We report the evolution and confinement of atomically precise and luminescent gold clusters in a small protein, lysozyme (Lyz) using detailed mass spectrometric (MS) and other spectroscopic investigations. A maximum of 12 Au0 species could be bound to a single Lyz molecule irrespective of the molar ratio of Lyz : Au3+ used for cluster growth. The cluster-encapsulated protein also forms aggregates similar to the parent protein. Time dependent studies reveal the emergence of free protein and the redistribution of detached Au atoms, at specific Lyz to Au3+ molar ratios, as a function of incubation time, proposing inter-protein metal ion transfer. The results are in agreement with the studies of inter-protein metal transfer during cluster growth in similar systems. We believe that this study provides new insights into the growth of clusters in smaller proteins. © 2013 The Royal Society of Chemistry.

Pradeep Thalappil

Department Of Chemistry

Ananya Baksi

Kamalesh Chaudhari

CLUSTER GROWTH

GOLD CLUSTERS

incubation time

ION TRANSFER

METAL TRANSFERS

Molar ratio

SMALL PROTEINS

Spectroscopic investigations

Time dependent

Aggregates

Enzymes

Gold

Metal ions

Proteins

Gold compounds

Lysozyme

Quantum Dot

article

chemistry

Circular dichroism

dimerization

Mass spectrometry

metabolism

protein secondary structure

Hydrogen-Ion Concentration

Muramidase

Protein Structure, Secondary

quantum dots

Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

IIT Madras is a public technical and research university located in Chennai, Tamil Nadu. Founded in 1959, it is recognised as an Institute of National Importance.

IIT Madras has been ranked as the top engineering institute in India for four years in a row by the National Institutional Ranking Framework of the MHRD

It currently offers undergraduate, postgraduate and research degrees across 16 disciplines in Engineering, Sciences, Humanities and Management. About 596 faculty belonging to science and engineering departments and centres of the Institute are engaged in teaching, research and industrial consultancy.

IIT Madras

Nanoscale

A new synthetic protocol is introduced which preserves the secondary structure of protecting proteins encapsulating a luminescent atomically-precise silver cluster. This was achieved by using a preformed triphenylphosphine (TPP)-protected silver cluster as the precursor forming bovine serum albumin (BSA)- and human serum albumin (HSA)-protected Ag18 clusters. This is the first example of the formation of luminescent protein-protected clusters in a neutral medium, without using any reducing agent, which results in minimal alteration of the protein structure during cluster growth. The cluster formed showed exceptional stability, unlike other silver clusters of this class. The formation of these red luminescent clusters was visualized by UV-vis and photoluminescence spectroscopy. The identification of Ag18 core was made through matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), and a plausible mechanism of the formation was identified by monitoring the systematic growth of the cluster core by time-dependent MALDI MS experiments and electrospray ionization mass spectrometry (ESI MS) of the reaction mixture. The cluster was successfully employed as a luminescent probe for cancer cell imaging. Retention of protein conformation in the clusters was confirmed through circular dichroism (CD) spectroscopy, and the same was reflected in the retention of 89% of the esterase activity of BSA in the Ag18@BSA clusters synthesized by this method, compared to only 28.7% for AgQC@BSA clusters synthesized using previous protocols, conducted in basic medium. © 2019 American Chemical Society.

Journal of Physical Chemistry C

Internalization of a Preformed Atomically Precise Silver Cluster in Proteins by Multistep Events and Emergence of Luminescent Counterparts Retaining Bioactivity

Hydrogen/deuterium exchange mass spectrometry was employed to probe the conformational changes in lysozyme (Lyz) during the course of formation of a protein-protected atomically precise Au8 cluster. MALDI MS showed the protein, Lyz, to be present in a denatured state in the cluster. Detailed ESI MS analysis of the Au-attached Lyz adducts, an intermediate of cluster formation, confirmed that these conformational changes are brought about by Au-S bond formation and a similar conformation is retained in the final cluster. These results were supported by computational results, which showed an increase in solvent accessible surface area upon the formation of the adducts. Infrared spectroscopy established that change in the rate as well as the extent of the hydrogen/deuterium exchange observed in the cluster was due to the change in the amide II region of the encapsulating protein. Hydrogen/deuterium exchange ESI MS of Cu adducts of Lyz showed a lower degree of denaturation than their Au counterparts. XPS analysis revealed that Cu binds differently to Lyz than it does with Au, which is likely because of the stronger soft-soft Au-S interaction. Alkali metal ion binding, on the other hand, does not affect the protein conformation because such ions do not affect the disulfide bonds. © 2019 American Chemical Society.

