenge to characterize the structure of the resulting heterogeneous condensates. We used cryo-electron tomography and deep-learning-based 3D reconstruction/segmentation to determine the molecular organization of condensates at various stages of LLPS. We show that nucleosomal LLPS involves a two-step process: a spinodal decomposition process yielding irregular condensates, followed by their unfavorable conversion into more compact, spherical nuclei that grow into larger spherical aggregates through accretion of spinodal materials or by fusion with other spherical condensates. Histone H1 catalyzes more than 10-fold the spinodal-to-spherical conversion. We propose that this transition involves exposure of nucleosome hydrophobic surfaces causing modified inter-nucleosome interactions. These results suggest a physical mechanism by which chromatin may transition from interphase to metaphase structures.

Structurally derived universal mechanism for the catalytic cycle of the tail-anchored targeting factor Get3.

Tail-anchored (TA) membrane proteins, accounting for roughly 2% of proteomes, are primarily targeted posttranslationally to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. For this complicated process, it remains unknown how the central targeting factor Get3 uses nucleotide to facilitate large conformational changes to recognize then bind clients while also preventing exposure of hydrophobic surfaces. Here, we identify the GET pathway in Giardia intestinalis and present the structure of the Get3-client complex in the critical postnucleotide-hydrolysis state, demonstrating that Get3 reorganizes the client-binding domain (CBD) to accommodate and shield the client transmembrane helix. Four additional structures of GiGet3, spanning the nucleotide-free (apo) open to closed transition and the ATP-bound state, reveal the details of nucleotide stabilization and occluded CBD. This work resolves key conundrums and allows for a complete model of the dramatic conformational landscape of Get3.

The effect of non-specific binding of Pd(II) complexes with N-heteroaromatic hydrazone ligands on the protein structure

Previously, the cytotoxic actions of five Pd(II) complexes with bidentate N-heteroaromatic chelators (complexes 1?5) on a palette of several cancer cell lines were investigated. However, the results of the cytotoxic activity did not correlate with the hydrophobic character of the complexes. To gain further insight into the structure?activity relationship, essential for the design of novel potential drugs, other factors, such as non-specific interactions with cellular proteins, have to be taken into account. To explore the potential non-specific influence of the complexes on protein structures, ovalbumin (OVA) was chosen as a model system to mimic cellular non-specific crowding environments with high protein concentrations. A Fourier-transform infrared spectroscopy study implied that the binding of 3 and 4 led to only moderate alternations in the secondary structures of the protein, without the possibility to penetrate into hydrophobic core of the protein and disruption of protein native fold. Contrary, the effect of complex 5 on OVA secondary structures was concentration- dependent. While the lower concentration of complex 5 had no effect on OVA structure, a doubled concentration of complex 5 led to complete disruption of the content native-like secondary structures. The concentration-dependent effect of complex 5 on the changes in secondary structures and considerable increase in the exposure of OVA hydrophobic surfaces to water may be related to a potential crosslinking that leads to OVA aggregation.

Promoting enzymatic hydrolysis of aggregated bamboo crystalline cellulose by fast microwave-assisted dicarboxylic acid deep eutectic solvents pretreatments

Deep eutectic solvents (DESs) have received considerable interests as pretreatment solvents for biorefinery. In the present work, five kinds of dicarboxylic acids based DESs were introduced to pretreatments on moso bamboo (MB) with microwave irradiation assistance. Factors influencing the enzymatic conversion of MB cellulose to glucose were determined. With the fast heating, pretreated samples all present significant delignification and hemicelluloses matrix removal, thus improving the enzymatic conversion yield from 15% of MB to ~60%. For the DESs, hydrogen donors with less carbon atoms (oxalic acid) and more hydroxyl groups (tartaric acid) displayed higher efficiency due to separation of aggregated cellulose microfibrils. The microwave assisted DESs (MW-DESs) pretreatments also contributed to cellulose crystal variations including decrystallization and more exposure of hydrophobic surfaces, which are beneficial for followed cellulase adsorption and hydrolysis. The exploration of fast MW-DESs pretreatments may expand the potentials of lignocellulose biomass on effective and applicable biorefinery.

