Blaber Lab Recent Publications - (Updated 3/24/11)

2011

In this publication Dr. Scarisbrick describes a novel molecular mechanism for lymphocyte survival involving kallikrein-related peptidase 6 (KLK6) activation of protease activated receptor 1 (PAR1). The results are important to understand immunological responses that depend on apotosis for immune clearance.

This report describes in detail the development of the novel "top-down symmetric deconstruction" method of protein design and its application in the successful creation of a 42-residue polypeptide able to symmetrically self-assemble into a b-trefoil protein architecture.

This report is the culmination of almost 18 years of effort to develop a strategy to successfully design purely-symmetric protein architecture. The method is termed "top-down symmetric deconstruction". Existing paradigm posits that pure symmetry of amino acid sequence is not compatible with foldability; thus, it is not tenable as a de novo design principle, and logically calls into question the feasibility of gene duplication and fusion in the evolution of symmetric protein architecture. Our report disproves this paradigm, as we successfully demonstrate excellent foldability, thermostability and solubility for a purely-3-fold symmetric form of the b-trefoil protein fold. The results support one of two competing models for the evolution of this symmetric architecture (i.e. the "conserved architecture model") and also opens the door to a new approach in protein design - one that takes maximum advantage of symmetry in simplifying the design process.

2010

This report describes how a novel disulfide bond mutant of FGF-1 increases the in vitro functional half-life of the protein by 14-fold. This mutant design approach may be applicable to "second-generation" forms of different members of the fibroblast growth factor family.

This is a review article describing the known interactions between the Kallikrein-related proteases and those proteases of the thrombogenesis/thrombolysis pathways.

2009

Gold particle conjugates with proteins is a novel area of biotechnology with many unanswered questions. In particular, are such conjugates deleterious to protein conformation? This report shows that gold nanoparticle conjugates of FGF-1 are compatible with the native folded structure.

The rabbit hind limb model is the de facto animal model for studies of ischemia; furthermore, FGF-1 is emerging as the leading growth factor for pro-angiogenic therapy. Despite its relevance to these types of investigations, the cDNA sequence, X-ray structure, biophysical properties (including thermostability and receptor-binding affinity) and mitogenic activity of rabbit FGF-1 have never been reported; this publication presents such data.

Neutron diffraction studies of proteins requires large crystals; however, this is often difficult to achieve. FGF-1 crystals are large in two dimensions, but the third dimension is typically thin. This report describes mutations to the crystal contact in the thin phyisical dimension, whereby solvent-mediated contacts are replaced by direct protein-protein contacts. This approach improves the crystal growth along the thin dimension.

The FGF family of proteins contains an absolutely conserved Cys residue (at position 83 in the 140 amino acid form of FGF-1) that is present as a buried free-cysteine (in the majority of FGF proteins), or as a half-cystine involving adjacent position 66. We present evidence that mutation of position 66 to Cys can produce a stabilizing disulfide bond in FGF-1 (where Cys83 is a free cysteine), suggesting that the conserved free cysteine residue in the FGF family of proteins is actually a vestigial half-cystine.

This study identifies a cooperative interaction between buried free cysteines and the thermostability of a protein that effectively regulates the functional half-life. A protein design principle is outlined whereby the functional half-life of a mutant protein may be modulated by several orders of magnitude; furthermore, the design principle suggests a means by which a desired half-life can be achieved with limited immunogenic potential.

We complete our study of the KLK "activome" (i.e. the ability of mature KLK proteases to cleave the KLK pro-peptide and thereby activate the pro-KLKs) by reporting data for profiles of KLK9, 10 and 15. This report contains a now-complete matrix of the 215 possible pairwise activation combinations for the 15-member KLK family.

2008

Kinetic hydrolysis information is presented to show how KLK1 and Plasma Kallikrein can process human kininogen if any of the Ser386, Ser390, or Ser391 are phosphorylated in physiological or pathological conditions, and a direct route of B1 agonist receptor [des-(Arg9)-Lys-Bk] release from kininogem is proposed.

This study shows that KLK6, but not KLK1 is an activator of CNS Protease Activated Receptors (PARs), and that both kallikreins are poised to signal in a B2 receptor-dependent fashion to regulate multiple signal transduction pathways relevant to CNS physiologic function and dysfunction.

