Biomedical subjects

Jenny J Yang

Publications and source records attributed to Jenny J Yang.

18 recordsLinked to original sources

PubMed2006

Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins.

The EF-hand protein with a helix-loop-helix Ca(2+) binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca(2+) in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF-hand and EF-like Ca(2+)-binding proteins. The canonical EF-hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N- and C-terminal EF-hands within S100 and S100-like proteins, a new signature profile has been established to allow for the identification of pseudo EF-hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF-hands. Furthermore, using the developed patterns, we have identified zero pseudo EF-hand motif and 467 canonical EF-hand Ca(2+) binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF-hand motifs are phylogenetically younger than canonical EF-hand motifs. Our prediction of Ca(2+) binding motifs provides not only an insight into the role of Ca(2+) and Ca(2+)-binding proteins in bacterial systems, but also a way to explore and define the role of Ca(2+) in other biological systems (calciomics).

Amino Acid Motifs↗

PubMed2006

Functional elements on SIRPalpha IgV domain mediate cell surface binding to CD47.

SIRPalpha and SIRPbeta1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPalpha with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPbeta1 shares highly homologous extracellular IgV structure with SIRPalpha, it does not bind to CD47. Here, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPalpha, but not SIRPbeta1, which determine the extracellular binding interaction of SIRPalpha to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPalpha directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPalpha extracellular binding mediated cell interactions and cell migration. Another SIRPalpha-specific residue, Met102, appears to assist SIRPalpha IgV binding through Gln67 and Ala/Val57. An essential role of these amino acid residues in SIRPalpha binding to CD47 was further confirmed by introducing these residues into the SIRPbeta1 IgV domain, which dramatically converts SIRPbeta1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPalpha binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses.

Amino Acid Sequence↗

PubMed2006

Predicting calcium-binding sites in proteins - a graph theory and geometry approach.

Identifying calcium-binding sites in proteins is one of the first steps towards predicting and understanding the role of calcium in biological systems for protein structure and function studies. Due to the complexity and irregularity of calcium-binding sites, a fast and accurate method for predicting and identifying calcium-binding protein is needed. Here we report our development of a new fast algorithm (GG) to detect calcium-binding sites. The GG algorithm uses a graph theory algorithm to find oxygen clusters of the protein and a geometric algorithm to identify the center of these clusters. A cluster of four or more oxygen atoms has a high potential for calcium binding. High performance with about 90% site sensitivity and 80% site selectivity has been obtained for three datasets containing a total of 123 proteins. The results suggest that a sphere of a certain size with four or more oxygen atoms on the surface and without other atoms inside is necessary and sufficient for quickly identifying the majority of the calcium-binding sites with high accuracy. Our finding opens a new avenue to visualize and analyze calcium-binding sites in proteins facilitating the prediction of functions from structural genomic information.

Algorithms↗

PubMed2006

Using protein design to dissect the effect of charged residues on metal binding and protein stability.

Ca2+ controls biological processes by interacting with proteins with different affinities, which are largely influenced by the electrostatic interaction from the local negatively charged ligand residues in the coordination sphere. We have developed a general strategy for rationally designing stable Ca2+- and Ln3+-binding proteins that retain the native folding of the host protein. Domain 1 of cluster differentiation 2 (CD2) is the host for the two designed proteins in this study. We investigate the effect of local charge on Ca2+-binding affinity based on the folding properties and metal-binding affinities of the two proteins that have similarly located Ca2+-binding sites with two shared ligand positions. While mutation and Ca2+ binding do not alter the native structure of the protein, Ca2+ binding specifically induced changes around the designed Ca2+-binding site. The designed protein with a -5 charge at the binding sphere displays a 14-, 20-, and 12-fold increase in the binding affinity for Ca2+, Tb3+, and La3+, respectively, compared to the designed protein with a -3 charge, which suggests that higher local charges are preferred for both Ca2+ and Ln3+ binding. The localized charged residues significantly decrease the thermal stability of the designed protein with a -5 charge, which has a T(m) of 41 degrees C. Wild-type CD2 has a T(m) of 61 degrees C, which is similar to the designed protein with a -3 charge. This decrease is partially restored by Ca2+ binding. The effect on the protein stability is modulated by the environment and the secondary structure locations of the charged mutations. Our study demonstrates the capability and power of protein design in unveiling key determinants to Ca2+-binding affinity without the complexities of the global conformational changes, cooperativity, and multibinding process found in most natural Ca2+-binding proteins.