Dual role of the active site 'lid' regions of protochlorophyllide oxidoreductase in photocatalysis and plant development.

Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light-dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR-Pchlide-NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a 'lid' to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR 'octamer' has been isolated and its structure investigated by cryo-electron microscopy at 7.7Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation - a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development.

Human αB-crystallin discriminates between aggregation-prone and function-preserving variants of a client protein

BackgroundThe eye lens crystallins are highly soluble proteins that are required to last the lifespan of an organism due to low protein turnover in the lens. Crystallin aggregation leads to formation of light-scattering aggregates known as cataract. The G18V mutation of human γS-crystallin (γS-G18V), which is associated with childhood-onset cataract, causes structural changes throughout the N-terminal domain and increases aggregation propensity. The holdase chaperone protein αB-crystallin does not interact with wild-type γS-crystallin, but does bind its G18V variant. The specific molecular determinants of αB-crystallin binding to client proteins is incompletely charcterized. Here, a new variant of γS, γS-G18A, was created to test the limits of αB-crystallin selectivity. MethodsMolecular dynamics simulations were used to investigate the structure and dynamics of γS-G18A. The overall fold of γS-G18A was assessed by circular dichroism (CD) spectroscopy and intrinsic tryptophan fluorescence. Its thermal unfolding temperature and aggregation propensity were characterized by CD and DLS, respectively. Solution-state NMR was used to characterize interactions between αB-crystallin and γS-G18A. ResultsγS-G18A exhibits minimal structural changes, but has compromised thermal stability relative to γS-WT. The placement of alanine, rather than valine, at this highly conserved glycine position produces minor changes in hydrophobic surface exposure. However, human αB-crystallin does not bind the G18A variant, in contrast to previous observations for γS-G18V, which aggregates at physiological temperature. ConclusionsαB-crystallin is capable of distinguishing between aggregation-prone and function-preserving variants, and recognizing the transient unfolding or minor conformers that lead to aggregation in the disease-related variant. General significanceHuman αB-crystallin distinguishes between highly similar variants of a structural crystallin, binding the cataract-related γS-G18V variant, but not the function-preserving γS-G18A variant, which is monomeric at physiological temperature.

Human islet amyloid polypeptide (hIAPP) aggregation in type 2 diabetes: Correlation between intrinsic physicochemical properties of hIAPP aggregates and their cytotoxicity

A large number of pathological diseases are known now to be associated with the misfolding and the aberrant oligomerization and deposition of peptides and proteins into various aggregates. One of these peptides is islet amyloid polypeptide (IAPP), which is responsible for amyloid formation in type 2 diabetes. The mechanism of IAPP amyloid formation in vivo and in vitro is not well understood and the factors behind the peptide aggregates toxicity are not fully defined. Therefore, the precise nature of toxic agents still remains to be elucidated. In this context, first we used a complementary biophysical approach to undertake a systematic study of the hIAPP aggregation process with focus on the lag phase, followed by the study of their degrees of toxicity when added to the extracellular medium of pancreatic cells.The structural properties of hIAPP aggregates are characterized by evaluating their size with DLS, their surface hydrophobicity with ANS, and the interactions between monomers through the intrinsic fluorescence of aromatic residues or by the quenching of these residues mainly the tyrosine in position 37. Our results indicate that despite the method used to study hIAPP aggregation, the obtained curve is easily well fitted in a sigmoidal curve but with some differences. In fact, the analysis of the kinetic parameters gives different information about the hIAPP aggregation process such as lag time and growth rate. Moreover, a high surface hydrophobicity and small size of the aggregates, mainly for the species formed during the lag time, shows strong correlation with the cytotoxicity. These findings provide new insights into the structural changes during hIAPP aggregation and are consistent with a model in which the exposure of hydrophobic surfaces and the small size of aggregates formed during the early stage of the process are crucial for their cytotoxicity.