The results of this study show that proteases of the thrombostasis family can efficiently activate specific pro-KLKs, demonstrating the potential for important regulatory interactions between these two major protease families.

Serum levels of KLK1 and KLK6 were elevated in Multiple Sclerosis (MS) patients, with highest levels associated with secondary progressive disease. Elevated KLK1 correlated with higher EDSS scores at the time of serum draw and KLK6 with future EDSS worsening in relapsing remitting patients. These novel findings suggest that KLK1 and KLK6 may serve as serological markers of progressive MS and contribute directly to the development of neurological disability by promoting axonal injury and neuron cell death.

FGF-1 crystal growth is limited along one axis, and this may be due to charge repulsion interactions at a crystallographic two-fold axis at position Glu81. Mutations at this position were evaluated for their ability to enhance FGF-1 crystal growth for the purpose of obtaining large crystals for neutron diffraction. A Glu81 to Ser mutation resulted in noticeably thicker crystals.

The substrate specificity of KLK1 and KLK6 was analyzed by substrate phage display using a random octapeptide library. Docking simulations of consensus peptide provide information on the identity of the enzyme residues that are responsible for substrate binding. Bioinformatic analysis suggested several putative KLK6 protein substrates, such as ionotropic glutamate receptor (GluR) and synphilin.

Type 1 b-turns contain a consensus sequence of Asx-Pro-Asx-Gly, however, the details of the contribution of the residues in this motif to the turn structure, folding and stability are poorly understood. This study, involving 28 mutants of FGF-1, undertakes a comprehensive study of Asp, Asn and Ala mutations at each of the two canonical Asx positions. The results demonstrate a type of "Logical OR" interaction between the Asx residues that serve to stabilize the turn structure, and can even protect it from deleterious mutations.

2007

The ability of the different KLK proteases to cleave the 15 different pro-KLK peptide sequences was characterized using a fusion protein method. The results demonstrate the activation potential of the KLK's, and permit construction of hypothetical KLK activation cascades.

The first and last beta-strands in FGF-1 form a beta-sheet and are known to be a key step in the folding pathway. Two positions in these regions, Lys12 and Pro134, are shown to destabilize the structure. Mutation to Val provides a substantial 6 kJ/mol of additional stability at each position. The results suggest that targeting the termini beta-strand interactions in a beta-barrel might be a generally useful approach to increase protein stability. Furthermore, although FGF-1 is characterized as a marginally-stable protein, the results suggest that a handful of mutations might shift the stability into the "thermophile" regime.

This study quantifies the ability of KLK6 to hydrolyze its pro-peptide activation sequence and internal autolytic inactivating sequence. The results show that KLK6 activity is two-orders of magnitude greater for autolytic inactivation as compared to pro-sequence activation. Furthermore, several other proteases are compared to KLK6 and shown to exhibit much greater efficiency in activating pro-KLK6. Overall, the results show that KLK6 is unlikely to effectively self-activate, and requires a distinct protease for this purpose (thus forming the basis for a proteolytic activation cascade).

2006

In this study by our collaborator, Dr. Morley Hollenberg, KLK14 is shown to be able to activate PAR1, 2 and 4; KLK5 and 6 are also shown to be able to activate PAR2 but do not cause PAR4-dependent aggregation. The results show that KLK's may represent important regulators of the PAR's, and exhibit differential activities.

In this study by our collaborator, Dr. Isobel Scarisbrick, data is presented to support the hypothesis that KLK6 activity in the CNS mediates events secondary to spinal cord trauma, including dynamic modification of the capacity for axon outgrowth.

In this study by our collaborator, Dr. Isobel Scarisbrick, the potential scope of action of KLK's in T cell-mediated immune response is characterized. Novel effector roles of specific KLK's in neurological disorders involving T cells (such as Multiple Sclerosis) are proposed..

In this study by our collaborator, Dr. Morley Hollenberg, KLK5, 6 and 14 are shown to cleave peptide sequences representing the activation motifs of plasminogen-activated receptor (PAR) 1 and 2. The results indicate that the KLK's must now be considered as "hormonal" regulators of tissue function via PAR activation.