Calcium↗

PubMed2006

Amyloid fibril formation by a domain of rat cell adhesion molecule.

Amyloid fibrils are protein aggregates implicated in several amyloidotic diseases. Cellular membranes with local decrease in pH and dielectric constant are associated with the amyloid formation. In this study, domain 1 of cell adhesion molecule CD2 (CD2-1) is used for studying amyloid fibril formation in a water/trifluoroethanol (TFE) mixture. CD2-1 is an all beta-sheet protein similar in topology to the amyloidogenic light chain variable domain, which deposits as amyloid in light chain amyloidosis at acidic pH. When incubated at pH 2.0 in the presence of 18% TFE, CD2-1 initiates the process to assemble into amyloid fibrils. It has been shown that TFE induces CD2-1 conformational change with a chemical transition (C(m)) of 18% (v/v). ANS (1-anilinonapthalene-8-sulfonic acid) binding was used to show that the hydrophobic surface becomes exposed under these solvent conditions. Our studies indicate that partial formation of a non-native conformation and the exposure of the hydrophobic interior could be the origins of oligomerization and fibril formation of CD2-1.

Amyloid↗

PubMed2005

Expression and optical properties of green fluorescent protein expressed in different cellular environments.

This study has investigated the expression of green fluorescent protein (GFP) variants in the cytosol and the endoplasmic reticulum (ER) of HeLa cells and evaluated the effects of the different cellular environments on the fluorescence properties of these GFP variants. Several GFP variants have been constructed by adding different N- or C-terminal signal sequences. These proteins were expressed and folded in distinct cellular compartments in HeLa cells. The localization of these GFP variants targeted to the endoplasmic recticulum was confirmed by the co-localization of DsRed2-ER as assessed by confocal microscopy. The addition of signal peptides targeting GFP variants to the ER or cytosol did not appear to alter the optical spectra of these GFP variants. However, the fluorescence intensity of these GFP variants in the ER was significantly less than that in the cytosol. Thus, the results clearly suggest that the cellular environment affects the formation and/or maturation of green fluorescence protein in vivo. These findings will be helpful in the future development and application of GFP technology aimed at investigating cellular functions performed in the ER and the cytosol.

Cytosol↗

PubMed2005

The effects of Ca2+ binding on the dynamic properties of a designed Ca2+-binding protein.

The effects of Ca(2+) binding on the dynamic properties of Ca(2+)-binding proteins are important in Ca(2+) signaling. To understand the role of Ca(2+) binding, we have successfully designed a Ca(2+)-binding site in the domain 1 of rat CD2 (denoted as Ca.CD2) with the desired structure and retained function. In this study, the backbone dynamic properties of Ca.CD2 have been investigated using (15)N spin relaxation NMR spectroscopy to reveal the effect of Ca(2+) binding on the global and local dynamic properties without the complications of multiple interactive Ca(2+) binding and global conformational change. Like rat CD2 (rCD2) and human CD2 (hCD2), residues involved in the recognition of the target molecule CD48 exhibit high flexibility. Mutations N15D and N17D that introduce the Ca(2+) ligands increase the flexibility of the neighboring residues. Ca(2+)-induced local dynamic changes occur mainly at the residues proximate to the Ca(2+)-binding pocket or the residues in loop regions. The beta-strand B of Ca.CD2 that provides two Asp for the Ca(2+) undergoes an S(2) decrease upon the Ca(2+) binding, while the DE-loop that provides one Asn and one Asp undergoes an S(2) increase. Our study suggests that Ca(2+) binding has a differential effect on the rigidity of the residues depending on their flexibility and location within the secondary structure.

Animals↗

PubMed2005

Probing site-specific calmodulin calcium and lanthanide affinity by grafting.