Conformational Changes of Protein upon Encapsulation of Noble Metal Clusters: An Investigation by Hydrogen/Deuterium Exchange Mass Spectrometry

Abstract: Materials emitting white luminescence are receiving increasing attention due to their potential applications in electroluminescent devices, information displays and fluorescent sensors. To produce white light, one must have either three primary colors, blue, green and red or two colors, blue and orange. In this paper, we have used thiol/phosphine protected red luminescent silver nanoclusters (Ag NCs), [Ag29(BDT)12(PPh3)4]3-(BDT=1,3-benzenedithiol), [AuxAg29-x(BDT)12(PPh3)4]3- and Ag 29(LA) 12 (LA = lipoic acid) as one of the fluorophores for white light emission. These clusters are mixed with blue luminescent silicon nanoparticles (Si NPs) and green luminescent fluorescein isothiocyanate (FITC). The mixtures show white luminescence with CIE coordinates of (0.31, 0.34), (0.33, 0.35) and (0.29, 0.31) which are in good agreement with pure white light (0.33, 0.33). The other clusters with yellow, blue, orange, etc., luminescence can also be used to make white light. This work provides a prospective pathway for white light emission based on atomically precise noble metal NCs. Graphical abstract: Synopsis Monolayer protected noble metal nanoclusters such as [Ag29(BDT)12(PPh3)4]3-(BDT=1,3-benzenedithiol), [AuxAg29-x(BDT)12(PPh3)4]3- and Ag 29(LA) 12 (LA = lipoic acid) are used as red luminophores which can produce white light emission when mixed with blue luminescent silicon nanoparticles (Si NPs) and green luminescent fluorescein isothiocyanate (FITC). The mixtures produce white luminescence with CIE (Commission Internationale d’Eclair) coordinates of (0.31, 0.34), (0.33, 0.35) and (0.29, 0.31), respectively. All-cluster-based white light emission is indeed possible.[Figure not available: see fulltext.]. © 2018, Indian Academy of Sciences.

Fulltext

Journal of Chemical Sciences

Atomically precise cluster-based white light emitters §

Ion mobility mass spectrometry studies on Aun-Lyz adducts showed gradual unfolding of the protein structure during binding of Au+ to the protein. The change of the charge state envelope in Aun-Lyz from that of Lyz in ESI MS data confirmed the relaxation of the protein structure. This Au+ binding occurs at cysteine sites through the breakage of disulfide bonds and this ruptures the H-bonded folded network structure of the protein leading to ∼30% change in helicity. Nearly 15% loss in the total H-bonding occurred during the attachment of 8 Au to the protein as calculated by a molecular dynamics simulation. Different Aun-Lyz structures were simulated, which confirmed significant unfolding of the protein. The structural insights were used to understand similar unfolding in the solution state as seen via circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. This open structure is indeed necessary to accommodate a cluster core inside a protein cavity during luminescent cluster synthesis. These studies unambiguously establish noble metal binding-induced conformational changes of protein structures to accommodate the clusters. © 2017 American Chemical Society.

Gold-Induced Unfolding of Lysozyme: Toward the Formation of Luminescent Clusters

In this paper, we report the studies relevant to controlling the size of protein-protected gold nanoclusters (AuNCs). In order to demonstrate this, we have chosen bovine apo α-Lactalbumin (apo-α-LA), which has a specific binding site for Ca2+, and La3+ occupies this position when apo-α-LA is treated with lanthanum trichloride. When the Apo-α-LA is treated with Au3+, it results in the formation of a protein-coated Au10 nanocluster where the protein reduces Au3+ to Au0 and protects the nanoclusters (apo-α-LA-AuNCs) which are luminescent. In these protein-protected luminescent gold nanoclusters, the protein is involved both in reduction as well as protection, thereby supporting green synthesis. As La3+ occupies the Ca2+ site in apo-α-LA, the size of AuNCs formed is reduced to smaller than the Au10, where the size is dependent on the extent to which La3+ is bound to the protein with a concomitant increase in luminescence. The apo-α-LA-AuNCs and the same formed in the presence of different concentrations of La3+-bound protein were all characterized by analytical, spectral, and microscopy techniques. Control of the size of AuNCs formed was also studied by using Gd3+ instead of La3+ and found similar results. In particular, the size variation of AuNCs was clearly demonstrated by MALDI-TOF-MS and HRTEM. Thus, the apo-α-LA protein-coated gold nanocluster is a useful green material for suitable applications. © 2017 American Chemical Society.

ACS Sustainable Chemistry and Engineering

Green Synthesis of Protein-Protected Fluorescent Gold Nanoclusters (AuNCs): Reducing the Size of AuNCs by Partially Occupying the Ca2+ Site by La3+ in Apo-α-Lactalbumin

Protein-encapsulated gold cluster aggregates: The case of lysozyme

Journal	Nanoscale
ISSN	20403364
Open Access	No