This paper describes a new nomenclature for the human kallikrein-related peptidases, supported by a consortium of investigators led by Dr. Ake Lundwall.

In this study by our collaborator, Dr. Luiz Juliano, the substrate specificity of KLK6 and effects of cosmotropic salts of the Hofmeister series are studied using internally-quenched fluorescent peptide substrates. The results identify AFRFSQ as the best synthetic substrate described so far for KLK6, and suggest possible modulation of ionotropic glutamate receptors and actvation of PAR 2 by KLK6.

FGF-1 contains fundamentally two types of beta-turns: type I 3:5 and type I 4:6. The role of the type I 4:6 turn was probed by mutations designed to convert the turn into type I 3:5. The results showed that a type I 3:5 turn can be implemented at each turn location in FGF-1 and yield a stable folded polypeptide. However, the mitogenic activity of such a mutant is substantially decreased, due to the functional requirement of a specfic type I 4:6 turn for receptor interaction. Thus, functional rather than folding considerations have resulted in the presence of a type I 4:6 turn in FGF-1.

2005

This is a contributed book chapter, detailing the solvent structure in the active site of human kallikrein 1, as presented at the International Workshop on "Hydrogen and Hydration in Proteins and Nucleic Acids", January 17-18, 2005, The University of Tokyo.

Symmetry-related hydrophobic packing groups in FGF-1 exhibit an asymmetric tolerance to side chain substitution. The packing environment of the positions in question is formed by specific surface loops. These loop regions vary in both sequence and length, and these differences dictate the asymmetric tolerance of the core substitutions. Thus, mutations in a specific surface loop region have "locked in" a requirement for a Cys residue at position 83. This residue cannot be mutated to a symmetrically-conserved Ile residue without first mutating the surrounding surface loop. Thus, this report highlights the co-evolutionary aspect of core and surface regions.

Only the second example of a human kallikrein structure to be deposited in the structural databank, we describe the x-ray structure of human apo- kallikrein 1. We detail the structural features, including solvent structure, of the S2 to S2' binding pockets. In comparison to porcine K1 with bound peptide inhibitors, the apo- kallikrein 1 structure exhibits a variety of induced-fit conformational changes upon substrate binding. These induced-fit changes include the active site serine, and indicate that the binding energy is utilized to position the active site serine for efficient catalysis.

Two b-hairpin structures in human fibroblast growth factor 1 are the same length but have different structures. Since they also differ in sequence, we thought this might be the reason for their different structures. However, sequence swapping mutations between the two turns did not result in a corresponding structural change. Thus, the turn structure is likely determined by the interaction of the turn residues with the local environment.

In this collaboration with Dr. Donald Caspar the structural and energetic consequences of mutations designed to alter the hydrophobic characteristics of the central cavity within interleukin 1-b are described. These mutations were designed to probe potential interactions with positionally-disordered solvent within the cavity (previously identified using low-resolution x-ray crystal data).

2004

This report is by Dr. Isobel Scarisbrick, our long-time collaborator at the Mayo Clinic, Rochester. In this report Isobel shows that part of the regulatory mechanism of human kallikrein 6 (K6) is determined by alternative promoters resulting in tissue-specific transcripts. Tissue-specific expression patterns and differential regulation in CNS disease indicates that each K6 5’ transcript is likely regulated by unique promoter elements and may serve as a molecular target to treat inflammatory demyelinating disease.

This paper is really important for the lab. It shows that mutations designed to increase both the tertiary and primary structure symmetry of human fibroblast growth factor 1 also result in a mutant form that is dramatically more stable than the wild type protein. Thus, we believe that the b-trefoil (symmetric) superfold can be designed with a symmetric constraint upon the primary structure. There are several important ramifications of this work, related to protein evolution and de novo design. We hypothesize that a fundamental property of the symmetric protein superfolds is the ability to adopt a symmetric primary structure with an intrinsically high thermal stability.

This report describes a 1.10Å resolution x-ray structure of human fibroblast growth factor 1. The data was of such high-resolution and quality that we were able to refine anisotropic thermal factors (i.e. the three-dimensional positional displacement for each atom). Using this information we asked whether certain regions of the protein exhibit correlated anisotropic motion (i.e. rigid body motion). We identified a region involving b-strands 5-12 that act as a rigid body distinct from the rest of the molecule. This demarcation also delineates heparin-binding from receptor-binding functionalities within the protein. It was selected as the cover article for the November 2004 issue of Proteins (the second cover article for the lab).