Ca2+ binding is essential for the biological functions of calmodulin (CaM) as a trigger/sensor protein to regulate many biological processes in the Ca2+ -signaling cascade. A challenge in understanding the mechanism of Ca2+ signaling is to obtain site-specific information about the Ca2+ binding properties of individual Ca2+ -binding sites of EF-hand proteins, especially for CaM. In this paper, we report the first estimation of the intrinsic Ca2+ affinities of the four EF-hand loops of calmoduin (I-IV) by individually grafting into the domain 1 of CD2. Taking advantage of the Trp residues in the host protein, we first determined metal-binding affinities for Tb3+, Ca2+, and La3+ for all four grafted EF-loops using Tb3+ aromatic resonance energy transfer. EF-loop I exhibits the strongest binding affinity for Ca2+, La3+, and Tb3+, while EF-loop IV has the weakest metal-binding affinity. EF-loops I-IV of CaM have dissociation constants for Ca2+ of 34, 245, 185, and 814 microM, respectively, with the order I > III approximately equal to II > IV. These findings support a charge-ligand-balanced model in which both the number of negatively charged ligand residues and the balanced electrostatic dentate-dentate repulsion by the adjacent charged residues are two major determinants for the relative Ca2+ -binding affinities of EF-loops in CaM. Our grafting method provides a new strategy to obtain site-specific Ca2+ binding properties and a better estimation of the cooperativity and conformational change contributions of coupled EF-hand proteins.

Animals↗

PubMed2005

Design of a calcium-binding protein with desired structure in a cell adhesion molecule.

Ca2+, "a signal of life and death", controls numerous cellular processes through interactions with proteins. An effective approach to understanding the role of Ca2+ is the design of a Ca2+-binding protein with predicted structural and functional properties. To design de novo Ca2+-binding sites in proteins is challenging due to the high coordination numbers and the incorporation of charged ligand residues, in addition to Ca2+-induced conformational change. Here, we demonstrate the successful design of a Ca2+-binding site in the non-Ca2+-binding cell adhesion protein CD2. This designed protein, Ca.CD2, exhibits selectivity for Ca2+ versus other di- and monovalent cations. In addition, La3+ (Kd 5.0 microM) and Tb3+ (Kd 6.6 microM) bind to the designed protein somewhat more tightly than does Ca2+ (Kd 1.4 mM). More interestingly, Ca.CD2 retains the native ability to associate with the natural target molecule. The solution structure reveals that Ca.CD2 binds Ca2+ at the intended site with the designed arrangement, which validates our general strategy for designing de novo Ca2+-binding proteins. The structural information also provides a close view of structural determinants that are necessary for a functional protein to accommodate the metal-binding site. This first success in designing Ca2+-binding proteins with desired structural and functional properties opens a new avenue in unveiling key determinants to Ca2+ binding, the mechanism of Ca2+ signaling, and Ca2+-dependent cell adhesion, while avoiding the complexities of the global conformational changes and cooperativity in natural Ca2+-binding proteins. It also represents a major achievement toward designing functional proteins controlled by Ca2+ binding.

Animals↗

PubMed2005

Calcium and lanthanide affinity of the EF-loops from the C-terminal domain of calmodulin.

To obtain site-specific information about individual EF-hand motifs, the EF-hand Ca(2+)-binding loops from site III and site IV of calmodulin (CaM) were inserted separately into a non-Ca(2+)-binding cell adhesion protein, domain 1 of CD2 (denoted as CaM-CD2-III-5G-52 and CaM-CD2-IV-5G-52). Structural analyses using various spectroscopic methods have shown that the host protein CD2 retains its native structure after the insertion of the 12-residue loops. The Tb(3+) fluorescence enhancement upon formation of a Tb(3+)-protein complex and the direct competition by La(3+) and Ca(2+) suggest that native Ca(2+)-binding pockets are formed in both engineered proteins. Moreover, as revealed by NMR, both Ca(2+) and La(3+) specifically interact with the residues at the grafted EF-loop. The CaM-CD2-III-5G-52 has stronger affinities to Ca(2+), Tb(3+) and La(3+) than CaM-CD2-IV-5G-52, indicating differential intrinsic metal-binding affinities of the EF-loops.

Amino Acid Sequence↗

PubMed2005

ATPase/helicase activities of p68 RNA helicase are required for pre-mRNA splicing but not for assembly of the spliceosome.