Insight into the Hydration of Cationic Surfactants: A Thermodynamic and Dielectric Study of Functionalized Quaternary Ammonium Chlorides.

Hydrophobic interactions are one of the main thermodynamic driving forces in self-assembly, folding, and association processes. To understand the dehydration-driven solvent exposure of hydrophobic surfaces, the micellization of functionalized decyldimethylammonium chlorides, XC10Me2N+Cl–, with a polar functional group, X = C2OH, C2OMe, C2OC2OMe, C2OOEt, together with the “reference” compound decyltrimethylammonium chloride, C10Me3N+Cl–, was investigated in aqueous solution by density measurements, isothermal titration calorimetry (ITC), and dielectric relaxation spectroscopy (DRS). From the density data, the apparent molar volumes of monomers and micelles were estimated, whereas the ITC data were analyzed with the help of a model equation, yielding the thermodynamic parameters and aggregation number. From the DRS spectra, effective hydration numbers of the free monomers and micelles were deduced. The comprehensive analysis of the obtained results shows that the thermodynamics of micellization are strongly affected by the nature of the functional group. Surprisingly, the hydration of micelles formed by surfactant cations with a single alkyl chain on quaternary ammonium is approximately the same, regardless of the alkyl chain length or functionalization of the headgroup. However, notable differences were found for the free monomers where increasing polarity lowers the effective hydration number.

Effects of Cu2+ on alkaline phosphatase from Macrobrachium rosenbergii

To gain insight into the effect of Cu2+ on the activity and structure of alkaline phosphatase (ALP) from Macrobrachium rosenbergii, the enzyme was purified using ammonium sulfate fractionation, Sephacryl S-200, and DEAE anion exchange chromatography. We studied Cu2+-mediated inhibition and aggregation of ALP, and found that Cu2+ significantly inactivated ALP activity with an IC50 of 1.47 ± 0.02 mM. We further revealed that Cu2+ reversibly inhibited ALP in a mixed-type manner with Ki = 0.41 ± 0.02 mM. Time-interval kinetics showed that the inhibition followed first-order reaction kinetics. This process was associated with conformational changes and significant transient free-energy change. Spectrofluorometry results showed that Cu2+ induced ALP tertiary structural changes, including the exposure of hydrophobic surfaces that directly induced ALP aggregation. The results provide new information regarding ALP from M. rosenbergii.

Structural perturbation by arsenic triggers the aggregation of hen egg white lysozyme by promoting oligomers formation

Arsenic trioxide is one of the most common metallic pollutants entering the food chain both by human activities and nature. Its entry inside the living organism through food, air and water results into the accumulation of heavy metal in several tissues which manifest several metabolic or hormonal disorders. Till now the effect of arsenic trioxide on protein misfolding and aggregation culminating into several neurodegenerative disorders is poorly understood. In the present study, we reveal the aggregation process of Hen Egg White Lysozyme (HEWL) in presence of arsenic trioxide (As2O3) at physiological conditions. We show that As2O3 promote the in vitro aggregation of HEWL in concentration dependent manner. Early phase of aggregation is observed to be induced by exposure of hydrophobic surfaces which later reorganized to promote further self-association leading to β sheet structure. Presence of lower ordered oligomers after two days and higher ordered oligomers along with amorphous aggregates after week long incubation indicate that As2O3 drives the self-assembly of lysozyme towards oligomeric form.

Characterizations of the Interactions between Escherichia coli Periplasmic Chaperone HdeA and Its Native Substrates during Acid Stress.