Another report by our long-time collaborator, Dr. Isobel Scarisbrick from the Mayo Clinic, Rochester. This paper is breakthrough work that shows that if the activity of kallikrein 6 (K6) is reduced (using a pre-immunization strategy) then animals that have been treated so as to develop a Multiple Sclerosis type of disease exhibit a delayed onset, and reduced severity. Thus, K6 represents a novel target in the treatment of inflammatory neurological diseases, like MS.

In this report we describe the x-ray structure of a mutant form of 2,5-diketo-D-gluconate reductase (the "vitamin C enzyme") which has an enhanced selectivity for NADH, versus NADPH, as a cofactor. The mutant was designed by our collaborator, Dr. Stephen Anderson at Rutgers University, and should prove useful in a new method for the industrial production of vitamin C.

This is a short invited review of a very interesting protein engineering study by Dr. Brian Matthews that investigates a possible mechanism for the evolution of allostery.

This is another short, invited review of myelencephalon specific protease (rat kallikrein 6) for the Handbook of Proteolytic Enzymes. The primary author of the review is Dr. Isobel Scarisbrick, our long time collaborator at the Mayo Clinic, Rochester.

2003

This is a report that details our NMR studies of turn mutants in human fibroblast growth factor 1 that were performed at the National High Magnetic Field Laboratory. Some mutants do not readily crystallize, and Jihun Lee and Jaewon Kim, two graduate students in the lab, are spearheading efforts to utilize NMR to obtain structural information for such mutants.

This is another very important paper for the lab. It details the ability to redesign the core region of human fibroblast growth factor 1 using a symmetric design constraint. Despite the imposed design constraint, the repacked core is among the most successful published and let to our hypothesis (developed in our later publications) that perhaps symmetric superfolds can be successfully designed using a symmetric primary structure. This article was selected as the cover for the Dec 2003 issue of Protein Science - the first cover article for the lab.

This paper details one of the more successful discoveries of a design principle for turn structures. We show that a glycine amino acid at the i+3 position in a type I 3:5 and 4:6 b-turn plays a critical role in stabilizing such structures. This discovery allowed us to reconcile conflicting published reports regarding the thermodynamic consequences of glycine substitutions at positions that lie within the L-a region of the Ramachandran plot.

This is an invited review article that arose from my graduate student, Gulsah Sanli, giving a poster at a scientific conference. In addition to presenting a review of known information, Gulsah also included a structural analysis of the apo- and holo-forms of 2,5-diketo-D-gluconate reductase and identified a hinge-bending motion associated with cofactor binding. The hinge-bending motion delineates two halves of the symmetric structure of the enzyme, and suggests that it may have evolved from an ancient homo-dimer.

2002

This report describes the x-ray structure determination for human kallikrein 6 (K6) - the first human kallikrein structure deposited into the Protein Data Bank. Unknown to us at the time, another group also solved a structure for K6 (the inactive pro-form). Their report came out only two weeks after ours, and in the same journal! K6 is shown to be structurally related to the digestive enzyme trypsin, although it is expressed primarily in the brain and spinal cord.

This is a report written by our long-term collaborator Dr. Isobel Scarisbrick (Mayo Clinic, Rochester). Isobel describes how kallikrein 6 (K6) is present in regions of active demyelination in Multiple Sclerosis lesions, and how exogenously added K6 causes cultured neurons to retract their processes. The results identify a role for K6 in the biology of myelin, and its potential implications in demyelinating disease.

In different crystal forms of human fibroblast growth factor a particular turn region exhibits distinctly different structures. Which one is correct, and which one may be a crystal packing artifact? This study describes a series of glycine mutations, in conjunction with stability studies, that identify one particular conformation as being likely populated in solution. The results also describe a statistical study of the structural data bank that suggests that a certain turn conformation may be present at higher than expected percentages due to crystal packing artifacts.

Our lab was the first to express recombinant kallikrein 6 (from the rat) and to determine its enzymatic properties. In this report, K6 is identified as a digestive-type protease, with broad substrate specificity. We also show that K6 has a built-in mechanism of autolytic inactivation, and this likely plays a key regulatory role.