We have previously demonstrated that p68 RNA helicase, as an essential human splicing factor, acts at the U1 snRNA and 5' splice site (5'ss) duplex in the pre-mRNA splicing process. To further analyze the function of p68 in the spliceosome, we generated two p68 mutants (motif V, RGLD to LGLD, and motif VI, HRIGR to HLIGR). ATPase and RNA unwinding assays demonstrated that the mutations abolished the RNA-dependent ATPase activity and RNA unwinding activity. The function of p68 in the spliceosome was abolished by the mutations, and the mutations also inhibited the dissociation of U1 from the 5'ss, while the mutants still interacted with the U1-5'ss duplex. Interestingly, the nonactive p68 mutants did not prevent the transition from prespliceosome to the spliceosome. The data suggested that p68 RNA helicase might actively unwind the U1-5'ss duplex. The protein might also play a role in the U4.U6/U5 addition, which did not require the ATPase and RNA unwinding activities of p68. In addition, we present evidence here to demonstrate the functional role of p68 RNA helicase in the pre-mRNA splicing process in vivo. Our experiments also showed that p68 interacted with unspliced but not spliced mRNA in vivo.

Adenosine Triphosphatases↗

PubMed2003

Rational design of a calcium-binding protein.

Calcium ions play key roles as structural components in biomineralization and as a second messenger in signaling pathways. We have introduced a de novo designed calcium-binding site into the framework of a non-calcium-binding protein, domain 1 of CD2. The resulting protein selectively binds calcium over magnesium with calcium-binding affinity comparable to that of natural extracellular calcium-binding proteins (K(d) of 50 microM). This experiment is the first successful metalloprotein design that has a high coordination number (seven) metal-binding site constructed into a beta-sheet protein. Our results demonstrate the feasibility of designing a single calcium-binding site into a host protein, taking into account only local properties of a calcium-binding site obtained by a survey of natural calcium-binding proteins and chelators. The resulting site exhibits strong metal selectivity, suggesting that it should now be feasible to understand and manipulate signaling processes by designing novel calcium-modulated proteins with specifically desired functions and to affect their stability.

Binding Sites↗

PubMed2003

A grafting approach to obtain site-specific metal-binding properties of EF-hand proteins.

The EF-hand calcium-binding loop III from calmodulin was inserted with glycine linkers into the scaffold protein CD2.D1 at three locations to study site-specific calcium binding properties of EF-hand motifs. After insertion, the host protein retains its native structure and forms a 1:1 metal-protein complex for calcium and its analog, lanthanum. Tyrosine-sensitized Tb3+ energy transfer exhibits metal binding and La3+ and Ca2+ compete for the metal binding site. The grafted EF-loop III in different environments has similar La3+ binding affinities, suggesting that it is largely solvated and functions independently from the host protein.

Animals↗

PubMed2003

Obtaining site-specific calcium-binding affinities of calmodulin.

Calmodulin (CaM) is an EF-hand Ca(II)-binding protein involved in the regulation of many important biological processes. To date, there is a wealth of information available concerning studies to obtain site-specific calcium binding affinities of CaM, and further to estimate the cooperativity of calcium binding using mutational studies, peptide models, and proteolytic fragmentation. In this paper, we will discuss the energetics of calcium binding and the strong relationship between calcium binding cooperativity and conformational change. We then explain the difficulty of studying key determinants of calcium binding affinity of CaM due to the large change of calcium binding affinity upon mutation. Subsequently, we will introduce "grafting" as a novel approach to obtain the site-specific metal binding properties of calmodulin.

Amino Acid Sequence↗

PubMed2002

Isolated EF-loop III of calmodulin in a scaffold protein remains unpaired in solution using pulsed-field-gradient NMR spectroscopy.