The bacterial acid-resistant chaperone HdeA is a "conditionally disordered" protein that functions at low pH when it undergoes a transition from a well-folded dimer to an unfolded monomer. The dimer dissociation and unfolding processes result in exposure of hydrophobic surfaces that allows binding to a broad range of client proteins. To fully elucidate the chaperone mechanism of HdeA, it is crucial to understand how the activated HdeA interacts with its native substrates during acid stress. Herein, we present a nuclear magnetic resonance study of the pH-dependent HdeA-substrate interactions. Our results show that the activation of HdeA is not only induced by acidification but also regulated by the presence of unfolded substrates. The variable extent of unfolding of substrates differentially regulates the HdeA-substrate interaction, and the binding further affects the HdeA conformation. Finally, we show that HdeA binds its substrates heterogeneously, and the "amphiphilic" model for HdeA-substrate interaction is discussed.

Towards Binding Mechanism of Cu2+ on Creatine Kinase from Pelodiscus sinensis: Molecular Dynamics Simulation Integrating Inhibition Kinetics Study.

Cu2+ is well known to play important roles in living organisms having bifacial distinction: essential microelement that is necessary for a wide range of metabolic processes but hyper-accumulation of Cu2+ can be toxic. The physiological function of Cu2+ in ectothermic animals such as Pelodiscus sinensis (Chinese soft-shelled turtle) has not been elucidated. In this study, we elucidated effect of Cu2+ on the energy producing metabolic enzyme creatine kinase (CK), which might directly affect energy metabolism and homeostasis of P. sinensis. We first conducted molecular dynamics (MD) simulations between P-CK and Cu2+ and conducted the inactivation kinetics including spectrofluorimetry study. MD simulation showed that Cu2+ blocked the binding site of the ATP cofactor, indicating that Cu2+ could directly inactivate P-CK. We prepared the muscle type of CK (P-CK) and confirmed that Cu2+ conspicuously inactivated the activity of P-CK (IC50 = 24.3 μM) and exhibited non-competitive inhibition manner with creatine and ATP in a first-order kinetic process. This result was well matched to the MD simulation results that Cu2+-induced non-competitive inactivation of P-CK. The spectrofluorimetry study revealed that Cu2+ induced tertiary structure changes in PCK accompanying with the exposure of hydrophobic surfaces. Interestingly, the addition of osmolytes (glycine, proline, and liquaemin) effectively restored activity of the Cu2+-inactivated P-CK. Our study illustrates the Cu2+-mediated unfolding of P-CK with disruption of the enzymatic function and the protective restoration role of osmolytes on P-CK inactivation. This study provides information of interest on P-CK as a metabolic enzyme of ectothermic animal in response to Cu2+ binding.

Stepwise unfolding of Ribonuclease A by a biosurfactant

The interaction of Ribonuclease A (RNase A) with the bile salt, sodium deoxycholate (NaDC) is meticulously investigated using various spectroscopic techniques. The binding isotherm constructed from the modulation of intrinsic tyrosine fluorescence of the protein following interaction with NaDC conspicuously reveals an intrinsically complex stepwise interaction process which proceeds through the formation of distinct conformational states of the protein. The conformational transitions are found to occur at c1=2.2mM, and c2=7.2mM of NaDC. These results are subsequently corroborated from the studies of excited-state relaxation dynamics of the intrinsic tyrosine residues of RNase A, and the modulations in fluorescence behavior of an extrinsic probe (ANS) bound to the protein. The far-UV circular dichroism spectral analyses unveil only nominal influence on the secondary structural element of the protein up to [NaDC]=c1=2.2mM, which is then followed by marked disruption of the secondary structure of RNase A following further addition of NaDC. This clearly accounts for differential interaction behaviors of RNase A with the monomeric and micellar forms of NaDC (CMC of NaDC in aqueous buffer is estimated to be ∼3.0mM). In Region-I (up to c1=2.2mM), the protein:surfactant interaction is argued to be predominantly governed by electrostatic/ionic interaction force. Subsequently, in Region-II (up to c2=7.2mM) the RNase A:NaDC interaction accompanies major denaturation of the protein (∼17% loss of the secondary structure of RNase A at c2=7.2mM) resulting in significant exposure of hydrophobic surfaces of the protein. However, the tertiary structure of the protein remains essentially unperturbed within the concentration regime of NaDC under study.