2001

This is our first report on efforts to redesign the core region of fibroblast growth factor 1 using a symmetric design constraint. We introduce three mutations into the 15 amino acid core and show that the mutations are well-tolerated. This study paved the way for further symmetric redesign studies.

Two outstanding undergraduate students in the lab, Sasha Grek and John Davis, undertook the Herculean task of writing a Windows™-based software program to deconvoluted data from differential scanning calorimetry studies. The program was a success, and currently 15 copies are downloaded each month from the lab's website.

We solved the x-ray structures of 2,5-diketo-D-gluconate reductase (the "vitamin C enzyme") in both holo- (i.e. with NADPH cofactor) and apo- forms (i.e. without) and discovered that the active sites is in a non-catalytically competent conformation in the absence of bound cofactor. Thus, the NADPH cofactor not only provides a hydride for catalysis, but also serves to organize the active site. This was the first time that such a role for the cofactor in this family of enzymes had been reported.

Genes from Corynebacterium are GC rich and prove problematic for DNA polymerase-based manipulations (i.e. PCR and sequencing). We designed a couple of synthetic genes that retained the amino acid sequence, but substantially reduced the GC content (and also improving E. coli codon bias). These genes were shown to be amenable to polymerase-based manipulations, and could be readily mutated, sequenced and expressed in E. coli. This study laid the groundwork for further mutagenesis studies to improve the stability and catalytic features of this enzyme.

2000

Previous reports suggested that cysteine to serine mutations in human fibroblast growth factor 1 (FGF1) resulted in substantially longer half-life in cell culture mitogenic assays. This observation was postulated to be due to either an increase in stability or elimination of thiol-based modifications. We had developed the methodology for determination of the DG unfolding for FGF1 and decided to test the stability hypothesis. Our results showed that the serine mutations destabilized the protein. Thus, the increase in half-life was due to elimination of thiol-based modifications. We postulate that a mechanism to limit protein half-life is to introduce free cysteine residues within the core region of a protein. Such proteins cannot refold if the cysteine forms thiol adducts or are chemically modified by oxidation.

This report is written by Dr. Isobel Scarisbrick, our long-time collaborator at the Mayo Clinic, Rochester. In this report Isobel describes the distribution of kallikrein 6 in the rat spinal cord, and shows that it is associated with white matter - i.e. the region of the CNS that is rich in myelin (the insulating sheath around nerve cells). The results suggest a potential role of K6 in myelin biology.

This report details our early implementation of the DSCFit software, designed to deconvoluted differential scanning calorimetry data.

Using bound solvent as a guide, this report details the modeling of substrate into the active site of 2,5-DKG-reducates, the "vitamin C enzyme". Although this enzyme was originally identified from screening of soil bacteria capable of reducing 2,5-diketo-D-gluconate, this study shows that this compound is unlikely to be the natural substrate.

1999 (partial listing)

This was an important publication for the lab. We detail the development of the methodology by which the reversible thermal denaturation of human fibroblast growth factor 1 can be achieved. The results allowed us to demonstrate the generally good agreement between isothermal equilibrium denaturation and differential scanning calorimetry, and showed that the 2-state models being invoked to analyze the data were valid.

This report was part of a collaboration with Dr. Don Caspar at Florida State University. Interleukin-1b has a large central cavity and there were conflicting reports in the literature on whether this cavity contained disordered solvent. By collecting low resolution data and scaling the electron density on an absolute scale, we were able to show that the central cavity contains approximately 1.8 water molecules worth of disordered electron density - suggesting that solvent is indeed rattling around within the core.

1998 (partial listing)

This was the first x-ray structure for a prokaryotic member of the aldo-keto reductase family. The structure provided details of the structural "seat belt", a pair of loops that covered the cofactor binding site and contributed to the kinetics of cofactor binding.

1996 (partial listing)

This is the first x-ray structure reported for human acidic fibroblast growth factor, and we beat another group by several months. A single crystal was used for both spacegroup determination (using precession photography) and data collection (using a multiwire detector). This was in the good old days when you needed to know the space group and cell dimensions prior to data collection. This is also the first crystal structure reported by our laboratory.