Calmodulin (CaM) is a trigger calcium-dependent protein that regulates many biological processes. We have successfully engineered a series of model proteins, each containing a single EF-hand loop but with increasing numbers of Gly residues linking the EF-hand loop to a scaffold protein, cluster of differentiation 2 (CD2), to obtain the site-specific calcium-binding ability of a protein with EF-hand motifs without the interference of cooperativity. Loop III of calmodulin with two Gly linkers in CD2 (CaM-CD2-III-5G) has metal affinities with K(d) values of 1.86 x 10(-4) and 5.8 x 10(-5) M for calcium and lanthanum, respectively. The oligomeric states of the CD2 variants were examined by pulsed-field-gradient nuclear magnetic resonance (PFG NMR). The diffusion coefficient values of CD2 variants are about 11.1 x 10(-7) cm(2)/s both in the presence and absence of metal ions, which are the same as that of wild-type CD2. This suggests that the isolated EF-loop III of calmodulin inserted in the scaffold protein is able to bind calcium and lanthanum as a monomer, which is in contrast to the previous observation of the EF-hand motif. Our results imply that additional factors that reside outside of the EF-loop III may contribute to the pairing of EF-hand motifs of calmodulin. This result is of interest as it opens up the way for studying the ion-binding properties of isolated EF-hands, which in turn can answer important questions about the properties of EF-hands, the large and important group of calcium-binding signaling proteins.

Binding Sites↗

PubMed2002

Structural analysis, identification, and design of calcium-binding sites in proteins.

Assigning proteins with functions based on the 3-D structure requires high-speed techniques to make a systematic survey of protein structures. Calcium regulates many biological systems by binding numerous proteins in different biological environments. Despite the great diversity in the composition of ligand residues and bond angles and lengths of calcium-binding sites, our structural analysis of 11 calcium-binding sites in different classes of proteins has shown that common local structural parameters can be used to identify and design calcium-binding proteins. Natural calcium-binding sites in both EF-hand proteins and non-EF-hand proteins can be described with the smallest deviation from the geometry of an ideal pentagonal bipyramid. Further, two different magnesium-binding sites in parvalbumin and calbindin(D9K) can also be identified using an octahedral geometry. Using the established method, we have designed de novo calcium-binding sites into the scaffold of non-calcium-binding proteins CD2 and Rop. Our results suggest that it is possible to identify calcium- and magnesium-binding sites in proteins and design de novo metal-binding sites.

Algorithms↗

PubMed2002

Metal-binding studies for a de novo designed calcium-binding protein.

To understand the key determinants in calcium-binding affinity, a calcium-binding site with pentagonal bipyramid geometry was designed into a non-calcium-binding protein, domain 1 of CD2. This metal-binding protein has five mutations with a net charge in the coordination sphere of -5 and is termed DEEEE. Fluorescence resonance energy transfer was used to determine the metal-binding affinity of DEEEE to the calcium analog terbium. The addition of protein concentration to Tb(III) solution results in a large enhancement of Tb(III) fluorescence due to energy transfer between terbium ions and aromatic residues in CD2-D1. In addition, both calcium and lanthanum compete with terbium for the same desired metal binding pocket. Our designed protein exhibits a stronger affinity for Tb(III), with a K(d) of 21 microM, than natural calcium-binding proteins with a similar Greek key scaffold.

Amino Acids↗

PubMed2002

Temperature-induced formation of a non-native intermediate state of the all beta-sheet protein CD2.

Domain 1 of the cell adhesion protein CD2 (CD2-1) has an all beta-structure typical of proteins belonging to the immunoglobin superfamily. It has a remarkable ability to fold as a native monomer or a metastable intertwined dimer. To understand the origin of structural rearrangements of CD2-1, we have studied equilibrium unfolding of the protein using various biophysical spectroscopic techniques. At temperatures above approx 68 degrees C, a partially folded state of CD2-1 (H state) with a distinct secondary structure, involving largely exposed aromatic and hydrophobic residues and a substantially perturbed tertiary structure, is observed. In contrast, an unfolded state (D state) of CD2-1 with random-coil-like secondary and tertiary structures is observed in 6 M GuHCl. This partially folded high-temperature state has increased negative molar ellipticity at 222 nm in far-ultraviolet CD spectra, implying formation of a non-native helical conformation. The existence of this non-native high-temperature intermediate is consistent with relatively high intrinsic helical propensities in the primary sequence of CD2-1. This conformational flexibility may be important in the observed domain swapping of CD2-1.

Anilino Naphthalenesulfonates↗