Hydrogen peroxide (H2O2) irreversibly inactivates creatine kinase from Pelodiscus sinensis by targeting the active site cysteine

Creatine kinase (EC 2.7.3.2, CK) plays an important role in cellular energy metabolism and homeostasis by catalysing the transfer of phosphate between ATP and creatine phosphate. In this study, we investigated the effects of H2O2 on PSCKM (muscle type creatine kinase from Pelodiscus sinensis) by the integrating method between enzyme kinetics and docking simulations. We found that H2O2 strongly inactivated PSCKM (IC50=0.25mM) in a first-order kinetic process, and targeted the active site cysteine directly. A conformational study showed that H2O2 did not induce the tertiary structural changes in PSCKM with no extensive exposure of hydrophobic surfaces. Sequential docking simulations between PSCKM and H2O2 indicated that H2O2 interacts with the ADP binding region of the active site, consistent with experimental results that demonstrated H2O2-induced inactivation. Our study demonstrates the effect of H2O2 on PSCKM enzymatic function and unfolding, and provides important insight into the changes undergone by this central metabolic enzyme in ectothermic animals in response to the environment.

The Hsp60 protein of helicobacter pylori displays chaperone activity under acidic conditions

The heat shock protein, Hsp60, is one of the most abundant proteins in Helicobacter pylori. Given its sequence homology to the Escherichia coli Hsp60 or GroEL, Hsp60 from H. pylori would be expected to function as a molecular chaperone in this organism. H. pylori is an organism that grows on the gastric epithelium, where the pH can fluctuate between neutral and 4.5 and the intracellular pH can be as low as 5.0. This study was performed to test the ability of Hsp60 from H. pylori to function as a molecular chaperone under mildly acidic conditions. We report here that Hsp60 could suppress the acid-induced aggregation of alcohol dehydrogenase (ADH) in the 7.0–5.0pH range. Hsp60 was found to undergo a conformational change within this pH range. It was also found that exposure of hydrophobic surfaces of Hsp60 is significant and that their exposure is increased under acidic conditions. Although, alcohol dehydrogenase does not contain exposed hydrophobic surfaces, we found that their exposure is triggered at low pH. Our results demonstrate that Hsp60 from H. pylori can function as a molecular chaperone under acidic conditions and that the interaction between Hsp60 and other proteins may be mediated by hydrophobic interactions.

Binding studies of caffeine and theophylline to bovine serum albumin: Calorimetric and spectroscopic approach

Binding studies of pharmacologically active drugs-caffeine and theophylline to bovine serum albumin (BSA) in phosphate buffer solution, pH=7.4 have been investigated using spectroscopic (UV–visible, fluorescence and nuclear magnetic resonance) and calorimetric (isothermal titration calorimetry) techniques. The derived parameters obtained from all the techniques suggest that the magnitude of binding affinity of BSA-drug follows the order: theophylline>caffeine, which may be attributed to greater acidity of former at physiological pH. UV–visible and fluorescence spectroscopy suggests 1:1 stoichiometry of drug-protein interactions, while calorimetric results support two binding sites. Apart from the tryptophan binding site, a low affinity site also exists which is assessed from the calorimetric measurements. The negative enthalpy and positive entropy, both favors the binding process, and the feasibility of binding is also supported by negative value of Gibbs free energy. The positive heat capacity, consistent with the positive entropy, may be attributed to the exposure of hydrophobic protein surfaces. The observed positive changes in chemical shifts, ∆δ may be due to the deshielding of drug protons owing to hydrophilic-ionic/hydrogen bonding interactions with BSA.